Sage Journals: Discover world-class research

Abstract

This mini-review will schematically update the progress of the expanding Mi-2/Nucleosome Remodeling Deacetylase (NuRD) complexes in cancer and in normal development such as stemness, with a focus on mammals and the increasingly popular and powerful model organism Caenorhabditis elegans. The Mi-2/NuRD complexes control gene activity during the development of complex organisms. Every Mi-2/NuRD complex contains many different core polypeptides, which form distinct multifunctional complexes with specific context-dependent regulators. The Mi-2/NuRD complexes have unique ATP-dependent chromatin remodeling, histone deacetylase, demethylase activities and higher order chromatin organization. They can regulate the accessibility of transcription factors or repair proteins to DNA. In this review, we summarize our current knowleges in the composition, interaction and function of the subunits within the Mi-2/NuRD complex, the methodology used for the identification of Mi-2/NuRD complexes, as well as the clinical and therapeutic implications targeting the Mi-2/NuRD subunits.

Keywords

Mi-2/NuRD complex cancer stemness mammals

Overview

To gain an insight into the functions of the Mi-2/ NuRD complex, we first need to understand the composition of this complexes. The Mi-2/NuRD complex is a expanding family of protein complexes, which control gene activity spatiotemorally during the development of complex organisms. In general, it has been regarded as one biochemical entity containing a number of core polypeptides but forming distinct multifunctional complexes with specific context-dependent regulators.^1,2 Some of these complexes are active during normal development such as stemness^3–7 and in cancer^8,9 (Table 1). These complexes are characterized by their unique ATP-dependent chromatin remodeling, histone deacetylase, demethylase activities and higher-order chromatin organization.^2,24 Nucleosomes form the building blocks of chromatin and, in general, these nucleosomes inhibit processes that require access to the DNA template, such as transcription and DNA repair. Mi-2/NuRDs use ATP hydrolysis to change the nucleosomes in DNA and they can regulate the accessibility of transcription factors or repair proteins to DNA.²⁵ The two highly homologous proteins Mi-2α/CHD3 and Mi-2β/CHD4 represent the catalytic ATP hydrolyzing subunits in the complex. The histone lysine-specific demethylase (LSD1/KDM1) was recently shown to be recruited to the NuRD complex via interaction between the Tower domain and metastasis-associated proteins MTA1-3 in breast cancer cells.⁸ This newly identified subunit demethylates both di- and mono-methylated K4 (H3K4me2/1).²⁶ In addition, the core of the Mi-2/NuRD complex contains:

•

at least two histone deacetylases, HDAC1 and HDAC2;

•

the retinoblastoma-associated tumor suppressors RbAp48 (RBBP4) and/or RbAp46 (RBBP7);

•

MTA1 and/or 2 and/or 3;

•

GATAD2b (p66α) and/or GATAD2a (p66β);

•

mCpG-binding domain proteins MBD2 and/or MBD3.²⁷

Table 1

The neoplasms associated with core subunits of the Mi-2/NuRD compelx.

Core subunit	Cancers associated with deregulation of core subunits of the Mi-2/NuRD complex	References
Mi-2 α	Breast and non-Hodgkin lymphomas.	Wang et al⁸
		Pencil et al¹¹
		Toh et al^12,13
Mi-2 β	Breast, ovarian, lung, pancreatic, stomach and colorectal cancers; Non-Hodgkin lymphomas.	Hill et al¹⁰
Mi-2 β		Wang et al⁸
MTA1	Breast, pancreatic and prostate cancers; Lymphoma; Thymoma; Head and Neck Squamous cell Carcinoma (HNSCC) and Non-Small Cell Lung Cancer (NSCLC); Gastrointestinal cancer and adenocarcinoma.	Wang et al⁸
		Toh et al⁹
		Mazumdar et al¹⁴
MTA2	Breast metastasis and ovarian cancer.	Wang et al⁸
		Ji et al¹⁶
		Cui et al¹⁵
		Toh et al⁹
MTA3	Breast cancer	Wang et al⁸
MTA3	Breast cancer	Toh et al⁹
RBBP4	Breast, oropharyngeal and prostate cancers; Primitive neuroectodermal tumor of the Central Neruous System(CNS); Retinoblastoma; Skin tumor	Wang et al⁸
RBBP7	Breast, lung and prostate cancers; Primitive neuroectodermal tumor of the CNS; Retinoblastoma; Skin tumor	Wang et al⁸
HDAC1	Breast, testicular, prostate, lung, colorectal, gastric, ovarian and pancreatic cancers; Hematopoietic disorders	Wang et al⁸
		Kim et al¹⁷
		Minucci et al¹⁸ Choi et al¹⁹
HDAC2	Breast, colon, pancreatic, cutaneous T-gastric, cervical, prostate and colorectal cancers; Cell lymphoma	Wang et al⁸
		Kim et al¹⁷
		Minucci et al¹⁸
LSD1	Breast, prostate, colon cancer and bladder cancers.	Wang et al⁸
		Shi et al²⁰
		Huang et al²¹
MBD2	Breast, prostate, colorectal, bladder and colon cancers; Oral carcinogenesis	Wang et al⁸
		Le Guezennec et al²²
		Zhu et al²³
MBD3	Breast, lung and colon cancers; Mouse lymphosarcoma; Oral carcinogenesis	Wang et al⁸
		Le Guezennec et al²²
		Zhu et al²³

Importantly, the stemmed Mi-2/NuRD complex emerges from these core components. The gene targeting mechanism of MBD2-recruitting Mi-2/NuRD (ie, the MeCP1 complex)²⁸ and the gene targeting mechanism of the transcriptional factor-interacting M-2/NuRD complex (eg, the lymphoid transcription factor Ikaros,²⁹ the transcriptional corepressor KAP-1,³⁰ the tumor suppressor p53³¹ and Bcl-6)^32,33 have been determined as the interactions that are mediated by Mi-2α (Ikaros and KAP-1), MTA-2 (p53) or MTA-3 (Bcl-6), and therefore different transcriptional regulators use distinct NuRD subunits for recruitment. However, the details of the mechanisms of many other co-regulators (Supplementary Table 1) interacting with Mi-2/NuRD complexes remain largely unknown. The distinct tissue- and cell-specific Mi-2/NuRD complexes can execute their multifunctional, fine and spatiotemporal regulation in a variety of fundamental biological processes. In principle, this diversity could challenge the capture of all or most interactors from one purification experiment using core subunits of the Mi-2/NuRD complex tagged with a single condition; on the other hand, for cancer, cell-specific transcriptional factors and co-regulators of the Mi-2/NuRD complexes offer unexpected therapeutical benefits for designing both systemic and local site treatments (eg, RNAi therapeutics) in the future and may enable different therapeutic strategies tailored to patients with different profiles. These studies have benefitted from the comparative advantages of interspecies Mi-2/NuRD complexes.^34,35 Finally, increasing our knowledge of the Mi-2/NuRD complex could significantly advance cancer therapy.

The Interacting Proteins and Functions of the Core Subunits within the Mi-2/NuRD Complex

The Mi-2α, β/CHD3, 4 proteins

The evolutionarily conserved chromatin remodeling factor Mi-2 protein was first identified as an autoantigen in patients with dermatomyositis^36,37 and belongs to the chromodomian CHD3-CHD4 ATPase subfamiliy (CHDs) II. Mi-2 has been isolated from human cell lines, mice, plant, fly, worm and Xenopus egg extracts^27,36–43 (Fig. 1). The human genome contains two genes encoding isoforms of Mi-2: Mi-2α/CHD-3 and Mi-2β/CHD-4.⁴⁴

Figure 1

The structural domains of Mi-2α,β/CHD3,4 proteins.

Mi-2 proteins contain several domains: two PHD zinc fingers, two chromodomains (chromatin organization modifier), a SWI2/SNF-like helicase domain and a telobox-related motif⁴³ (Fig. 1a). PHD zinc finger domains are thought to be involved in protein-protein interactions. In fact, the Mi-2 PHD domains were shown to be required for Mi-2's interaction with HDAC1.⁴³ At least part of the nucleosomal recognition is contributed by Mi-2 itself, since the ATPase activity of the recombinant enzyme is strongly stimulated by nucleosomes, but does not react to the presence of free histones or DNA.⁴⁵ Mi-2 and its complex regulate gene expression by modifying chromatin accessibility during development. The Mi-2 subunits are implicated in a wide variety of cancers (Table 1). Mi-2β acts as a transcriptional repressor and as an activator, which is required for hematopoietic stem cell self-renewal and differentiation.^46,47 The Mi-2/NuRD complex has significant roles in DNA damage and repair.^{25,35,48–50} TWIST1 interacts with several components of the Mi-2/NuRD complex, Mi-2β, RbAp46, MTA2 and HDAC2, and recruits them to the proximal regions of the E-cadherin promoter for transcriptional repression. Depletion of these TWIST1 complex components from cancer cell lines, which depend on TWIST1 for metastasis, efficiently suppresses cell migration and invasion in culture and lung metastasis in mice.⁵¹

Previous reports showed that Mi-2α and Mi-2β can coexist within the same complex.^39,42 So far, it remains unknown whether these Mi-2α and Mi-2β are forming heterodimers or whether they are assembled into distinct complexes.

But it is likely that all 3 types of complexes exist like ie, Mi-2α/NuRD,³⁰ Mi-2αβ/NuRD and Mi-2αβ/ NuRD. Furthermore, though the increasing number of published interactors of Mi-2 proteins show up (Fig. 2; supplementary Fig. 1), we are in await for a systematic screening of interactors of Mi-2 proteins via current proteomic assay, which has evolved and matured to level where it is able to assess the complexity of the human proteome.⁵²

Figure 2a.

The interactors of mammalian Mi-2β/CHD4.

Figure 2b.

The interactors of mammalian Mi-2α/CHD-3.

Figure 2c.

The interactors of C. elegans Mi-2β/LET-418.

Two Caenorhabditis elegans Mi-2 orthologs, LET-418 and CHD-3, have been cloned and characterized.⁵³ let-418 has proven to be an essential gene expressed in most, if not all, nuclei of the worm. Mutations in let-418 have pleiotropic phenotypes, including vulval defects and sterility and, without maternal contribution, result in L1 larval arrest.⁵³

Furthermore, let-418 is required for the maintenance of somatic differentiation in C. elegans.⁵⁴ By contrast, mutations in chd-3 do not display any obvious phenotype under standard culture. However, a requirement for chd-3 becomes apparent in let-418(lf); chd-3(lf) double mutants, which display strong vulval defects and L4 arrest or, without any let-418 maternal contribution, embryonic arrest. These results suggest that let-418 and chd-3 have essential and partially redundant functions during development.⁵³ Previously, we clarified the functions of LET-418/Mi-2β using genetic and biochemical techniques including Chromatin Immuno-Precipitation (ChIP), quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR), co-immunopreciptation, reporter assays and suppression subtractive hybridization (SSH); we demonstrated that LET-418/Mi-2β along with LIN-1/ ETS directly binds the promoter of the lin-39/Hox gene to repress its expression⁵⁵ and LET-418/Mi-2β directly regulates LAG-2/Delta in the LIN-12/Notch pathway.⁵⁶ LIN-39/Hox itself performs an auto-regulatory function as well as directly controlling EGL-17/ FGF5 and LAG-2/Delta^55,57,58 (Zhang and Mueller, unpublished; Fig. 3). The role of regulation of Mi-2/ NuRD in the LIN-12/Notch pathway has been demonstrated in different labs.^53,59,60 Furthermore, spr-5 encodes the C. elegans LSD1 homolog and is implicate in Notch signaling.^61–64 Mittal et al recently observed high-level expression of Notch receptors and ligands, and its increased activation in several human breast cancers and early precursors. This places Notch signaling as a key player in breast cancer pathogenesis. Notch signaling cooperates with the Ras/MAPK pathway in transformation and it offers combined inhibition of the two pathways as a new modality for breast cancer treatment.⁶⁵ The cross-talk and precise molecular cooperation between EGF/RAS and LIN-12/Notch cell signalling pathways have been clearly shown in C. elegans vulva development.⁶⁶

Figure 3

The downstream target genes of the Mi-2/NuRD complex in C. elegans. Upper: The LET-418/Mi-2/NuRD complex (blue star) represses lag-2 transcription (red dashed line). Middle: LIN-39/HoxD4 binds to the promoters of lag-2/Delta, egl-17/FGF5, lin-39/HoxD4 and possibly lin-12/ Notch, and then promote the activities of respective genes (green line). Bottom: A model for how NuRD antagonises vulval cell fate. 1) In the absence of EGF/RTK/Ras signaling, LIN-1/ETS (red rectangle) binds to the promoter of lin-39, and it recruits a LET-418/Mi-2/NuRD complex and represses lin-39 transcription by affecting histones and condensing the chromatin. The repression, however, is not complete, since basal levels of LIN-39 are required for normal vulval development. 2) EGF/RTK/Ras signaling results in LIN-1/ETS phosphorylation, which disrupts LIN-1/ETS interactions with the LET-48/Mi-2/NuRD complex and upregulates the target gene lin-39 transcription required for vulval cell fate induction.

The mCpG-binding domain (MBD) proteins

MBD2/Mi-2/NuRD and MBD3/Mi-2/NuRD

As Figure 4 shows, MBD proteins have the mCpG-binding domain. MBD3's MBD domain has two point mutations that abolish the binding. MBD1, MBD2 and the methyl CpG binding protein 2 (MeCP2) have the Transcriptional Repressive Domain (TRD). MBD2 and MBD3 can form both homo- and heterodimers because both have the Coil-Coil (CC) domain. MBD4 is involved in DNA repair. Nearly all cases of Rett Syndrome are caused by a mutation in the MeCP2. The MBDs bind sites of hypermethylation in human cancer cell lines. MBDs function in transcriptional repression and long-range interactions in chromatin, and also appear to play a role in genomic stability, neural signaling⁸⁰ and transcriptional activation.⁸¹ MBD3 was shown to be an integral component of the Mi-2/NuRD complex,⁸² whereas murine MBD2, which can bind methylated DNA,⁸³ targets the Mi-2/NuRD to methylated CpG dinucleotides.^82,84 In general, MBD2 and MBD3 have been reported in a common Mi-2/NuRD complex, but different sets of inertactive proteins of MBD2 or MBD3 have been reported (Fig. 5). MBD2/Mi-2/NuRD complex (ie, the MeCP1 complex) was shown to be present at very early stages of development and to inactivate methylated promoters in the mouse embryo prior to implantation.⁸⁵ However, in MBD2/MBD3 tagged complex purification, in oral carcinogenesis and colon cancer, the MBD2 eluate but not that of MBD3 contained the arginine methyltransferase PRMT5, MEP50 and importin α nuclear transport proteins. The association between MBD2 and importins may indicate that MBD2 shuttles between the cytoplasm and the nucleus. The over-expression of DOC-1 identified in purification results in a G1 arrest and significant growth retardation compared to wild-type cells, which is consistent with loss of the protein in tumors. A proposed model is shown in Figure 6c,d: the MBD2/NuRD complex deacetylates the nucleosomes surrounding the targeting site;the addition of transcriptional repressive arginine methyl marks the H4 tail by its associated PRMT5. The hypoacetylated and arginine methylated nucleosomes surrounding the MBD2/ PRMT5 targeting site in turn may provide a binding scaffold for the MBD3/NuRD complex. The MBD2/ NuRD and MBD3/NuRD complexes co-occur on some CpG islands. The MBD3/NuRD complex can further deacetylate the nucleosomes and then facilitate the spreading of deacetylation and maintenance of transcriptional repression.²² Unlike the other MBD members, MBD3 is unable to bind methylated DNA Human MBD3 and HDAC1 are localized at Aurora-A-positive centrosomes in the M phase.⁸⁶ MBD3 is transiently phosphorylated during the late G2 and early M phase, and Aurora-A is the candidate kinase for its phosphorylation.

Figure 4

The structural domains of the mCpG-binding domain (MBD) proteins.

Figure 5a.

The interactors of mammalian MBD2.

Figure 5b.

The interactors of mammalian MBD3.

Figure 6

Summary of MBD/Mi-2/NuRD complex models. A) The inhibition model of mammalian MBD3L2 on MBD2 binding of CpG. The direct binding of MBD2 and MBD3 recruits the MBD2/MeCP1/NuRD complex to CpG-methylated DNA MBD3L2 can interact with MBD3, then compete with the MBD2, potentially leading to a loss of mCpG binding in specific forms of the MeCP1 complex and gene reactivation. The interactions of MBD2 and MBD3L2 with MBD3 may be mutually exclusive. B) The mutual antagonistic inhibition model of mammalian MBD3L1 and MBD2 interacting with MBD2 binding on CpG. Because MBD3L1 interacts with MBD2 but not MBD3 in vivo, and MBD3L1 and MBD3 bind to overlapping regions on the MBD2 protein, MBD3L1 replaces MBD3 and may form a special “isoform” of the MeCP1 complex. C, D) Independent model of the mutual exclusion of mammalian MBD3 and MBD2 to bind to CpG.

In mice and humans, two proteins with high homology to MBD3 were identified: MBD3L1 and MBD3L2 (methyl-CpG-binding protein 3-like 1 and 2), which lack the MBD domain.⁸⁷ MBD3L1 can enhance MBD2-mediated repression in CpG-methylated promoters. MBD3L1 and MBD3 were shown to bind to overlapping regions of MBD2. MBD2 may interact with MBD3 or with MBD3L1 to form two different complexes with redundant function.⁸⁷ The Mi-2/NuRD complex may interact either with MBD2 or MBD3L2, via MBD3 (Fig. 3). MBD3L2 was shown to oppose MBD2/NuRD-mediated methylation silencing⁸⁸ by recruiting the complex away from methylated DNA and reactivating transcription. The hetero-oligomeric complexes can be formed between the four related proteins MBD2, MBD3, MBD3L1 and MBD3L2 and therefore different subforms of the Mi-2/NuRD complex (Fig. 6a,b).

Interestingly, the C. elegans genomic DNA is unmethylated, so C. elegans mbd-2⁸⁹ may exist in vestigial mammalian MBD3. The general role of MBD proteins in epigenetic regulation may be independent of methyl-DNA binding. Interestingly, while searching for the C. elegans homologs of components of the Mi-2/NuRD complex, we identified that CID-1, FLT-1 (now the C. elegans ACF1 homolog) adn R05D3.11 (now Met-2) have about 25% or better amino acid indentityl to the mammalian MBD domain, and knock-down (KD) animals with RNA interference against these genes had a low percentage of weak multivulvae, a phenotype of LET-418/Mi-2 deficiency. For the checkpoint protein CID-1, it remains tempting to know if it could transiently recruit Mi-2/NuRD complex in order to execute its function in longevity⁹⁰ (Zhang, unpublished).

The metastasis-associated (MTA) proteins

A decade ago, the function of the MTA (metastasis-associated gene) subunits of Mi-2/NuRD complex were largely unknown. At that time, MTA protein was a newly discovered family of cancer progression-related genes and their encoded products. The MTA gene family is clearly involved in oncogenesis and it is also used as an important marker of prognosis in cancer. The vertebrate MTA family of proteins is encoded by three genes that code for five isoforms: MTA1 and a splice variant known as MTA1s, MTA2, MTA3 and a longer isoform MTA3L, and the ZG29p (Fig. 7), an N-terminal truncated form of MTA1 that is present in the zymogen granules of the pancreas (Fig. 4a). MTA1 is the original member of a small family of metastasis-associated genes which also includes MTA2 and MTA3. The longer MTA1 isoform contains a bromo-adjacent homology (BAH) domain, an EGL-27 and MTA1 homology (ELM2) domain, a GATA-type zinc finger and a MYB domain. It contains four phosphorylation sites two serine and two threonine and two SRC homology-3 (SH3)-binding motifs.⁹³ MTA1s (short isoform) is missing the 285 AA C-terminal sequence and has a substituted 33 AA C-terminal compared to the long form. MTA1s contains an estrogen receptor binding motif (LRILL) not found in the long form. The distinct Mi-2/NuRD complexes are defined by different MTA variants with different composition and distinct functions that presumably target different sets of gene promoters.^27,93 Presumably, MTA2 is expressed constitutively and may be involved in housekeeping functions of the Mi-2/NuRD complex, whereas MTA1 and MTA3 could be involved in cell type-specific transcription, since they are expressed in a tissue-specific manner.²⁷

MTA1/Mi-2/NuRD

Through differential cDNA screening, MTA1 was initially identified as being abundantly overexpressed in highly metastatic rat mammary adenocarcinomas.^11–13 Elevated expression of MTA proteins has been associated with many types of metastatic cancers such as gastrointestinal and esophageal carcinomas, and mammary adenocarcinomas, along with increased tumor aggressiveness, invasiveness, metastasis and poor prognosis^27,91–94 (Tables 1 and 2; Fig. 7).

Figure 7

The structural domains of the metastasis-associated (MTA) family members. All major members can be a subunit of the Mi-2/NuRD complexes. The different role of each member of the family is still not clear but there is high similarity among them. The BAH domain is involved in protein-protein interaction, whereas the SANT domain binds to histone tails.

Table 2

A short list of target genes of the core subunits of the Mi-2/NuRD complex.

Highlighted subunit	Validated/hightlighted targets	Method	Complex	References
HSF1	Hsp70, c-Myc, pS2	ChIP, reporter assay	NuRD (MTA1, HDAC1/2, CHD3)	Khaleque et al⁶⁷
Mi-2β	Tek, Mpl, Kit receptor, Lxn, Ltbp3, Dmxl2, Il17re, Tgm2, Ndn, Dach1, Ddx4, Pkd2, sash1, Alox5ap, Il-27, Mamdc2, Tgm2, kit, Ccnd2, Csf1r, Il6Ra, IL6st, Egr1, Ccr1, Ccr2, Hba-a1, hbb-b1, Dntt, Thy1, Rag1, Dppa5, Hba-x, Tcfl5, etc.	microarray analysis, RT-PCR,	NuRD	Yoshida et al⁴⁷
let-418/Mi-2β	lin-39, lag-2, sqt-2, sqt-3 etc.	SSH, RT-PCR, ChIP	NuRD	Guerry et al⁵⁵ Zhang et al⁵⁶
MBD3	Pramel6, Pramel7, Dppa3, Gata6, Brachyury, Tbx2, Tpbpa etc.	RT-PCR	NuRD	Kaji et al⁶⁸
	Cdx2, Eomesodermin, Hand, Cadherin 3 etc.	RT-PCR, ChIP	NuRD	Zhu et al⁶⁹
KAP1	NANOG, OCT4 (POU5F1)		HP1, NuRD, SETB1	Rowe et al⁷⁰
SALL4	pten, sall1	RT-PCR, ChIP	NuRD	Lu et al⁷¹
LSD1	brachyury, hoxb7, hoxd8, RARgamma and the aberrant transcription of 588 genes,	ChIP, microarray analysis, RT-PCR,	CoREST	Foster et al⁷
	E cad, GLDN7, KRT8	RT-PCR, ChIP, reporter assay	LSD1-Snai1	Lin et al⁷²
	Mi-2β, MTA3, TGFB1, EGFR, RHOA, ANGPTL4, LAMININ ALPHA4, COLLAGEN VI, ENDOTHELIN-1, LMNB2, IGF1R, CCND1, ADK, PSEN1, RHOA, FGF21, APAF1, etc.	ChIP-DSL, RT-PCR, re-ChIP	LSD1/MTA1/NuRD LSD1/MTA2/NuRD LSD1/MTA3/NuRD	Wang et al⁸
MTA3	E cad, Snail, CCL3 etc. MTA, etc.	RT-PCR, ChIP ChIP-DSL, RT-PCR, re-ChIP	BCL-6/NuRD MTA3/LSD1/NuRD	Fujita et al^32,33 Wang et al⁸
MTA1	Six3, Rhodopsin Mi-2, etc.	RT-PCR, ChIP ChIP-DSL, RT-PCR, re-ChIP	HDAC-MTA1 complex MTA1/LSD1/NuRD	Manavathi et al⁷³ Wang et al⁸
	c-Myc, pS2	RT-PCR, ChIP	NuRD (HDAC1/2, CHD4)	Mazumdar et al¹⁴
	Ac-p53, BAX Pax5, XBP-1, mAqp1, mStat1, mMaoa, mCdh1, mFabp1, mEps8, mEla1, mCdr2, mUbd, mSlamf8, mKLF4, mEmp1, mPik3r, mSerpinb1, mCcL-9, mLck, mPou2af, mSpint2, mMefc, etc.	RT-PCR, ChIP Double ChIP, microarray analysis, RT-PCR, Northern blot	p53/NuRD HDAC2-MTA1	Kai et al^74,75 Balasenthil et al⁷⁶
	AHF	Reporter assay	FOG-2/NuRD	Roche et al⁷⁷
	Gαi2	ChIP	MTA1-HDAC complex	Oshiro et al⁷⁸
	BCAS3 (breast cancer amplified sequence 3), cyclin D	ChIP cloning, RT-PCR, reporter assay	not available	Gururaj et al⁷⁹
HDA-1/HDAC1/2	lag-2, lon-1, cli-2/Cystain, and tissue-specific and extracellular matrix (ECM)-related genes	ChIP, microarray, RT-PCR	Not determined	Whetstine et al³⁴ Zhang et al⁵⁶ Dufourcq et al⁵⁹

Several studies have shown that these MTAs splicing variants form distinct protein complexes with Mi-2/NuRD components.^42,43,82,93 As mentioned above, the MTA1 gene yields two isoforms (MTA1 and MTA1s (Fig. 9a, b)) via alternative mRNA splicing and one novel zymogen granule protein (ZG29p) via alternative transcription initiation.^95,96 MTA1 is associated with the Mi-2/NuRD complex.⁴² The splice variant MTA1s is cytoplasmic because it lacks a nuclear localization sequence.²⁷ ZG29p mediates an interaction with amylase and is involved in condensation-sorting in the exocrine rat pancreas, and it could possibly be linked to the deadly pancreatic cancer^9,97 (Fig. 4a). MTA1s plays an important role in breast cancer malignancy by sequestering estrogen receptors in the cytoplasm. MTA1 could affect the transcription of disease-related genes via chromatin remodeling. However, Yao and Yang reported that MTA1 associates with specific NuRD complexes that contain HDAC1/2, RbAp46/48, and MBD3, but neither Sin3 nor Mi2.⁹³

Figure 8

The downstream paths of the Mi-2/NuRD complex in carcinogenesis and cancer progression. Upper left: The Sall4/Mi-2/NuRD complex represses the promoter activities of PTEN and SALL1. Upper right: The MTA1/Mi-2/NuRD complex represses the promoter activities of BRAC1, pS2 and c-Myc. Bottom: 1) x4 Tumor suppressor p53 protein is deacetylated and inactivated by both MTA1 and MTA2 proteins in NuRD complexes, prompting inhibition of growth arrest and apoptosis. 2) x4 A hypoxia inducible factor-1a (HIF-1a) is deacetylated and stabilized by MTA1 protein, leading to angiogenesis. 3) x4 Normally, MTA1/Mi-2/NuRD complexes repress transcriptional activation of the genes implicated in cell cycle and apoptosis (p21) through deacetylation, which results in cell cycle progression and survival. 4) x4 With downregulation by resveratrol, MTA1 has less stable interactions in Mi-2/NuRD complexes, decreasing Mi-2/NuRD complex activity, increasing acetylated p53. In parallel, resveratrol switch one the acetylation mechanisms by HATs increasing global protein acetylation. This favors the expression of genes implicated in cell cycle arrest and apoptosis.

Figure 9a.

The interactors of mammalian MTA1.

Figure 9b.

The interactors of mammalian MTA1s.

The NuRD-dependent mechanisms of tumorigenicity induced by MTA1 have been intensively studied by researchers such as Toh and Kumar.⁹ MTA1 is involved in regulation of genes such as the estrogen receptor (ER-α), c-Myc, BRCA1, p53 and many more (Fig. 8, Table 2 and Supplementary Table 1). Indeed, MTA1 converts breast cancer cells to a more aggressive phenotype by repression of the estrogen receptor (ER-α) trans-activation function through deacetylation of the chromatin in the ER-responsive element of ER-responsive genes. Furthermore, MTA1 plays an essential role in c-MYC-mediated cell transformation. Overexpressed MTA1 (the long isoform) in specific NuRD complexes may modulate tumor aggressiveness via interference with estrogen signaling. Activation of the heregulin-beta1/HER2 pathway¹⁴ of breast cancer cells induces MTA1-dependent repression of estrogen receptor transactivation, leading to enhanced anchorage-independent growth and hormone independence.^96–101 Since MICoA and NRIF3 are MTA1-binding partners, their coactivators and corepressors may coexist in a single super complex. c-Myc upregulates MTA1 expression (Fig. 8) and activation of NuRD complexes containing MTA1.¹⁰² Importantly, c-Myc is one of four induced pluripotent stem (iPS) cell factors and has been linked to the dedifferentiation of somatic cells to the iPS cell phenotype.^103,104 MTA1/Mi-2/NuRD complexes have been shown to repress the BRCA1 tumor suppressor gene, and this resulted in an abnormal centrosome number, and chromosomal instability and inhibition of p53-induced apoptosis.^105,106

MTA1s is upregulated by FGF-2 in breast cancer cells, and it sequesters ER-α in the cytoplasm and represses ER-α transcriptional activity, estrogen-induced proliferation and anchorage-independent growth of the human breast cancer cell line MCF-7.¹⁰⁷ In an animal model, MTA1s peptides block the tumor progression of MCF7 overexpressing ER-α, which promotes the malignant phenotypes.¹⁰⁸ MTA1s associates with casein kinase I-γ2, an estrogen-responsive kinase that phosphorylates and modulates the functions of MTA1s. The acetylated MTA1- histone deacetylase (HDAC) interaction facilitates the recruitment of the MTA1-HDAC complex to the Gαi2 regulatory element, and consequently for the repression of Gαi2 transcription and expression, which leads to activation of the Ras-Raf pathway.⁷⁸ MTA1 and MTA1s stimulate Wnt1 transcription through promoting its derepression from the Six3 corepressor.¹⁰⁹ Moreover, a hypoxia-inducible factor-1α (HIF-1α) is deacetylated and stabilized by MTA1, resulting in angiogenesis (Fig. 8). The HSF1, one interactor of MTA1 in the MCF7 cell line, has interactions with HIF-1α after stress (Li et al, unpublished). Thus, MTA1, and probably other MTA proteins, belong to the master co-regulatory molecules involved in carcinogenesis and the progression of various malignant tumors.

The ELM2 domain in vertebrate and invertebrate MTA1, -2 and -3^27,110 (Fig. 7) was first described in a C. elegans protein, EGL-27, which is a nuclear protein involved in embryonic patterning, cell polarity, cell migration and vulval development.¹¹¹ Based on sequence, EGL-27 is likely to be an ortholog of atrophin in the nematode C. elegans. Therefore, EGL-27 may be involved in nuclear receptor signaling in C. elegans. C. elegans has MTA1 homologs, egl-27 and egr-1, which are related to embryonic patterning. The Mi-2/NuRD complex including egr-1 antagonizes vulval development induced by the Ras signal transduction pathway, at least partly by promoting cell fusion between the vulval precursor cells and the hypodermal syncytium at an early larval stage. This inhibitory function of lin-40 might be carried out by downregulating lin-39 Hox expression^112,113 (Fig. 3). Thus, understanding their physiological functions will be absolutely necessary to understand the pathological functions of MTA proteins in human cancers. However, one RNAi/ egr-1 study⁶⁰ reported a failure to detect ectopic expression of lag-2::gfp, but this could be caused by a lower efficiency of RNAi, especially with the genomic RNAi construct; it would be interesting to examine the ectopic expression of the lag-2::gfp phenotype in egr-1 mutant and egl-27 mutant backgrounds (Fig. 3).

MTA2/Mi-2/NuRD

MTA2, along with MTA1 and MTA3 and their isoforms, is a member of the MTA family of novel nuclear receptor coregulators. MTA2 (Fig. 9c) appears in Mi-2/NuRD complexes composed of HDAC1/2, RbAp46/48, MBD3, Sin3 and Mi-2, and is associated with maintaining homeostasis of the cell. MTA2 is involved in the regulation of NuRD complexes, where it modulates the activity of the core histone deacetylase (HDAC) complex.⁸²

Figure 9c.

The interactors of mammalian MTA2.

MTA2 is in many cancers including breast cancer and epithelial ovarian cancer¹⁵ but it is not correlated as strongly with metastasis as MTA1. MTA1 and MTA2 both exert histone deacetylase and gene repressor activity. MTA2 (but not MTA1) forms complexes with the transcription factors YY1 and FKBP25.⁹³ YY1 is required for development. The MBD domain of MBD3 is necessary and sufficient for binding to HDAC1 and MTA2.¹¹⁴

MTA2 has normal physiological roles. In experiments with MTA2 null mice, MTA2 was shown to be important for embryonic survival and to be involved in modulating IL-4 and IFN-gamma expression in T-cell immune responses. These null mice develop lupus-like autoimmune symptoms.¹¹⁵ MTA1 and MTA2/PID expressiones repress p53-dependent transcriptional activity such as p53-mediated cell growth arrest and apoptosis via deacetylation of p53³¹ (Fig. 8). TWIST1 interacts with MTA2/NuRD to repress the E-cadherin promoter for transcription of E-cadherin and has essential roles as a component of the Mi2/NuRD complex in cancer metastasis; TWIST1's role is independent of another master regulator in “epithelial to mesenchymal transitions” (EMT), Snail, which is regulated by MTA3/NuRD, as discussed below.⁵¹ In addition, BMI1/PRC1and EZH2/PRC2 also interact with TWIST1, and they bind to promoters of E-cadherin and p16/INK4a. It would be interesting to know how these activities are coordinated. MTA2/NuRD complex has a role in mouse genomic imprinting, and is involved in proper imprinted expression of H19 and Peg3 during mouse preimplantation development.¹¹⁶

MTA3/Mi-2/NuRD

Both isoforms of MTA3 (Fig. 9d) are associated with the vertebrate Mi-2/NuRD complex.¹¹⁷ MTA3 has been shown to be part of an estrogen-dependent pathway that regulates growth and differentiation of mammary epithelial cells.¹¹⁷ MTA3 is induced by estrogen and represses the expression of the transcriptional repressor Snail, a master regulator of EMT, resulting in the expression of the cell adhesion molecule E-cadherin and maintenance of a differentiated normal epithelial phenotype in breast cells (Fig. 10).

Figure 9d.

The interactors of mammalian MTA3.

Figure 10

MTA3, a potent suppressor of “epithelial to mesenchymal transitions” (EMT). Through the promoter deacetylation of the Mi-2/NuRD complex, MTA3, an estrogen responsive gene, represses the expression of the transcriptional repressor Snail, thus enhancing the expression of the cell adhesion molecule E-cadherin and maintaining a differentiated normal epithelial status in breast cells.

Depletion of MTA3 leads to aberrant upregulation of the transcriptional repressor protein Snail. An increased expression of Snail is correlated with loss of differentiation and metastasis in breast tumors. MTA3 is also required to regulate B lymphocyte differentiation^32,33 by acting as a co-repressor for the transcriptional repressor BCL-6.

The systematic screening of the target genes of the MTA/Mi-2/NuRD complex is being developed. First, LSD1, MTA1/2/3 ChIP-DSL gives a list of genes, including components of the TGFβ signaling pathway, which is a key player in metastatic tumor invasion. Many of these targets are in the main cell signaling pathways, which are involved in apoptosis, Wnt pathways, MAPK, cell communication, cell cycle, focal adhesion, the actin cytoskeleton, etc.

The Histone Binding Proteins/ Retinoblastoma a Tumor Suppressor RbAp46/P48

The Mi-2/NuRD complex contains RbAp46 and RbAp48 (Fig. 11), two proteins that bind to the retinoblastoma A tumor suppressor.^39–42,118 They contain a number of WD repeats, a sequence motif that forms the basis for the beta-propeller structure of the beta-subunit of the G protein transducin.¹¹⁹ These two proteins are presumed to be structural subunits of the NuRD complex, with the potential for different blades of the propeller structure to act as protein interaction surfaces. Interestingly, RbAp46 and RbAp48 have been shown to be components of several multiprotein chromatin modification complexes,¹²⁰ in which they interact directly with core histones H3 and H4¹²¹ as an adaptor that attaches to tetramers.^122,123

Figure 11a.

The interactors of mammalian RBBP7/RbAp46/LIN-53.

Figure 11b.

The interactors of mammalian RBBP4/RbAp48.

The Rb complex component LIN-53 (mammalian RbAp48/46)^124,125 antagonizes a C. elegans Ras pathway and a synthetic multivulva B (synMuv B) pathway, which includes the homologs of the core subunits of Mi-2/NuRD complex, such as LET-418/ Mi-2β, HDA-1/HDAC1, dcp66, etc. The mutations in the Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes limited to germline-specific P granules. Furthermore, gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act via a similar mechanism to transform mammalian cells.¹²⁶ RBBP7 interacts with DOC1R, a MAP kinase substrate that controls microtubule organization of metaphase II mouse oocytes.^127,128

The histone deacetylases

Human histone deacetylases (HDACs) are targets for cancer therapy, and they are classified into three types.¹²⁹ The Class I histone deacetylases HDAC1 and HDAC2 are ubiquitously expressed but restricted to the nucleus. Histone deacetylases sometimes combine with histone acetyltransferases (HAT) to form a super HAT HDAC complex.¹³⁰

HDAC1 and HDAC2 have no preference for specific DNA sequences; therefore they associate with coactivators and co-repressors which bind to DNA in a specific manner. They both are part of the Mi-2/NuRD complex and bind to the histone binding proteins RbAp46/48 to form a HDAC core complex. HDAC1 and HDAC2 are also associated with other different co-regulators (Fig. 12; Fig. 13a, b Supplementary Table 1). Rb was shown to depend on HDAC1 for the transcriptional repression of E2F target genes.¹³¹ They promote nonhomologous DNA end-joining in the DNA damage response.⁵⁰

Figure 12a.

The interactors of mammalian HDAC1.

Figure 12b.

The interactors of mammalian HDAC2.

Figure 13a.

The pathways of interactors of mammalian HDAC1. Reprinted with permission from ingenuity^® systems, www.ingenuity.com

Figure 13b.

The pathways of interactors of mammalian HDAC2. Reprinted with permission from ingenuity^® systems, www.ingenuity.com.

The microarray analysis using C. elegans embryos identified tissue-specific and extracellular matrix (ECM)-related genes as major HDA-1 targets. Ectopic expression of HDA-1 or C. elegans cystatin, an HDA-1 target identified from the profiling, significantly affected mammalian cell invasion. Similarly, RNAi depletion or overexpression of human HDAC1 also affected cell migration. Therefore HDA-1/HDAC1 may play a critical evolutionarily conserved role in regulating the extracellular microenvironment.^34,59 The sumoylation of Smo-1 on HDA-1 inhibits LIN-12/ Notch signalling in the vulva.⁶⁰ HDA1 contributes to huntingin polyglutamine toxicity.¹³² HDA1 is required for cell migration and axon pathfinding.¹³³

GATAD2a/b (p66/p68)

Some versions of the NuRD complex are also thought to incorporate a second structural and/or regulatory subunit, p66a or p66b,^45,134 also known as Gatad2a (p66) and Gatad2b (p68) (Fig. 14). As previously mentioned, the p66 subunits interact with the MBD2 subunit and may be involved in interactions between the Mi-2/ NuRD complex and methylated DNA.²⁸ Like the RbAp46/48 subunits, both p66 isoforms have the capacity to interact directly with core histones.¹³⁵ Small ubiquitin-like modifier (SUMO)-modified forms of p66 efficiently interact with HDAC1, whereas RbAp46 binds to SUMO-p66.¹³⁶ The C. elegans p66 negatively regulates the expression of the Delta homolog lag-2 and RNAi knock-down has low penetrance of multiple Muv phenotypes⁶⁰ (Zhang and Mueller, unpublished).

Figure 14

The interactors of mammalian p66/68 (GATAD2a/b).

The Histone 3 Lysien-Specific Demethylase LSD1 independent, font big as the equivalents/other components of Mi-2/NuRD. Since its identification in 2004, LSD1 has been shown to be essential for many cellular processes. Significantly, Wang et al identified LSD1 as an integral component of the human Mi-2/ MTA1-3/NuRD complexes that had profound implications for oncogenesis.⁸ In breast cancer cells, LSD1 interacts directly with all MTA1-3 proteins and these different MTA/Mi-2/NuRD complexes have different genomic DNA landing signatures. MTA2 is required for nucleosome LSD1 demethylation. Furthermore, BRCA2-LSD1/Mi-2/NuRD complexes are associated with cancer susceptibility. LSD1/Mi-2/ NuRD complexes suppressed breast cancer metastasis. Overexpression of LSD1 has been correlated with prostate cancer and tumor recurrence during therapy. The association of LSD1 with the androgen receptor switches its substrate specificity from H3K4me/me2 to H3K9me/me2. Polyamine analogues inhibit LSD1 and result in re-expression of aberrantly silenced genes in colon carcinoma.^20,137–139 A 3’ domain of the first identified long intergenic noncoding RNAs (LincRNA), HOTAIR, binds the LSD1/CoREST/REST complex, and, consequently, CoREST prevents LSD1 degradation. This coordinated tethering of PRC2 and LSD1 to chromatin couples histone H3 lysine 27 methylation and lysine 4 demethylation of target genes.¹⁴⁰

LSD1 function is implicated in the DNA damage response by demethylating p53. LSD1 maintains DNA methyltransferase, Dnmt1. Loss of LSD1 demethylase activity results in reduced levels of Dnmt1 and global DNA methylation. Genetic ablation of LSD1 causes early embryonic lethality. Loss of LSD1 in embryonic stem (ES) cells reveals a reduction in CoREST levels and the aberrant transcription of 588 genes⁷ (Table 2). Like MTAs, LSD1 has SANT and ELM domains. It is unclear whether LSD1 and MTA proteins could have any redundant functions with such domains in a subset of target genes.

As mentioned earlier, spr-5 encodes the C. elegans ortholog of the human histone demethylase LSD1; loss of spr-5 activity can phenotypically suppress mutations in sel-12/presenilin and derepresses expression of hop-1, the second C. elegans presenilin in the LIN-12/Notch pathway. SPR-5 interacts with SPR-1, the C. elegans ortholog of CoREST.^61–63

Lamin a and the Mi-2/NuRD Complex

Hutchinson-Gilford Progeria Syndrome (HGPS) is caused by mutations in the LMNA gene, which encodes nuclear lamins A and C. The in vivo physical interaction of lamin A with RBBP4, RBBP7 and HDAC1 points to lamin A being a co-regulator for the nuclear lamina and the Mi-2/NuRD complex in normal cells. Multiple Mi-2/NuRD components are lost in HGPS. Similar to RBBP4/7KD, silencing of any subunit increased the percentage of cells lacking H3K9me3 and HP1γ heterochromatin foci. Furthermore, the KD of HDAC1, MTA3, CHD3 or CHD4 in primary human fibroblasts increased the percentage of cells containing phosphor-H2AX positive foci. Progerin causes the loss of RBB4/7, which is an early event in ageing-associated chromatin defects. Loss of any Mi-2/ NuRD component and reduction of HDAC1 activity is sufficient to recapitulate several ageing-associated chromatin defects. Induction of progerin or the KD of RBBP4 and RBBP7 results in changes within the heterochromatin structure, followed by accumulation of DNA damage. Loss of RBBP4/7 compromises the histone modifications and higher order chromatin structure, possibly making chromatin more susceptible to DNA damage. Thus the Mi-2/NuRD complex is a mediator of ageing-associated chromatin defects.¹⁹⁰

Figure 15

The interactors of mammalian LSD1.

Post-transcriptional modifications and upstream cell signaling pathways of Mi-2/NuRD

A large number of different posttranslational modifications were identified in all major Mi-2/NuRD subunits. These include phosphorylation sites in Mi-2α, Mi-2β, p66α, p66β, HDAC1 and HDAC2. The role of p66 in the NuRD complex arises from its sumoylation.

SUMO-modified forms of p66 efficiently interact with HDAC1, whereas RbAp46 binds to SUMO-p66.¹³⁶ MTA proteins display tissue-specific differential expression giving rise to distinct Mi-2/NuRD complexes. Sumolyation of HDA1 in C. elegans is evident. The Mi-2/NuRD complex has many phosphorylation sites, especially on sites in MBD2, MTA1, MTA2 and MTA3. Several post-translational modifications have been conserved, and these might therefore have a role in regulating protein -protein or protein DNA interactions, or in fine-tuning of enzymatic activities. More potential amino acid modifications in chromatin remodeling are determined by DNA methylation and by multiple histone modifications including methylation, ubiquitination, sumoylation, phosphorylation and acetylation.

From Table 1, we analyze the interactors of HDAC1 and HDAC2 or pooled interactors of the subunits of the Mi-2/NuRD complex, except for HDAC2, by using the Ingenuity Pathways Analysis (IPA) program for functional pathway analysis (Fig. 13a, b; Fig. 17).

Figure 16

Model of upstream effects on the Mi-2/NuRD complexes. Upper left and right: The upstream processes of the MTA1, such as hypoxia, oncogenic growth factor heregulin and oncogene c-Myc, induce MTA1 expression. MTA1 protein is included in NuRD complex that represses the transactivation function of ER-α, rendering breast cancer cells more phenotypically aggressive. Moreover, the MTA1 protein in NuRD complexes enhances the stability and functions of HIF1, and also increases metastasis. MTA1 itself could promote the activities of BCAS3 and Wnt4, which is antagonized by the repression of the estrogen-activated MTA3/Mi-2/NuRD. Middle: sp1, ETS and Kaiso can upregulate the activity of MTA2, and promote the cancinopgenesis. Bottom: UV treatment increases the protein level of Mi-2.

Figure 17

The pathways of pooled interactors of the core subunits of Mi-2/NuRD (excluding those of HDAC2). Reprinted with permission from ingenuity^® systems, www.ingenuity.com.

Finally, the expression of MTA2 is partly regulated by SP1 and ETS elements in its promoter¹⁴¹ and by Kaiso, a component of the human N-CoR complex.¹⁴² Kaiso recruits the NCoR complex to the MTA2 promoter in a DNA methylation-dependent manner, resulting in hypoacetylation and methylation at H3K9 at the promoter region.¹⁴² Therefore, this establishes that Kaiso is a DNA methylation-dependent transcriptional repressor of the MTA2 gene (Fig. 16, middle).

The Methodology for Identifying of Mi-2/NuRD Complex Composition

Classical biology has become “big biology”, ie, high-tech, high throughput and high computation. Identifying protein protein interaction is becoming increasingly important in gaining a molecular understanding of protein function and regulation in cells, in organisms under normal development and in diseased states. However, conventional chromatography has led to the discovery of the Mi-2/NuRD complex.^28,82,83 Moreover, single tagging studies like GST contributed significant insights for interacting proteins of the cell or tissue-specific Mi-2/NuRD^67,143 (Table 3). The yeast two-hybrid system further identified fruitful co-regulators of the Mi-2/NuRD complex^{9,67,73,101,144} (Table 3).

Table 3

Advantages and disadvantages of the methods used for the identification of Mi-2/NuRD complexes.

	Advantages	Disadvantages
Yeast two-hybrid	1. Routinely performed in many labs. 2. Able to provide first hint for the identification of interaction partners. 3. Scalable to large scale screen for interactions among many proteins. 4. Sometimes generate a similar quality to data generated by the co-affinity purification and mass spectrometry (AP/MS). 5. Able to capture some transient interactions. 6. Having well-established bioinformatic platform, esp. for functional modules and networking.	1. A high number of false positive (and false negative) identifications as high as 50%. The reason for this high error rate lies in i. Over-expression can result in non-specific interactions; ii. Fusion proteins; iii. In the Yeast. A mammalian protein is sometimes not correctly post-transcriptionally modified in yeast eg, missing phosphorylation; iv. In the nucleus. Some proteins might specifically interact when they are co-expressed in the yeast and nucleus, although in reality they are never present in the same cell at the same time. 2. All interactions should be confirmed by a high confidence assay, for example co-immunoprecipitation of the endogenous proteins.
GST tag	1. Commonly used to create fusion proteins. 2. Simple and straightforward 3. Many commercial GST-tagged plasmids include a thrombin domain for cleavage of the GST tag during protein purification. 4. Increase the solubility of insoluble or semi-soluble proteins expressed in E. coli. 5. Others i. Efficient translation; ii. Initiation; iii. Inexpensive affinity resin; iv. Mild elution conditions.	1. The tag is roughly 26 KDa, which, compared to other tags like the FLAG-tag, is quite big and possible to affect the native functions and structure of protein of interest 2. generally fused to the N-terminus of a protein. 3. The over-expression of GST fusion proteins can result in non-specific interactions. 4. The conformation of the GST after expressing might be challenge, esp. for membrane proteins. 5. Others i. high metabolic burden; ii. Homodimeric protein; iii. Does not enhance solubility.
HIS tag	1. The most widely used fusion tags. 2. Small in size, less immunogenically active, and suitable for both purifying protein and downstream applications. 3. A large number of commercial vectors ready for expressing His-tagged proteins. 4. Be placed at either the amino-terminus (N) or carboxy-terminus (c), or in association with other tags. 5. The interaction of the His-tag does not rely on the tag structure, possible to purify insoluble proteins using denaturing conditions. 6. If solubility is not an issue, the constructs already exist in 6X or 8x HIS based vectors for the characterization of interactions. 7. It was used for FLAG–HIS (FH) TAP and have a high yield in both drosophila and mammalian systems. 8. Relatively high purity, and yield. 9. Others: i. Low metabolic burden; ii. Inexpensive affinity resin; iii. Mild elution conditions; iv. Tag works under both native and denaturing conditions.	1. Specificity of IMAC is not as high as other affinity methods. 2. Does not enhance solubility.
FLAG tag	1. Very small size; 2. Many high quality antibody is commercially available; 3. Widely used; 4. Be placed at either the amino-terminus (N) or carboxy-terminus (c), or in association with other tags. 5. It will not usually interfere with the fusion protein expression, proteolytic maturation or activity. 6. Relatively high purity, and yield. 7. Others: i. Low metabolic burden; ii. High specificity.	1. Expensive affinity resin. 2. Harsh elution conditions.
STREP tag	1. A recombinant form of streptavidin with a near-neutral pI is commercially available. With no carbohydrate modification and a near-neutral pI, it has the advantage of much lower nonspecific binding than avidin. 2. It was used for FLAG–STREP (FS) TAP and have a high yield. 3. Relatively high purity, and yield 4. Others i. Low metabolic burden; ii. High specificity; iii. Mild elution conditions.	1. Expensive affinity resin; 2. Does not enhance solubility.
CBP	1. Low metabolic burden. 2. High specificity. 3. Mild elution conditions. 4. Relatively high purity, and yield.	1. Expensive affinity resin. 2. Does not enhance solubility.
TAP	1. Low false positive. 2. The novel modified TAPs get much improvements. 3. High purity, moderate yield and specificity in comparison with single epitope tag affinity purification.	1. Relatively low yield in mammalian classic TAP tagged purification. 2. A big size and possible to affect the functions of fusion proteins. 3. Stringent conditions to discriminate nonspecific interactor, loss of weak interaction, TEV remnant.
Chromatin immunoprecipitation and mass spectrometry	Gain insights for both real time protein-protein and protein-DNA interactions	The quality of fixation reversal is a critical challenge.

Mass spectrometry-based proteomics, combined with tandem affinity (TAP)-tag-based protein purification,¹⁴⁵ is one of the most effective strategies for isolating and identifying protein complexes and has revolutionized proteomic experiments (Table 3). Generally speaking, mass spectrometry can identify medium or high abundance proteins. Therefore, previously infrequently expressed proteins could need a crosslink fixation¹⁴⁶ (Zhang, unpublished). Now it has become routine to allow protein identification with high sensitivity and accuracy, and it recently produced several protein interaction network reports. The classic TAP using Protein A and CBP tags has proven successful in yeast to allow rapid purification of protein complexes and minimize the background. However, this classic TAP is not suitable for the purification and identification of proteins from tissues and cell lines. The large tags on either the N-terminus or the C-terminus destabilize the protein (Table 3). Some alternative other tags (eg, FLAG and His (FH-TAP), FLAG and CBP (FC-TAP), protein G and the streptavidin-binding peptide (GS-TAP), and FLAG and STREP (SF-TAP)) provide protein yields from tissues and mammals that are about an order of magnitude higher than the classic one^147–149 (Table 3; Zhang et al in preparation). Semi-quantitative specificity filters that are based on peptide spectral count measurements could derive an accurate complex composition.¹⁵⁰ Furthermore, to be extended to targeted therapy, such proteomics-based strategies could identify novel biomarkers that indicate a response of cancer cells to PI3K pathway inhibitors.¹⁵¹ For Mi-2/ NuRD, Le Guezennee et al. succeeded in identifying MBD2/NuRD and MBD3/NuRD complex compositions; to purify Ikaros/NuRD complexes under reduced stringency conditions, a two-step TAP procedure was successfully used.¹⁵²

However, some studies in mammals and C. elegans rely on cDNA overexpression driven by exogenous promoters or transgenic random integration approaches. These transgenes can lack alternate promoters, enhancer elements, and 3 untranslated region (UTR) elements, which play critical roles in the control of gene expression in vivo. In C. elegans, for example, both the daf-12 and fah-1 genes have important enhancer elements that lie outside of the proximal promoter which were missed in promoter-only constructs. Furthermore, many transgene constructs use the unc-54 3'UTR, which prevents regulation by the appropriate microRNA genes. Consequently, generating transgenes with large segments of mammalian and worm genomic DNA would be ideal for capturing all of promoters, splice variants, and 3'UTR control elements. Along with mammalian Bacterial Artificial Chromosome (BAC) or fosmid library, a C. elegans fosmid library, which consists of ~40 kb regions of genomic DNA and covers almost all of the genome, has been recently constructed. Previously, we developed fosmid DNA recombineering technology to carry out profiling studies of the DAF-12 targets by powerful genome-wide screening techniques including ChIP-tiling arrays and assay of interactors by proteomic analysis on transgenic C. elegans lines^153,154 (Hochbaun et al in preparation). Importantly, we could capture most, if not all, previously identified targets, and thus their related mechanisms, as well as thousands of novel targets. For the proteins expressed with low abundance, like DAF-12, the integration of a few copies rather than a single copy in transgenic animals significantly increased the capture of bona fide target genes (unpublished).

Recently, an epitope-tagging strategy for the purification with recombination of a full-length BAC that contains Oct4 has pulled down the stemned Mi-2/ NuRD complexes in mouse ESCs. As previously mentioned, fosmid recombinant cloning has the advantage of maintaining the endogenous promoter and therefore natural transcriptional regulation. The technology is amenable to high-throughput delivery, as demonstrated by random integration of tagged BAC transgenes,¹⁵⁵ and should considerably facilitate systematic tagging of genes and analysis of protein complexes with roles in development in different contexts, be it in stem cells, differentiated cell types or even mouse tissue.¹⁵⁶ Previous proteomic studies of Oct4 protein complexes needed lengthy single or tandem purifications from nuclear extracts with streptavidin capture¹⁵⁷ or anti-Oct4 antibodies,³ and yielded small datasets; in contrast, this novel approach has produced by far the most extensive analysis of Oct4-associated proteins to date. The advantage of using whole extracts and nuclear extracts is that the analysis is not restricted to the nuclear environment, and the dataset encompasses diverse aspects of the life of Oct4, both nuclear and non-nuclear. With such a broad dataset, Oct4 obviously is involved in diverse cellular processes that can have an impact on many aspects of stem cell biology.

For the dissection and assay of different tissue/ organ cancers, going small is the new big, ie, it is able to achieve comprehensive integrated molecular views of such defined cell populations. For the downstream sequences of target genes, low-quantity digital gene expression (LQ-DGE)¹⁵⁸ allows mRNA profiles of as few as 250 cells to be characterized without an amplification step. The new application of chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) from a limited number of cells (10,000) hematopoietic mouse progenitors.^159,160 The mass spectrometry-based proteomics assays have become ready for human proteomes.¹⁶¹ Such a strategy is certainly a hypothesis generator,¹⁶¹ with some of the most interesting connections revealed being completely unanticipated.

As high-sensitivity and high throughput mass spectrometry, microarray transcriptional profiling and DNA sequencing become more common, assays that scan an entire genome, proteome, ribonome or metabolome will generate a huge amount of data. Bioinformatics tools such as Cytoscape, DAVID, Metacore and Ingenuity Pathway Analysis (IPA) could certainly provide us with functional modules and/or genetic cause-and-effect regulatory networking.^162,163

The emerging stemnessed Mi-2/NuRD complexes

Oct-4 expression must be tightly regulated; too much or too little will cause differentiation of the cell. Oct4 can both activate and repress transcriptional targets in mouse and human ESCs.^164,165 To date, Oct4 has been shown to be associated mainly with members of repressor Mi-2/NuRD complexes.^3,5,6,157 MTA1/Mi-2/ NuRD may have an essential role in normal development as a mediator of pluripotency. MTA1 appears to be the preferred core component of NuRD complexes for Nanog and OCT4 interaction³ as a potential regulator of pluripotency. Recently, Sall4, a well-known Oct4 partner, and other members of the Spalt-like family of transcriptional cofactors have been shown to associate with NuRD.¹⁶⁶ One of the stemness factors for iPS, c-Myc, is also associated with the Mi-2/ NuRD complex.

Clinical and therapeutic implications

Human cancer is heterogeneous, and this is driven by progressive genetic and epigenetic abnormalities. The Mi-2/NuRD complex is the master regulator of epigenetic alterations in changed patterns of histone modification. In contrast to genetic changes, the epigenetic alterations in cancer cells are reversible by the epi-drugs, for example, the inhibitors of histone deacetylases, DNA methyltransferases and histone demethylases.

MTAs: targets for drug development

MTA1 protein, as a master co-regulator, or its gene, could be an excellent molecular target for cancer therapy, as well as being useful in cancer diagnosis or prognosis. The antisense silencing against MTA1 mRNA had a growth-inhibitory effect on human metastatic breast cancer cell lines.^167,168 RNAi knock-down of MTA1 in a human esophageal squamous cell carcinoma cell line resulted in significant inhibition of in vitro invasion and migration properties of the cancer cells. In malignant melanoma cells, knock-down of MTA1 by RNAi successfully suppressed the growth in vitro and experimental metastasis of mouse melanoma cells in vivo. MTA1s may also be a useful target for fighting breast cancer. In an animal model, the effect of the MTA1s peptide blocks the tumor progression of MCF-7, which overexpresses ER-α. MTA1 is a promising antigen for tumor rejection in that it is overexpressed in many different tumors and is expressed at lower levels in normal tissues. An initial study demonstrated the presence of immunogenic MHC class I-restricted peptides of MTA1.¹⁶⁹

Figure 18a.

The core-NuRD and transcriptional factor-NuRD.

Figure 18b.

The core-NuRD, transcriptional factor-NuRD and stemmed Nanog-NuRD.

Figure 18c.

The core-NuRD, transcriptional factor-NuRD, Stemmed Nanog-NuRD and stemmedOrchestra Oct4-NuRD.

Figure 18d.

The core-NuRD, transcriptional factor-NuRD, stemmed Nanog-NuRD stemmed Oct4-NuRD and stemmed SALL4-NuRD.

Figure 18e.

The core-NuRD, transcriptional factor-NuRD, stemmed Nanog-NuRD, stemmed c-Myc-NuRD and stemmed Oct4-NuRD.

MBDs: targets for drug development

MBD proteins may be important modulators of tumorigenesis through the gene silencing mechanism of DNA hypermethylation and may be excellent novel therapeutic targets.¹⁷⁰ Some intensively-investigated DNA methylation inhibitors include the pyrimidine nucleoside analogs decitabine (Dacogen, SuperGen, Inc.) and azacitidine (Vidaza, Celgene), and the nonnucleoside inhibitor hydralazine. Azacitidine and decitabine are both US Food and Drug Administration (FDA) approved for the treatment anemia and chronic myelogenous leukemia (CML). Hydralazine was originally approved for use as an antihypertensive and recently used to reactivate the expression of tumor suppressor genes in cancer. Pharmacologic inhibition of DNA methylation blocks DNMTs and their targeted degradation, and desilences genes that have been aberrantly silenced by hypermethylation. Finally, it causes inhibition of clonal expansion and tumor cell growth, induction of cell differentiation, and cancer cell death. Only rapidly dividing cells such as tumor cells will be targeted as these agents function by being incorporated into newly replicated DNAs.

HDACs: targets for drug development

Epigenetic therapy tries to reverse the aberrations with natural compounds and/or synthetic molecules that are active on specific epi-targets. Vorinostat (Zolinza, Merck) is the first HDAC inhibitor that has been licensed for clinical use. HDAC inhibitors have been tested as promising agents in treating blood-borne cancers, and also in treating polyglutamine diseases.^171–174 Two HDACs, hydralazine and magnesium valproate, can sensitize tumor cells to chemotherapy in patients with advanced and solid refractory tumors.¹⁷⁵ Inhibition of histone deacetylation enhances tumor radiosensitization.¹⁷⁶ So far, more than 11 HDAC inhibitors are in clinical development for epigenetic cancer therapy.

G9a: a target for drug development

The MTA proteins are associated with HDAC1/2 and methyltransferase G9a, and specific G9a inhibitors have been recently reported.¹⁷⁷ This raises the possibility that G9a inhibitors such as BIX-01294 might also be used, alone or together with the current FDA-approved HDAC inhibitors, for treating MTA-involved cancers and neurological diseases.¹⁷⁸

LSD1: a target for drug development

The development of LSD1 inhibitors may represent a significant weapon against cancer. To date, only two drugs, pargyline¹⁷⁹ and tranylcypromine,^20,180 have been described as LSD1 inhibitors, but their action is not as specific as the well-known anti-Monoamine Oxidase (MAO) agents. Pargyline blocks demethylation by LSD1, and consequently it blocks androgen-receptor-dependent transcription. Thus, modulation of LSD1 activity offers a new strategy to regulate androgen receptor functions, which may turn out to be important in prostate cancer models.¹⁷⁹

RNAi and potential combined therapy

Hypoxia inducible factor (HIF)-1 accumulation favors tumor angiogenesis.¹⁸¹ 5-AZA-CdR represses the hypoxia response pathway by downregulation of HIF-1 targets vascular endothelial growth factor (VEGF).¹⁸² HDACi also plays an important role in inhibiting tumor angiogenesis by concomitantly upregulating antiangiogenetic factors such as VHL and by downregulating pro-angiogenic factors such as HIF-1 and VEGF in cancer cells.¹⁸³ MTA1/NuRD has a role in tissue maintenance, via HIF-1a/VEGF expression against hypoxic condition.

Recently, ALN-VSP02 (KSP/VEGF siRNAs), a lipid nanoparticle containing two small interfering RNAs (siRNAs) for kinesin spindle protein (KSP) and vascular endothelial growth factor (VEGF), has demonstrated its potential antitumor activity. Upon intravenous administration, the siRNAs in KSP/ VEGF siRNAs ALN-VSP02ALN bind to both KSP and VEGF messenger RNAs (mRNAs), preventing translation of KSP and VEGF proteins; this may result in growth inhibition of tumor cells that overexpress KSP and VEGF.¹⁸⁴

Interestingly, as already mentioned, some components of the Mi-2/NuRD complex (eg, LIN-53/ RbAP48) in C. elegans could enhance RNAi. Furthermore, the long-term RNAi of Transcriptional Gene Silencing (TGS) with a chromatin remodeling-related mechanism might potentially be used in the future for therapeutic applications of RNAi for prolonged, epigenetic gene silencing.¹⁸⁵ Thus, it may be useful to apply such RNAi against components of the Mi-2/NuRD complex combined with thermo- or radiation- therapy in preclinical cancer.

Furthermore, the Mi-2/NuRD complex plays a crucial role in DNA damage and repair.^{25,35,48–50} Curcumin (diferuloylmethane) targets DNA damage and repair.¹⁸⁶ A combined strategy of the HDAC inhibitors (eg, Vorinostat, ie, suberoylanilide hydroxamic acid (SAHA) or the safe dietary compound resveratrol) and curcumin therapy provides new hope for cancer patients.

Future Mi-2/NuRD-based approaches

It is highly likely that greater understanding if Mi2/ NuRD subunit structure could contribute to a break-though in anti-cancer drug development since our knowledge in this field and the interdependence among the subunits is in its infancy (Tables 4 and 5). In the future, further understanding of the relationships between the Mi-2/NuRD complex structure and activity, as well as their biological mechanism(s) of action, will hopefully provide novel epigenetic approaches for fighting human cancer.

Table 4

Crystal structure of subunit of Mi-2/NuRD complex.

Subunit	Structure	References
CHD4/Mi-2β	+	PDB IDs: 2EE1, 1MM2, 1MM3
CHD3/Mi-2α	n.a
MTA1	+	PDB
MTA2	n.a
MTA3	+	PDB IDs: 2CRG
MBD2	+ (for MECP2)	PDB IDs: 1QK9
MBD3	n.a
GATAD2a/p68	n.a
GATAD2b/p66	n.a
RBBP4/RbAp48	n.a
RBBP7/RbAp46	n.a
LSD1	+	Chen Y et al
PNAS¹⁸⁷
2COM
2DW4
2EJR
2HKO
2IW5
2UXN
2UXX
2V1D
2W9I
2X0L
2XAF
2XAG
2XAH
2XAJ
2XAQ
2XAS
2Z3Y
2Z5U
3ABT
3ABU
HDAC1/HDAC2	+	Vannini et al¹⁸⁸
		PDB IDs:
		3HGT
		3HGQ

Abbreviations: n.a., not available; +, available; PDB, protein data bank.

Table 5

Interdependence of subunits of Mi-2/NuRD complex.

	Mi-2α	Mi-2β	MTA1	MTA2	MTA3	MBD2	MBD3	HDAC1	HDAC2	RBBP4	RBBP7	LSD1	p66/68	References
MTA2^-/-	n.d.	=	n.d.	↓	n.d.	=	=	=	=	n.d.	n.d.	n.d.	n.d.	Ma et al¹¹⁵
MBD3^-/-	n.d.	n.d.	n.d.	↓	n.d.	=	↓	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	Reese KJ et al¹⁸⁹
Lamin A^-/-	↓	↓	n.d.	n.d	↓	n.d.	n.d.	↓	n.d.	↓	↓	n.d.	n.d.	Pegoraro¹⁹⁰
CHD4^-/-	n.d.	↓	n.d.	=	n.d.	n.d.	n.d.	=	n.d.	n.d.	n.d.	n.d.	n.d	Polo SE et al¹²⁵

Abbreviations: n.d., not determined; =, no change; ↓, Down.

Future Mi-2/NuRD stemness-based treatment

A deeper understanding the origin of cancer stem cells (CSC)¹⁹⁰ and their epigenetic characteristics is important for the rational design of cancer treatment. Cancer derived from CSC may be more prone to drug resistance. However, epigenetic changes are mainly reversible through the administration of epi-drugs. Such drugs have been developed for targeting total cell populations and not CSC's specifically. Finally, a stem cell-based vaccine has been shown to successfully address colon cancer.¹⁹² An iPS-based vaccine has also been developed too.^192,193 Advancing our knowledge of the Mi-2/NuRD complex could possibly contribute to an improved design to achieve better effects.

The significance of cross-validation with interspecies (Mammals and C. elegans): a personal view

C. elegans has almost the same number of protein-coding genes as a human–-about 20,000 is the latest estimate–-and most of those genes encode similar functions.^34,35 So the basic parts set for animal development was established several hundred million years ago. Furthermore, C. elegans has two small evolution-distance nematode sister species, C. briggsae and C. remanei, and similar studies could provide very strong mutual cross-species validation.^{58, 195} This study of the Mi-2/NuRD complex in C. elegans proved that such cross-research can be a fruitful and probably cost-effective way to study cancer.¹⁹⁶ Besides, we are now able to perform more high-tech, high throughput assays, such as ChIP-seq, quantitative proteomics¹⁹⁷ and TAP on Mi-2/NuRD complexes and their interactors to learn more about them and their clinic implications for beating cancer. Screening all the genes or proteins in an organism is not much more difficult than analyzing a small subset, and robotics and high-throughput screening techniques are now within the reach of most labs. Second, the cost of systems biology scales sub-linearly while the payoffs scale super-linearly.¹⁶² A huge amount of information will need to be analalyzed with current powerful bioinformatic tools¹⁶³ so we can obtain information on the behavior of gene groups at a system level, as well as their functional modules and networks. This could be done quickly with genetically tractable organisms like C. elegans, and then projecting its mechanisms to human beings, as has already been demonstrated successfully for C elegans originals such as RNAi, programmed cell death and miRNAs. Put simply, we should be ready to worm our way into elucidating differential regulatory networks between healthy and cancerous tissues.

Discussion

We are towel on the road from “big biology” to “bigger biology”¹⁹⁸ with both “data first”¹⁹⁹ and “hypothesis first”²⁰⁰ approaches being used together rather than alone; from HGP and NextGen Sequencing to the NextGen mass-spectrometry and HUPO initiatives. Since the international Human Genome Project and Celera Genomics Corporation completing the initial draft of the human genome, the past decade has witnessed remarkable progress in our understanding of the expanding family of Mi-2/NuRD complexes and their powerful master roles in gene regulation during normal development and carcinogenesis across species. This rapid trajectory can continue with the emergence of the human proteome project. The Cancer Genome Atlas, a comprehensive platform to accelerate our understanding of the genetics of cancer using innovative genome analysis technologies, is now available. Weekly, even daily developments are emerging from this interesting Mi-2/NuRD complex family. Finally, all these new findings could be promisingly pave the way for a highly effective genetic and epigenetic therapy in the near future to cure cancer.

Disclosure

This manuscript has been read and approved by all authors. This paper is unique and is not under consideration by any other publication and has not been published elsewhere. The authors and peer reviewers of this paper report no conflicts of interest. The authors confirm that they have permission to reproduce any copyrighted material.

Footnotes

Acknowledgements

The authors thank the working groups of Dr. Mueller, Dr. Fisher and Dr. Calderwood for their personal communications and insightful discussions. Also thanks to the Swiss Federal Government for their fellowship support. We apologize to our many colleagues whose work could not be cited here owing to space considerations.

References

Denslow

S.A.

, Wade

P.A.

The human Mi-2/NuRD complex and gene regulation.

Oncogene. 2007; 26(37): 5433–8.

Ramírez

, Hagman

The Mi-2/NuRD complex: a critical epigenetic regulator of hematopoietic development, differentiation and cancer.

Epigenetics. 2009 Nov 16; 4(8): 532–6.

Liang

, Wan

, Zhang

Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells.

Nat Cell Biol. 2008 Jun; 10(6): 731–9.

, Crabtree

G.R.

Chromatin remodelling during development.

Nature. 2010 Jan 28; 463(7280): 474–84.

Van den Berg

D.L.

, Snoek

, Mullin

N.P.

An Oct4-centered protein interaction network in embryonic stem cells.

Cell Stem Cell. 2010 Apr 2; 6(4): 369–81.

Pardo

, Lang

, Yu

An expanded Oct4 interaction network: implications for stem cell biology, development, and disease.

Cell Stem Cell. 2010 Apr 2; 6(4): 382–95.

Foster

C.T.

, Dovey

O.M.

, Lezina

Lysine specific demethylase 1 (LSD1) regulates the embryonic transcriptome and CoREST stability.

Mol Cell Biol. 2010 Aug 16. [Epub ahead of print].

Wang

, Zhang

, Chen

LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer.

Cell. 2009 Aug 21; 138(4): 660–72.

Toh

, Nicolson

G.L.

The role of the MTA family and their encoded proteins in human cancers: molecular functions and clinical implications.

Clin Exp Metastasis. 2009; 26(3): 215–27.

10.

Hill

C.L.

, Zhang

, Sigurgeirsson

Frequency of specific cancer types in dermatomyositis and polymyositis: a population-based study.

Lancet. 2001 Jan 13; 357(9250): 96–100.

11.

Pencil

S.D.

, Toh

, Nicolson

G.L.

Candidate metastasis-associated genes of the rat 13762NF mammary adenocarcinoma.

Breast Cancer Res Treat. 1993; 25: 165–74.

12.

Toh

, Pencil

S.D.

, Nicolson

G.L.

Novel candidate metastasis-associated gene, mta1, differentially expressed in highly metastatic mammary adenocarcinoma cell lines. cDNA cloning, expression, and protein analyses.

J Biol Chem. 1994 Sep 16; 269(37): 22958–63.

13.

Toh

, Pencil

S.D.

, Nicolson

G.L.

Analysis of the complete sequence of the novel metastasis-associated candidate gene, mta1, differentially expressed in mammary adenocarcinoma and breast cancer cell lines.

Gene. 1995; 159: 97–104.

14.

Mazumdar

, Wang

R.A.

, Mishra

S.K.

Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor.

Nat Cell Biol. 2001; 3: 30–7.

15.

, Zhang

, Lu

, Ma

Expression of MTA2 gene in ovarian epithelial cancer and its clinical implication.

J Huazhong Univ Sci Technolog Med Sci. 2006; 26(3): 359–62.

16.

Cui

, Niu

, Pestell

Metastasis-associated protein 2 is a repressor of estrogen receptor alpha whose overexpression leads to estrogen-independent growth of human breast cancer cells.

Mol Endocrinol. 2006 Sep; 20(9): 2020–35. Epub 2006 Apr 27.

17.

Kim

, Park

, Lim

Decreased syndecan-2 expression correlates with trichostatin-A induced-morphological changes and reduced tumorigenic activity in colon carcinoma cells.

Oncogene. 2003 Feb 13; 22(6): 826–30.

18.

Minucci

, Nervi

, Lo Coco

, Pelicci

P.G.

Histone deacetylases: a common molecular target for differentiation treatment of acute myeloid leukemias?

Oncogene. 2001 May 28; 20(24): 3110–5.

19.

Choi

J.H.

, Kwon

H.J.

, Yoon

B.I.

Expression profile of histone deacetylase 1 in gastric cancer tissues.

Jpn J Cancer Res. 2001 Dec; 92(12): 1300–4.

20.

Shi

Histone lysine demethylases: emerging roles in development, physiology and disease.

Nat Rev Genet. 2007 Nov; 8(11): 829–33.

21.

Huang

, Greene

, Murray Stewart

Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes.

Proc Natl Acad Sci U S A. 2007; 104: 8023–8.

22.

Le Guezennec

, Vermeulen

, Brinkman

A.B.

MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties.

Mol Cell Biol. 2006 Feb; 26(3): 843–51.

23.

Zhu

, Harrison

D.J.

, Bader

S.A.

Genetic and epigenetic analyses of MBD3 in colon and lung cancer.

Br J Cancer. 2004 May 17; 90(10): 1972–5.

24.

Peric-Hupkes

, Meuleman

, Pagie

Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation.

Mol Cell. 2010 May 28; 38(4): 603–13.

25.

Polo

S.E.

, Kaidi

, Baskcomb

, Galanty

, Jackson

S.P.

Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4.

EMBO J. 2010 Aug 6. [Epub ahead of print].

26.

Shi

, Lan

, Matson

Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.

Cell. 2004; 119: 941–53.

27.

Bowen

N.J.

, Fujita

, Kajita

, Wade

P.A.

Mi-2/NuRD: multiple complexes for many purposes.

Biochim Biophys Acta. 2004 Mar 15; 1677(1-3): 52–7.

28.

Feng

, Zhang

The MeCP1 complex represses transcription through preferential binding, remodeling, and deacetylating methylated nucleosomes.

Genes Dev. 2001 Apr 1; 15(7): 827–32.

29.

Kim

, Sif

, Jones

Ikaros DNA-binding proteins direct formation of chromatin remodeling complexes in lymphocytes.

Immunity. 1999 Mar; 10(3): 345–55.

30.

Schultz

D.C.

, Friedman

J.R.

, Rauscher

3rd

Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD.

Genes Dev. 2001 Feb 15; 15(4): 428–43.

31.

Luo

, Su

, Chen

, Shiloh

, Gu

Deacetylation of p53 modulates its effect on cell growth and apoptosis.

Nature. 2000 Nov 16; 408(6810): 377–81.

32.

Fujita

, Jaye

D.L.

, Geigerman

MTA3 and the Mi-2/NuRD complex regulate cell fate during B lymphocyte differentiation.

Cell. 2004 Oct 1; 119(1): 75–86.

33.

Fujita

, Kajita

, Taysavang

, Wade

P.A.

Hormonal regulation of metastasis-associated protein 3 transcription in breast cancer cells.

Mol Endocrinol. 2004 Dec; 18(12): 2937–49.

34.

Whetstine

J.R.

, Ceron

, Ladd

, Dufourcq

, Reinke

, Shi

Regulation of tissue-specific and extracellular matrix-related genes by a class I histone deacetylase.

Mol Cell. 2005 May 13; 18(4): 483–90.

35.

Smeenk

, Wiegant

W.W.

, Vrolijk

, Solari

A.P.

, Pastink

, van Attikum

The NuRD chromatin-remodeling complex regulates signaling and repair of DNA damage.

J Cell Biol. 2010 Aug 30. [Epub ahead of print].

36.

, Nilasena

D.S.

, O'Brien

C.A.

Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen.

J Clin Invest. 1995; 96(4): 1730–7.

37.

Seelig

H.P.

, Moosbrugger

, Ehrfeld

, Fink

, Renz

, Genth

The major dermatomyositis-specific Mi-2 autoantigen is a presumed helicase involved in transcriptional activation.

Arthritis Rheum. 1995 Nov; 38(10): 1389–99.

38.

Marhold

, Kramer

, Kremmer

, Lyko

The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A-methylated DNA.

Development. 2004 Dec; 131(24): 6033–9.

39.

Tong

J.K.

, Hassig

C.A.

, Schnitzler

G.R.

, Kingston

R.E.

, Schreiber

S.L.

Chromatin deacetylation by an ATP-dependent nucleosome remodelling complex.

Nature. 1998 Oct; 395(6705): 917–21.

40.

Wade

P.A.

, Gegonne

, Jones

P.L.

, Ballestar

, Aubry

, Wolffe

A.P.

Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation.

Nat Genet. 1999 Sep; 23(1): 62–6.

41.

Wade

P.A.

, Jones

P.L.

, Vermaak

, Wolffe

A.P.

A multiple subunit Mi-2 histone deacetylase from Xenopus laevis cofractionates with an associated Snf2 superfamily ATPase.

Curr Biol. 1998 Jul 2; 8(14): 843–6.

42.

Xue

, Wong

, Moreno

G.T.

, Young

M.K.

, Côté

, Wang

NuRD, a novel complex with both ATP-dependent chromatin-remodeling and histone deacetylase activities.

Mol Cell. 1998 Dec; 2(6): 851–61.

43.

Zhang

, LeRoy

, Seelig

H.P.

, Lane

W.S.

, Reinberg

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

Cell. 1998 Oct 16; 95(2): 279–89.

44.

Woodage

, Basrai

M.A.

, Baxevanis

A.D.

, Hieter

, Collins

F.S.

Characterization of the CHD family of proteins.

Proc Natl Acad Sci U S A. 1997 Oct 14; 94(21): 11472–7.

45.

Guschin

, Wade

P.A.

, Kikyo

, Wolffe

A.P.

ATP-Dependent histone octamer mobilization and histone deacetylation mediated by the Mi-2 chromatin remodeling complex.

Biochemistry. 2000 May 9; 39(18): 5238–45.

46.

Williams

C.J.

, Naito

, Arco

P.G.

The chromatin remodeler Mi-2beta is required for CD4 expression and T cell development.

Immunity. 2004; 20(6): 719–33.

47.

Yoshida

, Hazan

, Zhang

The role of the chromatin remodeler Mi-2beta in hematopoietic stem cell self-renewal and multilineage differentiation.

Genes Dev. 2008 May 1; 22(9): 1174–89.

48.

Schmidt

D.R.

, Schreiber

S.L.

Molecular association between ATR and two components of the nucleosome remodeling and deacetylating complex, HDAC2 and CHD4.

Biochemistry. 1999 Nov 2; 38(44): 14711–7.

49.

Larsen

D.H.

, Poinsignon

, Gudjonsson

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage.

J Cell Biol. 2010 Aug 30. [Epub ahead of print].

50.

Miller

K.M.

, Tjeertes

J.V.

, Coates

Human HDAC1 and HDAC2 function in the DNA-damage response to promote DNA nonhomologous end-joining.

Nat Struct Mol Biol. 2010 Aug 29. [Epub ahead of print].

51.

, Qin

, He

The TWIST1/Mi2/NuRD protein complex and its essential role in cancer metastasis.

Cell Res. 2010 Aug 17. [Epub ahead of print].

52.

Nilsson

, Mann

, Aebersold

, Yates

3rd , Bairoch

, Bergeron

J.J.

Mass spectrometry in high-throughput proteomics: ready for the big time.

Nat Methods. 2010 Sep; 7(9): 681–5.

53.

Von Zelewsky

, Palladino

, Brunschwig

, Tobler

, Hajnal

, Müller

The C. elegans Mi-2 chromatin-remodelling proteins function in vulval cell fate determination.

Development. 2000 Dec; 127(24): 5277–84.

54.

Unhavaithaya

, Shin

T.H.

, Miliaras

, Lee

, Oyama

, Mello

C.C.

MEP-1 and a homolog of the NuRD complex component Mi-2 act together to maintain germline-soma distinctions in C. elegans.

Cell. 2002 Dec 27; 111(7): 991–1002.

55.

Guerry

, Marti

C.O.

, Zhang

, Moroni

P.S.

, Jaquiéry

, Müller

The Mi-2 nucleosome-remodeling protein LET-418 is targeted via LIN-1/ETS to the promoter of lin-39/Hox during vulval development in C. elegans.

Dev Biol. 2007 Jun 15; 306(2): 469–79.

56.

Zhang

Identification of differentially expressed target genes of human nucleosome remodelling Mi-2 orthologue LET-418 in “C. elegans”. 2006, PhD thesis.

57.

Cui

, Han

Cis regulatory requirements for vulval cell-specific expression of the Caenorhabditis elegans fibroblast growth factor gene egl-17.

Dev Biol. 2003 May 1; 257(1): 104–16.

58.

Takács-Vellai

, Vellai

, Chen

E.B.

Transcriptional control of Notch signaling by a HOX and a PBX/EXD protein during vulval development in C. elegans.

Dev Biol. 2007 Feb 15; 302(2): 661–9. Epub 2006 Oct 4.

59.

Dufourcq

, Victor

, Gay

, Calvo

, Hodgkin

, Shi

Functional requirement for histone deacetylase 1 in Caenorhabditis elegans gonadogenesis.

Mol Cell Biol. 2002 May; 22(9): 3024–34.

60.

Poulin

, Dong

, Fraser

A.G.

, Hopper

N.A.

, Ahringer

Chromatin regulation and sumoylation in the inhibition of Ras-induced vulval development in Caenorhabditis elegans.

EMBO J. 2005 Jul 20; 24(14): 2613–23.

61.

Jarriault

, Greenwald

Suppressors of the egg-laying phenotype of sel-12 presenilin mutants implicate the CoREST co-repressor complex in LIN-12/Notch signalling in C. elegans.

Genes & Development. 2002; 16(20): 2713–28.

62.

Eimer

, Lakowski

, Donhauser

, Baumeister

Loss of spr-5 bypasses the requirement for the C. elegans presenilin sel-12 by derepressing hop-1.

EMBO J. 2002; 21: 5787–96.

63.

Greenwald

LIN-12/notch signaling in C. elegans.

WormBook. 2005 Aug 8: 1–16.

64.

Shi

, Whetstine

J.R.

Dynamic regulation of histone lysine methylation by demethylases.

Mol Cell. 2007; 25: 1–14.

65.

Mittal

, Subramanyam

, Dey

, Kumar

R.V.

, Rangarajan

Cooperation of notch and Ras/MAPK signaling pathways in human breast carcinogenesis.

Mol Cancer. 2009 Dec 23; 8: 128.

66.

Sternberg

P.W.

Developmental biology. A pattern of precision.

Science. 2004 Jan 30; 303(5658): 637–8.

67.

Khaleque

M.A.

, Bharti

, Gong

Heat shock factor 1 represses estrogen-dependent transcription through association with MTA1.

Oncogene. 2008 Mar 20; 27(13): 1886–93.

68.

Kaji

, Caballero

I.M.

, MacLeod

, Nichols

, Wilson

V.A.

, Hendrich

The NuRD component Mbd3 is required for pluripotency of embryonic stem cells.

Nat Cell Biol. 2006 Mar; 8(3): 285–92.

69.

Zhu

, Fang

, Li

, Zhang

Mbd3, a component of NuRD/Mi-2 complex, helps maintain pluripotency of mouse embryonic stem cells by repressing trophectoderm differentiation.

PLoS One. 2009 Nov 3; 4(11): e7684.

70.

Rowe

H.M.

, Jakobsson

, Mesnard

KAP1 controls endogenous retroviruses in embryonic stem cells.

Nature. 2010 Jan 14; 463(7278): 237–40.

71.

, Jeong

H.W.

, Kong

Stem cell factor SALL4 represses the transcriptions of PTEN and SALL1 through an epigenetic repressor complex.

PLoS One. 2009; 4(5): e5577. Epub 2009 May 18.

72.

Lin

, Ponn

, Hu

, Law

B.K.

, Lu

Requirement of the histone demethylase LSD1 in Snai1-mediated transcriptional repression during epithelial-mesenchymal transition.

Oncogene. 2010 Sep 2; 29(35): 4896–904. Epub 2010 Jun 21.

73.

Manavathi

, Peng

, Rayala

S.K.

Repression of Six3 by a corepressor regulates rhodopsin expression.

Proc Natl Acad Sci U S A. 2007 Aug 7; 104(32): 13128–33.

74.

Kai

, Samuel

S.K.

, Levenson

A.S.

Resveratrol enhances p53 acetylation and apoptosis in prostate cancer by inhibiting MTA1/NuRD complex.

Int J Cancer. 2010 Apr 1; 126(7): 1538–48.

75.

Kai

, Wang

, Ivanovic

Targeting prostate cancer angiogenesis through metastasis-associated protein 1 (MTA1).

Prostate. 2010 Aug 17. [Epub ahead of print].

76.

Balasenthil

, Gururaj

A.E.

, Talukder

A.H.

Identification of Pax5 as a target of MTA1 in B-cell lymphomas.

Cancer Res. 2007 Aug 1; 67(15): 7132–8.

77.

Roche

A.E.

, Bassett

B.J.

, Samant

S.A.

, Hong

, Blobel

G.A.

, Svensson

E.C.

The zinc finger and C-terminal domains of MTA proteins are required for FOG-2-mediated transcriptional repression via the NuRD complex.

J Mol Cell Cardiol. 2008 Feb; 44(2): 352–60.

78.

Ohshiro

, Rayala

S.K.

, Wigerup

Acetylation-dependent oncogenic activity of metastasis-associated protein 1 co-regulator.

EMBO Rep. 2010 Jul 23. [Epub ahead of print].

79.

Gururaj

A.E.

, Singh

R.R.

, Rayala

S.K.

MTA1, a transcriptional activator of breast cancer amplified sequence 3.

Proc Natl Acad Sci U S A. 2006 Apr 25; 103(17): 6670–5. Epub 2006 Apr 14.

80.

Macdonald

J.L.

, Verster

, Berndt

, Roskams

A.J.

MBD2 and MeCP2 regulate distinct transitions in the stage-specific differentiation of olfactory receptor neurons.

Mol Cell Neurosci. 2010 May; 44(1): 55–67.

81.

Bogdanovi

, Veenstra

G.J.

DNA methylation and methyl-CpG binding proteins: developmental requirements and function.

Chromosoma. 2009 Oct; 118(5): 549–65.

82.

Zhang

, Ng

H.H.

, Erdjument-Bromage

, Tempst

, Bird

, Reinberg

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.

Genes Dev. 1999 Aug 1; 13(15): 1924–35.

83.

H.H.

, Zhang

, Hendrich

MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex.

Nat Genet. 1999 Sep; 23(1): 58–61.

84.

Hendrich

, Guy

, Ramsahoye

, Wilson

V.A.

, Bird

Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development.

Genes Dev. 2001 Mar 15; 15(6): 710–23.

85.

Kantor

, Makedonski

, Shemer

, Razin

Expression and localization of components of the histone deacetylases multiprotein repressory complexes in the mouse preimplantation embryo.

Gene Expr Patterns. 2003 Dec; 3(6): 697–702.

86.

Sakai

, Urano

, Ookata

MBD3 and HDAC1, two components of the NuRD complex, are localized at Aurora-A-positive centrosomes in M phase.

J Biol Chem. 2002 Dec 13; 277(50): 48714–23.

87.

Jiang

C.L.

, Jin

S.G.

, Pfeifer

G.P.

MBD3L1 is a transcriptional repressor that interacts with methyl-CpG-binding protein 2 (MBD2) and components of the NuRD complex.

J Biol Chem. 2004 Dec 10; 279(50): 52456–64.

88.

Jin

S.G.

, Jiang

C.L.

, Rauch

, Li

, Pfeifer

G.P.

MBD3L2 interacts with MBD3 and components of the NuRD complex and can oppose MBD2-MeCP1-mediated methylation silencing.

J Biol Chem. 2005 Apr 1; 280(13): 12700–9.

89.

Gutierrez

, Sommer

R.J.

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae.

Nucleic Acids Res. 2004 Dec 2; 32(21): 6388–96.

90.

Olsen

, Vantipalli

M.C.

, Lithgow

G.J.

Checkpoint proteins control survival of the postmitotic cells in Caenorhabditis elegans.

Science. 2006 Jun 2; 312(5778): 1381–5.

91.

Mahoney

M.G.

, Simpson

, Jost

Metastasis-associated protein (MTA)1 enhances migration, invasion, and anchorage-independent survival of immortalized human keratinocytes.

Oncogene. 2002 Mar 28; 21(14): 2161–70.

92.

Hofer

M.D.

, Tapia

, Browne

T.J.

, Mirlacher

, Sauter

, Rubin

M.A.

Comprehensive analysis of the expression of the metastasis-associated gene 1 in human neoplastic tissue.

Arch Pathol Lab Med. 2006 Jul; 130(7): 989–96.

93.

Yao

Y.L.

, Yang

W.M.

The metastasis-associated proteins 1 and 2 form distinct protein complexes with histone deacetylase activity.

J Biol Chem. 2003 Oct 24; 278(43): 42560–8. Epub 2003 Aug 13.

94.

Manavathi

, Kumar

Metastasis tumor antigens, an emerging family of multifaceted master coregulators.

J Biol Chem. 2007; 282: 1529–33.

95.

Kleene

, Zdzieblo

, Wege

, Kern

H.F.

A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat.

J Cell Sci. 1999 Aug; 112(Pt 15): 2539–48.

96.

Khaleque

M.A.

, Bharti

, Gong

A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat.

J Cell Sci. 2008; 112: 2539–48.

97.

Kleene

, Classen

, Zdzieblo

, Schrader

SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas.

Biochemistry. 2000 Aug 15; 39(32): 9893–900.

98.

Kumar

, Wang

R.A.

, Bagheri-Yarmand

Emerging roles of MTA family members in human cancers.

Semin Oncol. 2003; 30: 30–7.

99.

Mishra

S.K.

, Mazumdar

, Vadlamudi

R.K.

MICoA, a novel metastasis-associated protein 1 (MTA1) interacting protein coactivator, regulates estrogen receptor-alpha transactivation functions.

J Biol Chem. 2003; 278: 19209–19.

100.

Talukder

A.H.

, Mishra

S.K.

, Mandal

MTA1 interacts with MAT1, a cyclin-dependent kinase-activating kinase complex ring finger factor, and regulates estrogen receptor transactivation functions.

J Biol Chem. 2003; 278: 11676–85.

101.

Talukder

A.H.

, Gururaj

, Mishra

S.K.

, Vadlamudi

R.K.

, Kumar

Metastasis-associated protein 1 interacts with NRIF3, an estrogen-inducible nuclear receptor coregulator.

Mol Cell Biol. 2004; 24: 6581–91.

102.

Zhang

X.Y.

, DeSalle

L.M.

, Patel

J.H.

Metastasis-associated protein 1 (MTA1) is an essential downstream effector of the c-MYC oncoprotein.

Proc Natl Acad Sci U S A. 2005; 102: 13968–73.

103.

Takahashi

, Yamanaka

Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.

Cell. 2006; 126: 663–76.

104.

Wernig

In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state.

Nature. 2007; 448: 318–24.

105.

Molli

P.R.

, Singh

R.R.

, Lee

S.W.

, Kumar

MTA1-mediated transcriptional repression of BRCA1 tumor suppressor gene.

Oncogene. 2008; 27: 1971–80.

106.

Moon

H.E.

, Cheon

, Lee

M.S.

Metastasis-associated protein 1 inhibits p53-induced apoptosis.

Oncol Rep. 2007; 18: 1311–4.

107.

Singh

R.R.

, Kaluarachchi

, Chen

Solution structure and antiestrogenic activity of the unique C-terminal, NR-box motif-containing region of MTA1s.

J Biol Chem. 2006 Sep 1; 281(35): 25612–21.

108.

Kumar

, Wang

R.A.

, Mazumdar

A naturally occurring MTA1 variant sequesters oestrogen receptor-alpha in the cytoplasm.

Nature. 2002; 418: 654–7.

109.

Kumar

, Balasenthil

, Pakala

S.B.

, Rayala

S.K.

, Sahin

A.A.

, Ohshiro

Metastasis-associated protein 1 short form stimulates Wnt1 pathway in mammary epithelial and cancer cells.

Cancer Res. 2010 Aug 15; 70(16): 6598–608.

110.

Ding

, Gillespie

L.L.

, Paterno

G.D.

Human MI-ER1 alpha and beta function as transcriptional repressors by recruitment of histone deacetylase 1 to their conserved ELM2 domain.

Mol Cell Biol. 2003 Jan; 23(1): 250–8.

111.

Herman

M.A.

, Ch'ng

, Hettenbach

S.M.

, Ratliff

T.M.

, Kenyon

, Herman

R.K.

EGL-27 is similar to a metastasis-associated factor and controls cell polarity and cell migration in C. elegans. Oncogene. 23: 4422–9.

Development. 1999 Feb; 126(5): 1055–64.

112.

Chen

, Han

Role of C. elegans lin-40 MTA in vulval fate specification and morphogenesis.

Development. 2001 Dec; 128(23): 4911–21.

113.

Chen

, Han

C. elegans Rb, NuRD, and Ras regulate lin-39-mediated cell fusion during vulval fate specification.

Curr Biol. 2001 Nov 27; 11(23): 1874–9.

114.

Saito

, Ishikawa

The mCpG-binding domain of human MBD3 does not bind to mCpG but interacts with NuRD/Mi2 components HDAC1 and MTA2.

J Biol Chem. 2002 Sep 20; 277(38): 35434–9. Epub 2002 Jul 17.

115.

, Kovalev

G.I.

, Chang

Inactivation of NuRD component Mta2 causes abnormal T cell activation and lupus-like autoimmune disease in mice.

J Biol Chem. 283: 13825–33.

116.

, Lin

, Bartolomei

, Schultz

Metastasis Tumor Antigen 2 (MTA2) is involved in proper imprinted expression of H19 and Peg3 during mouse preimplantation development.

Biol Reprod. 2010 Aug 18. [Epub ahead of print].

117.

Fujita

, Jaye

D.L.

, Kajita

, Geigerman

, Moreno

C.S.

, Wade

P.A.

MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer.

Cell. 2003 Apr 18; 113(2): 207–19.

118.

Qian

Y.W.

, Lee

E.Y.

Dual retinoblastoma-binding proteins with properties related to a negative regulator of ras in yeast.

J Biol Chem. 1995 Oct 27; 270(43): 25507–13.

119.

Sondek

, Bohm

, Lambright

D.G.

, Hamm

H.E.

, Sigler

P.B.

Crystal structure of a G-protein beta gamma dimer at 2.1A resolution.

Nature. 1996 Jan 25; 379(6563): 369–74.

120.

Kadonaga

J.T.

Eukaryotic transcription: an interlaced network of transcription factors and chromatin-modifying machines.

Cell. 1998 Feb 6; 92(3): 307–13.

121.

Kuzmichev

, Nishioka

, Erdjument-Bromage

, Tempst

, Reinberg

Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of Zeste protein.

Genes Dev. 2002 Nov 15; 16(22): 2893–905.

122.

Verreault

, Kaufman

P.D.

, Kobayashi

, Stillman

Nucleosomal DNA regulates the core-histone-binding subunit of the human Hat1 acetyltransferase.

Curr Biol. 1998 Jan 15; 8(2): 96–108.

123.

Henikoff

Versatile assembler.

Nature. 2003 Jun 19; 423(6942): 814–5, 817.

124.

, Horvitz

H.R.

lin-35 and lin-53, two genes that antagonize a C. elegans pathway, encode proteins similar to Rb and its binding protein RbAp48.

Cell. 1998 Dec 23; 95(7): 981–91.

125.

Walhout

A.J.

, Sordella

, Lu

Protein interaction mapping in C. elegans using proteins involved in vulval development.

Science. 2000 Jan 7; 287(5450): 116–22.

126.

Wang

, Kennedy

, Conte

Jr . Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants. Nature. 2005 Jul 28; 436(7050): 593–7.

127.

Terret

M.E.

, Lefebvre

, Djiane

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes.

Development; 2003.

128.

Malovannaya

, Li

, Bulynko

Streamlined analysis schema for high-throughput identification of endogenous protein complexes.

Proc Natl Acad U S A. 2010 Feb 9; 107(6): 2431–6.

129.

Yang

X.J.

, Seto

The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.

Nat Rev Mol Cell Biol. 2008 Mar; 9(3): 206–18.

130.

Yamagoe

, Kanno

Interaction of histone acetylases and deacetylases in vivo.

Mol Cell Biol. 2003 Feb; 23(3): 1025–33.

131.

Van den Heuvel

, Dyson

N.J.

Conserved functions of the pRB and E2F families.

Nat Rev Mol Cell Biol. 2008 Sep; 9(9): 713–24.

132.

Bates

E.A.

, Victor

, Jones

A.K.

, Shi

, Hart

A.C.

Differential contributions of Caenorhabditis elegans histone deacetylases to huntingtin polyglutamine toxicity.

J Neurosci. 2006 Mar 8; 26(10): 2830–8.

133.

Zinovyeva

A.Y.

, Graham

S.M.

, Cloud

V.J.

, Forrester

W.C.

The C. elegans histone deacetylase HDA-1 is required for cell migration and axon pathfinding.

Dev Biol. 2006 Jan 1; 289(1): 229–42.

134.

Brackertz

, Gong

, Leers

, Renkawitz

p66alpha and p66beta of the Mi-2/NuRD complex mediate MBD2 and histone interaction.

Nucleic Acids Res. 2006 Jan 13; 34(2): 397–406.

135.

Brackertz

, Boeke

, Zhang

, Renkawitz

Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3.

J Biol Chem. 2002 Oct 25; 277(43): 40958–66.

136.

Gong

, Brackertz

, Renkawitz

SUMO modification enhances p66-mediated transcriptional repression of the Mi-2/NuRD complex.

Mol Cell Biol. 2006 Jun; 26(12): 4519–28.

137.

Nottke

, Colaiácovo

M.P.

, Shi

Developmental roles of the histone lysine demethylases.

Development. 2009 Mar; 136(6): 879–89.

138.

Lan

, Nottke

A.C.

, Shi

Mechanisms involved in the regulation of histone lysine demethylases.

Curr Opin Cell Biol. 2008 Jun; 20(3): 316–25. Epub 2008 Apr 25.

139.

S.C.

, Zhang

Active DNA demethylation: many roads lead to Rome.

Nat Rev Mol Cell Biol. 2010 Sep; 11(9): 607–20.

140.

Tsai

M.C.

, Manor

, Wan

Long noncoding RNA as modular scaffold of histone modification complexes.

Science. 2010 Aug 6; 329(5992): 689–93.

141.

Xia

, Zhang

Sp1 and ETS family transcription factors regulate the mouse Mta2 gene expression.

Gene. 2001; 268: 77–85.

142.

Yoon

H.G.

, Chan

D.W.

, Reynolds

A.B.

, Qin

, Wong

N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso.

Mol Cell. 2003 Sep; 12(3): 723–34.

143.

Hong

, Nakazawa

, Chen

Y.Y.

FOG-1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA-1.

EMBO J. 2005 Jul 6; 24(13): 2367–78.

144.

Sekimata

, Takahashi

, Murakami-Sekimata

, Homma

Involvement of a novel zinc finger protein, MIZF, in transcriptional repression by interacting with a methyl-CpG-binding protein, MBD2.

J Biol Chem. 2001 Nov 16; 276(46): 42632–8.

145.

Rigaut

, Shevchenko

, Rutz

, Wilm

, Mann

, Séraphin

A generic protein purification method for protein complex characterization and proteome exploration.

Nat Biotechnol. 1999 Oct; 17(10): 1030–2.

146.

Kaiser

, Meierhofer

, Wang

, Huang

Tandem affinity purification combined with mass spectrometry to identify components of protein complexes.

Methods Mol Biol. 2008; 439: 309–26.

147.

Yang

, Sampson

H.M.

, Krause

H.M.

A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor dHNF4.

Proteomics. 2006 Feb; 6(3): 927–35.

148.

Collins

M.O.

, Choudhary

J.S.

Mapping multiprotein complexes by affinity purification and mass spectrometry.

Curr Opin Biotechnol. 2008 Aug; 19(4): 324–30.

149.

Gloeckner

C.J.

, Boldt

, Schumacher

, Roepman

, Ueffing

A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.

Proteomics. 2007 Dec; 7(23): 4228–34.

150.

Malovannaya

, Li

, Bulynko

Streamlined analysis schema for high-throughput identification of endogenous protein complexes.

Proc Natl Acad Sci U S A. 2010 Feb 9; 107(6): 2431–6.

151.

Andersen

J.N.

, Sathyanarayanan

, di Bacco

Pathway-based identification of biomarkers for targeted therapeutics: personalized oncology with PI3K pathway inhibitors.

Sci Transl Med. 2010 Aug 4; 2(43): 43ra55.

152.

Sridharan

, Smale

S.T.

Predominant interaction of both Ikaros and Helios with the NuRD complex in immature thymocytes.

J Biol Chem. 2007 Oct 12; 282(41): 30227–38.

153.

Zhang

, Nash

, Fisher

A.L.

A simplified, robust, and streamlined procedure for the production of C. elegans transgenes via recombineering.

BMC Dev Biol. 2008 Dec 30; 8: 119.

154.

Zhang

, Kashyap

, Ferguson

A.A.

, Fisher

A.L.

The production of C. elegans transgenes via recombineering with the galk selectable marker.

J Vis Exp. 2010. (in press).

155.

Poser

, Sarov

, Hutchins

J.R.

BAC Transgeneomics: a high-throughput method for exploration of protein function in mammals.

Nat Methods. 2008 May; 5(5): 409–15.

156.

Fernández

, Collins

M.O.

, Uren

R.T.

Targeted tandem affinity purification of PSD-95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins.

Mol Syst Biol. 2009; 5: 269.

157.

Wang

, Rao

, Chu

A protein interaction network for pluripotency of embryonic stem cells.

Nature. 2006 Nov 16; 444(7117): 364–8.

158.

Ozsolak

, Ting

D.T.

, Wittner

B.S.

Amplification-free digital gene expression profiling from minute cell quantities.

Nat Methods. 2010 Aug; 7(8): 619–21.

159.

Adli

, Zhu

, Bernstein

B.E.

Genome-wide chromatin maps derived from limited numbers of hematopoietic progenitors.

Nat Methods. 2010 Aug; 7(8): 615–8.

160.

Polyak

Going small is the new big.

Nat Methods. 2010 Aug; 7(8): 597, 599–600.

161.

Behrends

, Sowa

M.E.

, Gygi

S.P.

, Harper

J.W.

Network organization of the human autophagy system.

Nature. 2010 Jul 1; 466(7302): 68–76.

162.

Fischbach

M.A.

, Krogan

N.J.

The next frontier of systems biology: higher-order and interspecies interactions.

Genome Biol. 2010; 11(5): 208. Epub 2010 May 5.

163.

Zhang

Rough set soft computing cancer classification and network: one stone, two birds.

Cancer Inform. 2010 Jul 15; 9: 139–45.

164.

Babaie

, Herwig

, Greber

Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells.

Stem Cells. 2007 Feb; 25(2): 500–10.

165.

Loh

Y.H.

, Wu

, Chew

J.L.

The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.

Nat Genet. 2006 Apr; 38(4): 431–40.

166.

Lauberth

S.M.

, Rauchman

A conserved 12-amino acid motif in Sall1 recruits the nucleosome remodeling and deacetylase corepressor complex.

J Biol Chem. 2006 Aug 18; 281(33): 23922–31. Epub 2006 May 17.

167.

Nawa

, Nishimori

, Lin

Tumor metastasis-associated human MTA1 gene: its deduced protein sequence, localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligonucleotides.

J Cell Biochem. 2000 Aug 2; 79(2): 202–12.

168.

Nicolson

G.L.

, Nawa

, Toh

, Taniguchi

, Nishimori

, Moustafa

Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation.

Clin Exp Metastasis. 2003; 20(1): 19–24.

169.

Assudani

D.P.

, Ahmad

, Li

, Rees

R.C.

, Ali

S.A.

Immunotherapeutic potential of DISC-HSV and OX40L in cancer.

Cancer Immunol Immunother. 2006 Jan; 55(1): 104–11.

170.

Ellis

, Atadja

P.W.

, Johnstone

R.W.

Epigenetics in cancer: targeting chromatin modifications.

Mol Cancer Ther. 2009 Jun; 8(6): 1409–20. Epub 2009 Jun 9.

171.

Carey

, la Thangue

N.B.

Histone deacetylase inhibitors: gathering pace.

Curr Opin Pharmacol. 2006 Aug; 6(4): 369–75. Epub 2006 Jun 14.

172.

Hockly

, Richon

V.M.

, Woodman

Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease.

Proc Natl Acad Sci U S A. 2003 Feb 18; 100(4): 2041–6. Epub 2003 Feb 7.

173.

Minamiyama

, Katsuno

, Adachi

Sodium butyrate ameliorates phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy.

Hum Mol Genet. 2004 Jun 1; 13(11): 1183–92. Epub 2004 Apr 21.

174.

Ying

, Xu

, Wu

Sodium butyrate ameliorates histone hypoacetylation and neurodegenerative phenotypes in a mouse model for DRPLA.

J Biol Chem. 2006 May 5; 281(18): 12580–6. Epub 2005 Dec 28.

175.

Candelaria

, Gallardo-Rincón

, Arce

A phase II study of epigenetic therapy with hydralazine and magnesium valproate to overcome chemotherapy resistance in refractory solid tumors.

Ann Oncol. 2007 Sep; 18(9): 1529–38.

176.

Camphausen

, Tofilon

P.J.

Inhibition of histone deacetylation: a strategy for tumor radiosensitization.

J Clin Oncol. 2007 Sep 10; 25(26): 4051–6.

177.

Kubicek

, O'Sullivan

R.J.

, August

E.M.

Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase.

Mol Cell. 2007 Feb 9; 25(3): 473–81.

178.

Wang

, Charroux

, Kerridge

, Tsai

C.C.

Atrophin recruits HDAC1/2 and G9a to modify histone H3K9 and to determine cell fates.

EMBO Rep. 2008 Jun; 9(6): 555–62. Epub 2008 May 2.

179.

Metzger

, Wissmann

, Yin

LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription.

Nature. 2005 Sep 15; 437(7057): 436–9.

180.

Schmidt

D.M.

, McCafferty

D.G.

Trans-2-Phenylcyclopropylamine is a mechanism-based inactivator of the histone demethylase LSD1.

Biochemistry. 2007 Apr 10; 46(14): 4408–16.

181.

Herman

J.G.

, Latif

, Weng

Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.

Proc Natl Acad Sci U S A. 1994 Oct 11; 91(21): 9700–4.

182.

Alleman

W.G.

, Tabios

R.L.

, Chandramouli

G.V.

. The in vitro and in vivo effects of re-expressing methylated von Hippel-Lindau tumor suppressor gene in clear cell renal carcinoma with 5-aza-2‘-deoxycytidine. Clin Cancer Res. 2004 Oct 15; 10(20): 7011–21.

183.

Liu

L.Z.

, Hu

X.W.

, Xia

Reactive oxygen species regulate epidermal growth factor-induced vascular endothelial growth factor and hypoxia-inducible factor-1alpha expression through activation of AKT and P70S6K1 in human ovarian cancer cells.

Free Radic Biol Med. 2006 Nov 15; 41(10): 1521–33.

184.

Kim

D.H.

, Rossi

J.J.

Strategies for silencing human disease using RNA interference.

Nat Rev Genet. 2007 Mar; 8(3): 173–84.

185.

Vastenhouw

N.L.

, Brunschwig

, Okihara

K.L.

, Müller

, Tijsterman

, Plasterk

R.H.

Gene expression: long-term gene silencing by RNAi.

Nature. 2006 Aug 24; 442(7105): 882.

186.

Targeting DNA damage and repair by curcumin.

Breast Cancer (Auckl). 2010 Feb 16; 4: 1–3.

187.

Chen

, Yang

, Wang

Crystal structure of human histone lysine-specific demethylase 1 (LSD1).

Proc Natl Acad Sci U S A. 2006 Sep 19; 103(38): 13956–61. Epub 2006 Sep 6.

188.

Vannini

, Volpari

, Filocamo

Crystal structure of a eukaryotic zinc-dependent histone deacetylase, human HDAC8, complexed with a hydroxamic acid inhibitor.

Proc Natl Acad Sci U S A. 2004 Oct 19; 101(42): 15064–9.

189.

Reese

K.J.

, Lin

, Verona

R.I.

, Schultz

R.M.

, Bartolomei

M.S.

Maintenance of paternal methylation and repression of the imprinted H19 gene requires MBD3.

PLoS Genet. 2007 Aug; 3(8): e137. Epub 2007 Jun 29.

190.

Pegoraro

, Kubben

, Wickert

, Göhler

, Hoffmann

, Misteli

Ageing-related chromatin defects through loss of the NuRD complex.

Nat Cell Biol. 2009 Oct; 11(10): 1261–7. Epub 2009 Sep 6.

191.

Lapidot

, Sirard

, Vormoor

A cell initiating human acute myeloid leukaemia after transplantation into SCID mice.

Nature. 1994 Feb 17; 367(6464): 645–8.

192.

, Zeng

, Xu

R.H.

, Liu

, Li

Vaccination with human pluripotent stem cells generates a broad spectrum of immunological and clinical responses against colon cancer.

Stem Cells. 2009 Dec; 27(12): 3103–11.

193.

Senju

, Hirata

, Motomura

Pluripotent stem cells as source of dendritic cells for immune therapy.

Int J Hematol. 2010 Apr; 91(3): 392–400. Epub 2010 Feb.

194.

Mathews

L.A.

, Crea

, Farrar

W.L.

Epigenetic gene regulation in stem cells and correlation to cancer.

Differentiation. 2009 Jul; 78(1): 1–17. Epub 2009 May 14.

195.

De Wit

, Linsen

S.E.

, Cuppen

, Berezikov

Repertoire and evolution of miRNA genes in four divergent nematode species.

Genome Res. 2009 Nov; 19(11): 2064–74. Epub 2009 Sep 15.

196.

Kirienko

N V.

, Mani

, Fay

D.S.

Cancer models in Caenorhabditis elegans.

Dev Dyn. 2010 May; 239(5): 1413–48.

197.

Sharma

, Weber

, Bairlein

Proteomics strategy for quantitative protein interaction profiling in cell extracts.

Nat Methods. 2009 Oct; 6(10): 741–4. Epub 2009 Sep 13.

198.

De la Fuente

From ‘differential expression’ to ‘differential networking‘—identification of dysfunctional regulatory networks in diseases.

Trends Genet. 2010 Jul; 26(7): 326–33.

199.

Golub

Counterpoint: data first.

Nature. 2010 Apr 1; 464(7289): 679.

200.

Weinberg

Point: hypotheses first.

Nature. 2010 Apr 1; 464(7289): 678.

The Expanding Mi-2/NuRD Complexes: A Schematic Glance

Abstract

Keywords

Overview

The Interacting Proteins and Functions of the Core Subunits within the Mi-2/NuRD Complex

The Mi-2α, β/CHD3, 4 proteins

The mCpG-binding domain (MBD) proteins

MBD2/Mi-2/NuRD and MBD3/Mi-2/NuRD

The metastasis-associated (MTA) proteins

MTA1/Mi-2/NuRD

MTA2/Mi-2/NuRD

MTA3/Mi-2/NuRD

The Histone Binding Proteins/ Retinoblastoma a Tumor Suppressor RbAp46/P48

The histone deacetylases

GATAD2a/b (p66/p68)

Lamin a and the Mi-2/NuRD Complex

Post-transcriptional modifications and upstream cell signaling pathways of Mi-2/NuRD

The Methodology for Identifying of Mi-2/NuRD Complex Composition

The emerging stemnessed Mi-2/NuRD complexes

Clinical and therapeutic implications

MTAs: targets for drug development

MBDs: targets for drug development

HDACs: targets for drug development

G9a: a target for drug development

LSD1: a target for drug development

RNAi and potential combined therapy

Future Mi-2/NuRD-based approaches

Future Mi-2/NuRD stemness-based treatment

The significance of cross-validation with interspecies (Mammals and C. elegans): a personal view

Discussion

Disclosure

Footnotes

Acknowledgements

References