LS² Annual Meeting 2023

It is clear that the cause of obesity is a result of eating more than you burn.  It is physics. What is more complex to answer is why some people eat more than others? Differences in our genetic make-up mean some of us are slightly more hungry all the time and so eat more than others. In contrast to the prevailing view, obesity is not a choice. People who are obese are not bad or lazy; rather, they are fighting their biology.

The endosomal protein complex required for transport-III (ESCRT-III) participates in membrane remodelling processes of essentially all cellular organelles and catalyses membrane fission from within membrane necks in many crucial cellular functions, from cell division to lysosome degradation and autophagy. The simple protein structure shared by all ESCRT-III subunits can assemble into a large variety of filament shapes, however understanding of how these filaments achieve membrane remodelling and fission is limited. We characterized a sequential polymerization of ESCRT-III subunits that, driven by a continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of subunits induces first a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filaments to drive the formation of membrane protrusions and ends with the formation of a highly constricted co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of gradual changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to drive ESCRT-III membrane remodelling activity.

Hunger is a basic survival state that shapes much of the activities in the living world. As in other goal directed behaviors, food intake involves decisions resulting from neural integration of signals from the external environment (e.g. sight, taste, smell) and interosensory information that signals internal state to the brain. Interosensory information is conveyed to key circuit nodes responsible for goal directed behaviors by a complex system of neural connections, and the activity of these pathways has a significant impact on prioritization of external cues and adaptive responses. Hypothalamic neural networks maintain energy homeostasis by coordinating endocrine signals with behavioral and autonomic functions to ensure that behaviors and physiological responses remain in tune with environmental demands. Because the architecture of neural circuits determines how they function, a comprehensive understanding of how neural systems responsible for neuroendocrine integration are organized is essential, and we are working to determine how developmental events impact their construction and functional properties. By evaluating the impact of early hormonal and nutritional challenges on the brain wide organization of these essential neural systems, and by profiling neuronal responses to varied interosensory stimuli in vivo, we are gaining insight into neurobiological mechanisms underlying developmental programming of neuroendocrine integration, within the functional context of feeding behavior.

Segregation of genomic regions into accessible euchromatin and inaccessible heterochromatin is essential for temporal and tissue-specific gene transcription. In C. elegans, the SETDB1 homolog MET-2 promotes heterochromatic silencing of satellite repeats, transposable elements and tissue-specific genes, by promoting the demethylation of histone H3 lysine 9. The segregation of heterochromatin from euchromatin helps maintain tissue integrity by restricting gene expression and stabilizing the genome. Animals lacking the H3K9 HMT show temperature-dependent phenotypes including a loss of fertility, developmental delay, and shortened lifespan. MET-2’s ability to preserve heterochromatin repression coincides with concentration in nuclear foci through physical interaction with the intrinsically disordered protein LIN-65. These foci have a second, non-catalytic function that contributes to gene repression to a limited extent. Catalytically deficient MET-2 was sufficient to reduce H3K9  and H3K27 acetylation at a subset of promoters and enhancers, and to restore fertility to a met-2 strain. We suggest that germline integrity stems in part from an appropriate organization of heterochromatic vs euchromatic domains.

The sensitivity of peripheral pain-receptor neurons to noxious thermal and chemical stimuli is tuned by a variety of receptors and second messengers, in part through tuning the sensitivity and number of TRPV1 ion channels that act as receptors for thermal and chemical stimuli. The gain can be decreased, producing desensitization, or increased, producing hyperalgesia. Over the last decade, significant progress has been made in understanding both desensitization and hyperalgesia at the cellular and molecular levels. This talk will focus on the mechanisms regulating plasma membrane density of TRPV1 ion channels. Nerve growth factor released onto sensory neurons during inflammation triggers a signaling cascade that increases the number of TRPV1 ion channels in the neuronal plasma membrane. The increased number of channels make the cell more sensitive to noxious TRPV1 activators. We have recently developed new click chemistry tools to label TRPV1 channels on the cell surface with unprecedented speed and specificity. Together with genetic code expansion to incorporate noncanonical amino acid click substrates into TRVP1 and optogenetic methods to manipulate the nerve growth factor signaling cascade, these tools allow us to dissect the steps of TRPV1 trafficking– or any membrane protein – in living cells in real time.

Cellular stress triggers re-programming of transcription, which is fundamental for the maintenance of protein homeostasis, also called proteostasis, under adverse growth conditions. Stress-induced changes in transcription include induction of cytoprotective genes and repression of genes related to the regulation of the cell cycle, transcription programs, and metabolism. Induction of transcription is mediated through the activation of stress-responsive transcriptions factors that facilitate the release of stalled RNA polymerase II, thereby allowing for transcriptional elongation. Repression of transcription, in turn, involves components that retain RNA polymerase II in a paused state in gene promoters. Moreover, transcription during stress is regulated by a massive activation of enhancers and complex changes in chromatin organization. Heat shock has provided an excellent model to investigate the mechanisms of nascent transcription, but less is known about the transcriptional regulation upon other types of cellular stress. Therefore, we examined re-programming of genes and enhancers in response to two distinct stresses, i.e. oxidative stress and heat shock by combining different genome-wide analyses (PRO-seq and ChIP-seq). This approach allowed determining the target repertoire of specific members of the heat shock factor (HSF) family. We found that HSF1 and HSF2 drive stress-type specific transcription programs and that besides functioning as promoter-binding transcription factors, both HSFs activate genes through enhancers in response to oxidative stress and heat shock. Intriguingly, in contrast to the promoter-bound HSF1, which regulates classical chaperone genes, recruitment of HSF1 to enhancers is required for the induction of genes encoding proteins that reside in the plasma membrane. It is also plausible that the capacity of HSFs to orchestrate transcription via enhancers is not limited to stress responses, since HSFs play important roles also in developmental and pathological processes, such as progression of cancer.

The brain is a highly complex and fascinating organ and we are interested in understanding how cellular heterogeneity emerges during brain development and how brain cells can regenerate upon injury. We are tackling these questions by applying and further developing integrative, multi-modal single-cell technologies. 
In the first part of my talk, I will present our work on human pluripotent stem cell (PSC) derived organoids that model human brain development in vitro. We generated a single-cell multiomic atlas and developed a novel computational tool to infer a gene regulatory network underlying early human brain organoid development. We then used pooled genetic perturbation with single-cell transcriptome readout to assess transcription factor requirement for cell fate and state regulation in organoid and identified an important role of GLI3 during human telencephalon dorso-ventral patterning. Further, we developed single-cell methodologies to directly track developmental lineages in brain organoids and could identify clonality of brain organoid regions as well as a temporal window of regional fate specification.
In the second part of my talk, I will present our work on understanding the organization and regeneration of the telencephalon in the axolotl salamander using single-cell genomics. We first generated a single-cell multiomic atlas of the axolotl telencephalon, identified evolutionary conservation of neuronal cell types and reconstructed trajectories of post-embryonic neurogenesis. We then showed that upon major injury, all neuronal cell types reemerge through regenerative neurogenesis and neuronal projections to other brain regions are re-established. Finally, we identified a regeneration specific state of neural progenitor cells that is characterized by expression of wound healing genes.
Together, our work highlights the power of single-cell technologies to understand the gene regulatory logic underlying brain development and regeneration.