Abstract
What a shock when we learned that Steven A. Brown, 52, succumbed to injuries resulting from a crash landing near the airport in Happy Valley-Goose Bay, Canada, on 14 December 2022. Steve, professor of chronobiology and sleep research at the Faculty of Medicine, University of Zürich, was an excellent scientist, a dear friend, a wonderful collaborator, and an incredibly courageous adventurer. His sudden death immediately reminded us of the Billy Joel song: Only the good die young.
Steve owned a Piper PA-46 Malibu aircraft that he had to bring to Florida for an annual control and update. In company with his wife Patrycja Paruch, he then piloted his airplane to Nashua, NH, where they visited Steve’s mother. The trip was to continue to Nuuk, Greenland, with an intermediate stop in Goose Bay, Canada, where the tragic incident occurred. Patrycja, a highly successful physics professor at the University of Geneva and a great friend of ours, survived the crash. We would like to express our wishes of a full and prompt recovery and offer our sincere condolences to Patrycja. Our deepest sympathy goes to family members, friends, and colleagues of Steve.
Steve, The Graduate Student and Wine Connoisseur
Born on 1 April 1970, in Boston, Steve studied biochemistry at Harvard College, Cambridge, Massachusetts, where he received his Bachelor of Arts in 1990 and his PhD in 1997. During his PhD thesis work in Robert (Bob) Kingston’s laboratory, he dissected basic mechanisms involved in the control of transcription and transcriptionally induced chromatin transitions. To this end, he used heat shock–stimulated gene expression in mammalian cells as a model system. At elevated temperatures proteins denature, thereby exposing hydrophobic peptide regions. This leads to protein-protein interactions that form irreversible protein precipitates that are harmful to cells. Upon heat shock, cells rapidly express several chaperones, also known as heat shock proteins (HSPs), which are expressed as a programmed reaction to hyperthermia. They bind to misfolded proteins and either prevent or resolve protein aggregates. The temperature-dependent transcription of HSPs is controlled by heat shock transcription factors (HSFs). As shown by Rougvie and Lis (1988), RNA polymerase II is associated with the promoters of HSP genes even in non-stressed cells, where these genes are only weakly or not at all transcribed. This situation, called polymerase pausing, was later observed for many if not most genes. Steve made important contributions toward the understanding of HSF-mediated transcription. Thus, he established an in vitro system mimicking polymerase pausing on the Hsp70 promoter. This allowed him to conclude that nucleosome formation downstream of this promoter was required for efficient pausing. Furthermore, in the presence of the chromatin remodeler SWI/SNF, a chimeric transcription factor carrying the HSF1 transactivation domain could release stalled RNA polymerase II from the pause site (Brown et al., 1996). In an additional study, Steve demonstrated that HSF1 activation disrupts HSP70 promoter-proximal chromatin involved in pausing in a transcription-independent step. In contrast, transcription elongation by RNA polymerase II was required to modify promoter-distal chromatin within the HSP70 gene body (Brown and Kingston, 1997). By using different transcription factor chimeras, Steve made an original and unexpected discovery: while a variety of transactivation domains could stimulate transcription initiation at the HSP70 promoter, only the ones of HSF1 (and that of the Herpes virus activator VP16) were able to also induce transcription elongation. Detailed mutagenesis experiments with the HSF1 transactivation domain revealed that different amino acid residues were involved in the boosting of initiation and elongation (Brown et al., 1998).
When we asked Bob Kingston for his reminiscences of Steve as a PhD student in his lab, he wrote the following:
Steve was a precocious intellect in his youth who was accepted to and started Harvard College at the age of 16. He had worked as an undergraduate in Fred Winston’s group at Harvard Medical School and had contributed significantly to a paper from Fred’s lab that provided the first evidence that the SWI/SNF complex altered nucleosome structure (Hirschhorn et al., 1992). This was based on work done primarily by Joel Hirschhorn, an undergraduate and then a graduate student with Fred and now a tenured faculty member in Genetics at Harvard Medical School.
The interest in possible roles for chromatin in gene regulation was what attracted him to do a rotation in my group as he started graduate school. We had developed biochemical protocols to study the impact of nucleosomal structures on transcription processes. Steve chose to develop an in vitro system to study the regulation of transcriptional elongation on nucleosomal templates. He developed a protocol to do so, which required multiple steps as it was, by necessity, very complicated. One of Steve’s defining characteristics was the precision with which he approached complicated protocols (also echoed in his outstanding cooking.) He was an unusually consistent graduate student. He worked hard, his demeanor was always pleasant and more formal than the average American student of his era. He did hard experiments, he did them well, and he took setbacks with equanimity. He had strong opinions on pretty much all topics and was transparent concerning those opinions. I enjoyed working with him very much because I knew I would hear what he really thought, and later in life I enjoyed visiting with him because again I would get his unvarnished opinion. He was fine with disagreements, rarely defensive but also rarely backing away from a viewpoint if he felt strongly about it.
I once asked him why he had decided to join our lab. He told me that when he was a rotating student, he attended the group meeting of an especially volatile student I had in the group at the time. The student and I got into an argument about the strategy of the student project. It escalated until the student screamed “F. . . you Bob.” I of course remembered that incident vividly, as thankfully it was not a normal occurrence. I asked Steve why that helped him decide to join our group. Steve said that I handled the confrontation calmly, that we moved on with group meeting, so therefore Steve felt that I would have no problem handling Steve’s opinions on any topic. Ironically, Steve was the politest student I have ever had in the group and the least likely to swear at me in any setting (U.S., C.D. and J.R. can vividly confirm this assessment: Steve was the only one in our lab never swearing when things went wrong).
I am passionate about wine and started collecting a large wine cellar in 1985. I would always do a wine tasting with bottles that I had collected at all group functions. Steve especially enjoyed those tastings and developed a passion for wine that, in very little time, greatly exceeded mine. While he was in Geneva, he spent a large amount of time driving to Burgundy and getting to know some of the great producers of red Burgundy. I visited Geneva and we left a day open, and Steve drove me there and we visited a few producers and tasted with them. The highlight was a visit with Charles Rousseau, one of the great wine producers in the world. For the next decade, Steve would come by to visit on his way to visit his mother and would bring me six bottles of Rousseau red Burgundy and charged me the price that he paid for the wine purchased directly from the Château. Three to four times lower than I would have paid in the US even if I could find the bottles (which were and still are highly allocated). I commented that this was incredibly generous of him, and he always replied that he was repaying me for stimulating his interest in wine. I still have many of those bottles in my cellar and open them to mark special occasions. I have always raised a glass to Steve in the past and will do so in a different way going forward.
Steve, The Postdoc: An Unplanned Journey from Chromatin/Transcription Research to Chronobiology
After receiving a PhD degree from Harvard University, Steve decided to pursue his research career by doing postdoctoral work abroad. As a passionate mountaineer, he selected Switzerland or Austria as appropriate places and asked Bob for advice concerning the choice of his future mentor. Bob recommended to join “Uli’s” lab at the Department of Molecular Biology, University of Geneva. So Steve applied for a postdoctoral position to Ueli Schibler and was invited for an interview. The decision of accepting Steve was a no-brainer: he immediately impressed all members of the lab with his intellectual sharpness, his broad knowledge in molecular biology, and his pleasant, polite, and unpretentious personality. Postdoctoral candidates of that caliber belong to a rare and highly sought-after species. His transition from chromatin research to molecular chronobiology was swift and painless, and as described below, he made impressive contributions in the lab. Later, it turned out that Steve misinterpreted Bob Kingston’s advice in choosing a mentor for his postdoctoral work: Bob meant Uli Laemmli, one of the world leaders in chromatin research—and not the much less famous Ueli Schibler, who, coincidentally, was also a professor in the same department.
Just around the time when Steve arrived in the Schibler lab (1997), Aurelio Balsalobre, another postdoc, discovered that cultured fibroblasts are equipped with self-sustained and cell-autonomous circadian clocks. These can be transiently synchronized by a serum shock (Balsalobre et al., 1998) and drugs activating a puzzling variety of well-known signaling cascades (Balsalobre et al., 2000b). Steve added a novel phase-resetting pathway to the list. His experiments demonstrated that square-wave rhythms oscillating between 33 °C and 37 °C efficiently synchronized fibroblast clocks. This was, perhaps, not surprising, since square-wave temperature rhythms had previously been shown to function as strong zeitgebers for ectotherm uni- and multicellular organisms. The more relevant question then arose of whether natural, smoothly scaled body temperature cycles of endotherm animals could also synchronize circadian timekeepers. Our department was in the favorable position of hosting a mechanical workshop run by highly gifted precision engineers. They designed and built an incubator for Steve, which could impose telemetrically recorded mouse body temperature rhythms on cultured cells. Using this sophisticated device, Steve demonstrated that natural body temperature rhythms fluctuating between 35 °C and 38 °C can indeed maintain phase coherence among fibroblast clocks (Brown et al., 2002). Thereby, he established the basis for several follow-up studies in the labs of U.S. and others on the impact of body temperature on rhythmic gene expression.
Taking advantage of his biochemistry training in Bob Kingston’s lab, Steve set out to characterize protein complexes containing Period (PER) proteins. PERs, together with Cryptochromes (CRYs), serve as the co-repressors in shaping the major negative feedback loop in clock gene expression. These endeavors revealed PER-CRY protein complexes larger than 1 Megadalton (MDa) that contained proteins hitherto not known to be associated with PERs or CRYs (Brown et al., 2005b). Very elegant studies by Chuck Weitz’s group confirmed the large size of nuclear PER-CRY complexes and provided more detailed information on their composition. The PER-CRY repressosomes isolated from liver nuclei in Chuck’s lab had a molecular mass of nearly 2 MDa and contained about 30 different polypeptides (Aryal et al., 2017).
Steve’s third major research effort in the Schibler lab was dedicated to the recording of molecular rhythms in primary skin fibroblasts obtained from punch biopsies of 19 human subjects. While the period lengths were highly reproducible for fibroblasts harvested from the same individuals, they showed a wide distribution in fibroblasts prepared from different subjects (Brown et al., 2005a). In mice, the period length of cultured tail tip fibroblasts correlated with that of wheel running activity, an output of the central clock in the suprachiasmatic nuclei (SCN). Hence, central and peripheral clocks shared common properties. As shown by Steve some years later in his own laboratory, the same conclusion could be reached for humans (see below). In addition to the studies described above, Steve collaborated with Aurelio Balsalobre and the Group of Günther Schütz, German Cancer Center in Heidelberg, on the synchronization of peripheral clocks by glucocorticoids. This work culminated in a highly cited Science paper (Balsalobre et al., 2000a).
Steve, The Professor and Mentor
To the delight of U.S. and his collaborators, Steve stayed in the lab for an extended period before he applied for assistant professor positions. Owing to his impressive track record and his extraordinary lecturing skills, he was offered several positions at Universities in Europe and the United States. Fortunately for Swiss science, he accepted a tenure track assistant professorship in the Institute of Pharmacology and Toxicology at the University of Zürich in 2004. The position became vacant after the retirement of Alex Borbély, one of the foremost figures in sleep research. However, Steve had to wait till 2006 until the laboratory space became available. In the meantime, he joined Achim Kramer’s lab as a Humboldt Fellow. This collaboration resulted in a close friendship between Steve and Achim and 4 high-impact publications during and after Steve’s sabbatical in Berlin (Brown et al., 2008; Kowalska et al., 2012; Azzi et al., 2014; Gaspar et al., 2017) that paved the way to further molecular studies of human oscillators and their outputs (Dallmann et al., 2012; Gaspar et al., 2014; Gaspar and Brown, 2015). Not surprisingly, Steve’s research career as an independent investigator was highly successful. His projects encompassed the genetics and physiology of circadian rhythms and sleep in humans and mice and, more recently, molecular neuroscience. Let us just illustrate the originality of his research endeavors by a few examples. As aforementioned, Steve was interested in the relationship between central and peripheral clocks in humans. In a study he conducted in collaboration with several other research teams, the period lengths of melatonin rhythms in sighted and blind human subjects were compared to those of oscillators operative in skin fibroblasts from the same individuals (Pagani et al., 2010). These experiments revealed convincingly that the period lengths of central and peripheral clocks are directly proportional in humans, similar to what Steve had previously documented for mice. A particularly striking observation was made by Steve on the so-called “after-effect” in mice (Pittendrigh and Daan, 1976). When he exposed young mice to shorter or longer than 24-h light-dark cycles (T-cycles) for some weeks, the mice free-ran with shorter or longer period, respectively, for months thereafter (Azzi et al., 2014). Therefore, the SCN must have “memorized” the entrained period length for extended time periods. What mechanisms could account for such a long-term memory in SCN neurons? Steve’s group identified an unexpected culprit: the DNA methylome. The CpG methylation patterns within clock gene promoters were found to be altered in the SCN after T-cycle exposure. Moreover, pharmacological inhibition of DNA methylation abolished the “after effect” in free-running period. In old mice, no or little “after effect” could be observed after the exposure to short and long T-cycles, and this lack of period plasticity was accompanied by low levels of the DNA methyl transferases DNMT3a. In a more recent study, Steve’s group presented some evidence for a pathway involving neuronal network dynamics, rather than cell-autonomous mechanisms, in the shaping of the DNA methylome in the SCN (Azzi et al., 2017).
Steve’s latest work addressed the molecular basis of synaptic transmission in the forebrain and the SCN. In collaboration with Charo Robles’ lab, he examined the impact of sleep-wake cycles and circadian clocks on synaptic activities in the forebrain across the 24-h day. About 50% of synaptic phosphoproteins, including protein kinases, displayed high-amplitude cycles, and the rhythmic protein phosphorylation affected cytoskeleton dynamics and the balance between excitatory and inhibitory interneuronal signaling. As sleep deprivation abolished virtually all synaptic phosphorylation cycles, it appeared likely that sleep-wake cycles, rather than the circadian clock, controlled synaptosomal protein phosphorylation rhythms and their consequences on neuronal outputs (Bruning et al., 2019). In the same issue of Science, the teams of Steve and Charo demonstrated that nearly 70% of synaptic mRNAs accumulate in a circadian fashion. Of these, more than 90% cycle exclusively in axonal nerve terminals and attached postsynaptic structures (synaptoneurosomes). Since only about 6% of forebrain transcripts oscillate, the strong enrichment of rhythmic mRNAs in synaptoneurosomes must be accomplished by a posttranscriptional mRNA transport mechanism, shown to be gated by the circadian clock. Interestingly, sleep deprivation eliminated daily protein oscillations in synaptosomes, but did not affect rhythmic mRNA accumulation in these structures. Thus, whereas the clock drives cyclic synaptosomal transcript expression, sleep-wake cycles orchestrate rhythmic synaptosomal protein accumulation (Noya et al., 2019). In conclusion, Steve’s research was always guided by his ambition of making novel and original discoveries, rather than by adding more of the same to what we already know.
During his postdoctoral period, Steve already excelled in mentoring graduate and undergraduate students in U.S.’s team. He even perfected this virtue when supervising his own collaborators as a professor. Beyond being a constant inspiration to his close colleagues, Steve influenced many young scientists during the trainee days and PhD schools that he organized in a remarkably original way.
Steve, The Daring Adventurer
Steve’s temperate and polite personality was in stark contrast to his adventurous hobbies. As a passionate mountaineer, he climbed many challenging routes and several major peaks. The physicist and mountain climber Bruce Normand, one of Steve’s friends, wrote:
As an undergraduate Steve was an activist in the famous Harvard Mountaineering Club (HMC), and in addition to climbing rock and ice in his native New England, through his graduate years he was a frequent visitor to Yosemite Valley and to Red Rocks in Nevada. In 1990, he and his father made the trip to Denali in Alaska, and the HMC was where he met his future wife, Patrycja. For this side of his character, Geneva was the perfect place for a postdoc, and Steve made regular visits to the Mont Blanc range. After settling in Zurich, he joined the Akademischer Alpenclub Zürich (AACZ), where he was an enthusiastic participant in a wide range of climbs, ski-tours and club events. In addition to the technical side of climbing, for Steve the exploration aspect and the big mountains and were a special passion, and after Denali he formed small teams to attempt difficult and aesthetic mountains in Kazakhstan (Khan Tengri, 1991), Pakistan (Muztagh Tower, 2005), Xinjiang (Xuelian, 2009), and Khan Tengri again (2014). In 2022 he contributed all his experience and enthusiasm to the 125th-anniversary expedition of the AACZ, where a large team of old friends and younger club members visited the remote Changla Himal in West Nepal.
But mountain climbing was just one manifestation of Steve’s daring character. When still in Geneva, he witnessed an act of vandalism late at night. Drunken youngsters were jumping up and down on the roofs of parked car. When he called the police, the delinquents tried to escape. He followed one of them and captured him after a 10-min run. Once Steve had safely immobilized him on the ground, he called the police department again. Two police officers arrived swiftly and arrested Steve, letting the culprit break free. Fortunately, the confusion was soon cleared, and Steve was released from the police office.
Steve also enjoyed some more innocuous avocations than ascending mountain tops and arresting criminals. As pointed out by Bob Kingston, he was an excellent cook and wine connoisseur, with a cellar harboring over 1000 bottles of the finest wines. In fact, he put tons of gravel in the basement in the house for the right climate when he first rented it and made his own wine with all the tools that biochemistry can offer. Also, his meat (game) was sometimes stored at −80 °C in the lab, because he believed it freezes in smaller crystals. On many unforgettable occasions, Steve spoiled us as a highly gifted cook and sommelier.
Steve’s tragic death has left an immense void in our hearts, and in the community of chronobiology and sleep science. We will miss Steve dearly as a generous and vivid personality, a caring friend, a brilliant, original, and inspiring scientist, and a role model for young researchers.