Co-Evolution of Transcriptional Silencing Proteins and the DNA Elements Specifying Their Assembly

Co-evolution of transcriptional regulatory proteins and their sites of action has been often hypothesized but rarely demonstrated. Here we provide experimental evidence of such co-evolution in yeast silent chromatin, a finding that emerged from studies of hybrids formed between two closely related Saccharomyces species. A unidirectional silencing incompatibility between S. cerevisiae and S. bayanus led to a key discovery: asymmetrical complementation of divergent orthologs of the silent chromatin component Sir4. In S. cerevisiae/S. bayanus interspecies hybrids, ChIP-Seq analysis revealed a restriction against S. cerevisiae Sir4 associating with most S. bayanus silenced regions; in contrast, S. bayanus Sir4 associated with S. cerevisiae silenced loci to an even greater degree than did S. cerevisiae's own Sir4. Functional changes in silencer sequences paralleled changes in Sir4 sequence and a reduction in Sir1 family members in S. cerevisiae. Critically, species-specific silencing of the S. bayanus HMR locus could be reconstituted in S. cerevisiae by co-transfer of the S. bayanus Sir4 and Kos3 (the ancestral relative of Sir1) proteins. As Sir1/Kos3 and Sir4 bind conserved silencer-binding proteins, but not specific DNA sequences, these rapidly evolving proteins served to interpret differences in the two species' silencers presumably involving emergent features created by the regulatory proteins that bind sequences within silencers. The results presented here, and in particular the high resolution ChIP-Seq localization of the Sir4 protein, provided unanticipated insights into the mechanism of silent chromatin assembly in yeast.National Science Foundation (U.S.) (GM31105)National Science Foundation (U.S.) (Predoctoral Fellowships

The Awesome Power of Yeast Evolutionary Genetics: New Genome Sequences and Strain Resources for the Saccharomyces sensu stricto Genus

High-quality, well-annotated genome sequences and standardized laboratory strains fuel experimental and evolutionary research. We present improved genome sequences of three species of Saccharomyces sensu stricto yeasts: S. bayanus var. uvarum (CBS 7001), S. kudriavzevii (IFO 1802T and ZP 591), and S. mikatae (IFO 1815T), and describe their comparison to the genomes of S. cerevisiae and S. paradoxus. The new sequences, derived by assembling millions of short DNA sequence reads together with previously published Sanger shotgun reads, have vastly greater long-range continuity and far fewer gaps than the previously available genome sequences. New gene predictions defined a set of 5261 protein-coding orthologs across the five most commonly studied Saccharomyces yeasts, enabling a re-examination of the tempo and mode of yeast gene evolution and improved inferences of species-specific gains and losses. To facilitate experimental investigations, we generated genetically marked, stable haploid strains for all three of these Saccharomyces species. These nearly complete genome sequences and the collection of genetically marked strains provide a valuable toolset for comparative studies of gene function, metabolism, and evolution, and render Saccharomyces sensu stricto the most experimentally tractable model genus. These resources are freely available and accessible through www.SaccharomycesSensuStricto.org

Role of psychiatric hospitals during a pandemic: introducing the Munich Psychiatric COVID-19 Pandemic Contingency Plan

BACKGROUND Psychiatry is facing major challenges during the current coronavirus disease 2019 (COVID)-19 pandemic. These challenges involve its actual and perceived role within the medical system, in particular how psychiatric hospitals can maintain their core mission of attending to people with mental illness while at the same time providing relief to overstretched general medicine services. Although psychiatric disorders comprise the leading cause of the global burden of disease, mental healthcare has been deemphasised in the wake of the onslaught of the pandemic: to make room for emergency care, psychiatric wards have been downsized, clinics closed, psychiatric support systems discontinued and so on. To deal with this pressing issue, we developed a pandemic contingency plan with the aim to contain, decelerate and, preferably, avoid transmission of COVID-19 and to enable and maintain medical healthcare for patients with mental disorders. AIMS To describe our plan as an example of how a psychiatric hospital can share in providing acute care in a healthcare system facing an acute and highly infectious pandemic like COVID-19 and at the same time provide support for people with mental illness, with or without a COVID-19 infection. METHOD This was a descriptive study. RESULTS The plan was based on the German national pandemic strategy and several legal recommendations and was implemented step by step on the basis of the local COVID-19 situation. In addition, mid- and long-term plans were developed for coping with the aftermath of the pandemic. CONCLUSIONS The plan enabled the University Hospital to maintain medical healthcare for patients with mental disorders. It has offered the necessary flexibility to adapt its implementation to the first and second waves of the COVID-19 pandemic in Germany. The plan is designed to serve as an easily adaptable blueprint for psychiatric hospitals around the world

Impact of Chromatin Structures on DNA Processing for Genomic Analyses

Chromatin has an impact on recombination, repair, replication, and evolution of DNA. Here we report that chromatin structure also affects laboratory DNA manipulation in ways that distort the results of chromatin immunoprecipitation (ChIP) experiments. We initially discovered this effect at the Saccharomyces cerevisiae HMR locus, where we found that silenced chromatin was refractory to shearing, relative to euchromatin. Using input samples from ChIP-Seq studies, we detected a similar bias throughout the heterochromatic portions of the yeast genome. We also observed significant chromatin-related effects at telomeres, protein binding sites, and genes, reflected in the variation of input-Seq coverage. Experimental tests of candidate regions showed that chromatin influenced shearing at some loci, and that chromatin could also lead to enriched or depleted DNA levels in prepared samples, independently of shearing effects. Our results suggested that assays relying on immunoprecipitation of chromatin will be biased by intrinsic differences between regions packaged into different chromatin structures - biases which have been largely ignored to date. These results established the pervasiveness of this bias genome-wide, and suggested that this bias can be used to detect differences in chromatin structures across the genome

The multimodal Munich Clinical Deep Phenotyping study to bridge the translational gap in severe mental illness treatment research

Introduction: Treatment of severe mental illness (SMI) symptoms, especially negative symptoms and cognitive dysfunction in schizophrenia, remains a major unmet need. There is good evidence that SMIs have a strong genetic background and are characterized by multiple biological alterations, including disturbed brain circuits and connectivity, dysregulated neuronal excitation-inhibition, disturbed dopaminergic and glutamatergic pathways, and partially dysregulated inflammatory processes. The ways in which the dysregulated signaling pathways are interconnected remains largely unknown, in part because well-characterized clinical studies on comprehensive biomaterial are lacking. Furthermore, the development of drugs to treat SMIs such as schizophrenia is limited by the use of operationalized symptom-based clusters for diagnosis. Methods: In line with the Research Domain Criteria initiative, the Clinical Deep Phenotyping (CDP) study is using a multimodal approach to reveal the neurobiological underpinnings of clinically relevant schizophrenia subgroups by performing broad transdiagnostic clinical characterization with standardized neurocognitive assessments, multimodal neuroimaging, electrophysiological assessments, retinal investigations, and omics-based analyzes of blood and cerebrospinal fluid. Moreover, to bridge the translational gap in biological psychiatry the study includes in vitro investigations on human-induced pluripotent stem cells, which are available from a subset of participants. Results: Here, we report on the feasibility of this multimodal approach, which has been successfully initiated in the first participants in the CDP cohort; to date, the cohort comprises over 194 individuals with SMI and 187 age and gender matched healthy controls. In addition, we describe the applied research modalities and study objectives. Discussion: The identification of cross-diagnostic and diagnosis-specific biotype-informed subgroups of patients and the translational dissection of those subgroups may help to pave the way toward precision medicine with artificial intelligence-supported tailored interventions and treatment. This aim is particularly important in psychiatry, a field where innovation is urgently needed because specific symptom domains, such as negative symptoms and cognitive dysfunction, and treatment-resistant symptoms in general are still difficult to treat

Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies

First published: 16 February 202

Evolutionary Genetic Studies of Mating Type and Silencing in Saccharomyces

This thesis describes studies exploring the evolution of the genetic circuits regulating yeast mating-type and silencing by Sir (Silent Information Regulator) proteins in the budding yeast Saccharomyces bayanus, a close relative of the laboratory workhorse S. cerevisiae (a.k.a., budding yeast, or brewer's yeast). The two central subjects of these studies, mating type and silencing, are textbook examples of "well understood" mechanisms of eukaryotic gene regulation: the former serves as a model for understanding the genetic control of cell-type differentiation, the latter serves as a model for understanding physically condensed, transcriptionally repressed portions of the genome, often referred to as "heterochromatin". The two subjects are intimately connected in the biology of the budding yeast life cycle, as explained below, and I argue that a deeper appreciation of this connection is necessary for further progress in the study of either subject. My thesis brings a critical evolutionary perspective to certain assumptions underlying current knowledge of mating-type regulation and silencing--in short, an appreciation of organismal biology that has been marginalized in the pursuit of understanding molecular mechanisms. The value of this perspective is in attempting to understand the purpose of a biological process--why is there such a thing as silencing, and why does it require the particular proteins and DNA elements that it does? To ask what silencing does for a yeast cell, we can start by asking how the silencing mechanism is constrained over evolutionary time. One of the surprising findings of my thesis is how unconstrained some elements of the silencing machinery are during evolution. At least three major findings arise from the comparative genetics studies described here: First, I describe the first new branch of the mating-type control circuit in almost 25 years. Although alpha-specific genes were previously thought to be "off" in MATa cells due to the absence of the alpha1 activator protein (i.e., by default), I show that these genes are, in fact, actively repressed by the Sum1 protein. This novel regulatory branch highlights the sophisticated control mechanisms necessary to coordinate the mating and mating-type switching processes. This finding has additional implications, including questioning the extent to which the "absence of activator" model is sufficient to explain the absence of a particular gene's expression; and that at least one subset of mating genes may be under environmental or metabolic regulation via the Sum1-associated NAD+-dependent histone deacetylase Hst1. Second, I show that at least two major genetic alterations to the Sir-based silencing machinery occurred in the recent ancestry of S. cerevisiae and its closest relative species. These changes reveal that our understanding of the silencing mechanism has been limited by the relative lack of comparative genetic sampling of the silencing process. That is, our understanding can improve via functional studies of silencing in close relatives of S. cerevisiae with variant silencing machinery, fueling new hypotheses about how silencing works. Although the identities of the major players (Sir1-4) largely remain the same, my discovery that certain silencing proteins are incompatible across closely related Saccharomyces species suggests evolutionary alterations in the genetic network of silencing--variation that could be tapped in future studies to understand better the way that silencing works. Of particular note are the rapid sequence evolution of SIR4, and the changes in copy number and sequence of SIR1, between S. bayanus and S. cerevisiae. SIR4 and SIR1 appear to rapidly evolve for interesting, though not completely overlapping, reasons. SIR4 appears to be under diversifying selection in modern yeast populations, and its coding sequence evolves rapidly across two rather distant clades spanning the Saccharomyces complex--the sensu stricto clade, and the Torulaspora clade. Third, I show that Sir4 and silencers are engaged in a remarkable pattern of co-evolution in Saccharomyces yeasts. I used a novel combination of classical genetic techniques in S. cerevisiae/S. bayanus hybrids to test cis versus trans contributions to a genetic incompatibility between S. cerevisiae SIR4 and the S. bayanus HMR locus. Comparative ChIP-Seq of Sir4 in these hybrids helped identify the molecular basis for this incompatibility. Critically, I show that the S. bayanus HMR locus, when transferred into S. cerevisiae, can be silenced only by the specific combination of S. bayanus Sir4 and Kos3 proteins, with potential contributions by S. bayanus ORC and the other Sir1 paralogs. A striking asymmetry in cross-species compatibility of S. bayanus versus S. cerevisiae SIR4 genes, and in each species' Sir4 ChIP-Seq profile, suggests that compensatory changes have occurred in SIR4 and in silencers along the S. cerevisiae lineage. Although the initial evolutionary pressure(s) driving these rapid changes remains uncertain, my results point to some pressure driving either the silencers' or Sir4's rapid sequence change, with the other factor subsequently changing to maintain compatibility within a species. From a practical standpoint, these results suggest that molecular studies of silencing using only S. cerevisiae suffer from a previously unrecognized bias. That S. bayanus has four Sir1-like proteins, each important for silencing, suggests additional dimensions (i.e., temporal and/or spatial components) to the interactions occurring at silencers between Sir1, Sir4, ORC, and Rap1. An interesting consequence of the comparative Sir4 ChIP-Seq experiments was the generation of a high-resolution picture of the architecture of silent chromatin in yeast. The unexpected non-uniform distributions of Sir4 protein across HML and HMR bring into question the standard "spreading" model for yeast silent chromatin formation, and will fuel future experiments to determine how Sir-based chromatin structures determine gene silencing and the epigenetic inheritance of gene expression states. I describe the novel ChIP-Seq picture of Sir protein association with silenced loci in Appendix A. Finally, in addition to these specific biological insights, my comparative genetic studies provide guidelines for using the genetic variation between S. bayanus and S. cerevisiae as a tool to learn more about conserved genetic circuits and gene regulation mechanisms in general. Two substantial advances in evolutionary genetic techniques are presented in Chapters 3 and 4, which involve the use of yeast hybrids. First, I show that the genetic facility of S. cerevisiae/S. bayanus hybrids can be used to tease apart interspecies genetic variation of functional consequence that resides in cis-regulatory DNA elements from that in trans-acting transcriptional regulatory proteins. Second, in the case of silencing, the very act of re-introducing genetic factors that have been independently evolving for millions of years leads to unexpected, emergent phenotypes in the hybrids that can be used to understand the silencing mechanism itself. Lessons from my work should inform principles of comparative genetics using organisms closely related to classical "model organism" species such as S. cerevisiae

Evolutionary Genetic Studies of Mating Type and Silencing in Saccharomyces

This thesis describes studies exploring the evolution of the genetic circuits regulating yeast mating-type and silencing by Sir (Silent Information Regulator) proteins in the budding yeast Saccharomyces bayanus, a close relative of the laboratory workhorse S. cerevisiae (a.k.a., budding yeast, or brewer's yeast). The two central subjects of these studies, mating type and silencing, are textbook examples of "well understood" mechanisms of eukaryotic gene regulation: the former serves as a model for understanding the genetic control of cell-type differentiation, the latter serves as a model for understanding physically condensed, transcriptionally repressed portions of the genome, often referred to as "heterochromatin". The two subjects are intimately connected in the biology of the budding yeast life cycle, as explained below, and I argue that a deeper appreciation of this connection is necessary for further progress in the study of either subject. My thesis brings a critical evolutionary perspective to certain assumptions underlying current knowledge of mating-type regulation and silencing--in short, an appreciation of organismal biology that has been marginalized in the pursuit of understanding molecular mechanisms. The value of this perspective is in attempting to understand the purpose of a biological process--why is there such a thing as silencing, and why does it require the particular proteins and DNA elements that it does? To ask what silencing does for a yeast cell, we can start by asking how the silencing mechanism is constrained over evolutionary time. One of the surprising findings of my thesis is how unconstrained some elements of the silencing machinery are during evolution. At least three major findings arise from the comparative genetics studies described here: First, I describe the first new branch of the mating-type control circuit in almost 25 years. Although alpha-specific genes were previously thought to be "off" in MATa cells due to the absence of the alpha1 activator protein (i.e., by default), I show that these genes are, in fact, actively repressed by the Sum1 protein. This novel regulatory branch highlights the sophisticated control mechanisms necessary to coordinate the mating and mating-type switching processes. This finding has additional implications, including questioning the extent to which the "absence of activator" model is sufficient to explain the absence of a particular gene's expression; and that at least one subset of mating genes may be under environmental or metabolic regulation via the Sum1-associated NAD+-dependent histone deacetylase Hst1. Second, I show that at least two major genetic alterations to the Sir-based silencing machinery occurred in the recent ancestry of S. cerevisiae and its closest relative species. These changes reveal that our understanding of the silencing mechanism has been limited by the relative lack of comparative genetic sampling of the silencing process. That is, our understanding can improve via functional studies of silencing in close relatives of S. cerevisiae with variant silencing machinery, fueling new hypotheses about how silencing works. Although the identities of the major players (Sir1-4) largely remain the same, my discovery that certain silencing proteins are incompatible across closely related Saccharomyces species suggests evolutionary alterations in the genetic network of silencing--variation that could be tapped in future studies to understand better the way that silencing works. Of particular note are the rapid sequence evolution of SIR4, and the changes in copy number and sequence of SIR1, between S. bayanus and S. cerevisiae. SIR4 and SIR1 appear to rapidly evolve for interesting, though not completely overlapping, reasons. SIR4 appears to be under diversifying selection in modern yeast populations, and its coding sequence evolves rapidly across two rather distant clades spanning the Saccharomyces complex--the sensu stricto clade, and the Torulaspora clade. Third, I show that Sir4 and silencers are engaged in a remarkable pattern of co-evolution in Saccharomyces yeasts. I used a novel combination of classical genetic techniques in S. cerevisiae/S. bayanus hybrids to test cis versus trans contributions to a genetic incompatibility between S. cerevisiae SIR4 and the S. bayanus HMR locus. Comparative ChIP-Seq of Sir4 in these hybrids helped identify the molecular basis for this incompatibility. Critically, I show that the S. bayanus HMR locus, when transferred into S. cerevisiae, can be silenced only by the specific combination of S. bayanus Sir4 and Kos3 proteins, with potential contributions by S. bayanus ORC and the other Sir1 paralogs. A striking asymmetry in cross-species compatibility of S. bayanus versus S. cerevisiae SIR4 genes, and in each species' Sir4 ChIP-Seq profile, suggests that compensatory changes have occurred in SIR4 and in silencers along the S. cerevisiae lineage. Although the initial evolutionary pressure(s) driving these rapid changes remains uncertain, my results point to some pressure driving either the silencers' or Sir4's rapid sequence change, with the other factor subsequently changing to maintain compatibility within a species. From a practical standpoint, these results suggest that molecular studies of silencing using only S. cerevisiae suffer from a previously unrecognized bias. That S. bayanus has four Sir1-like proteins, each important for silencing, suggests additional dimensions (i.e., temporal and/or spatial components) to the interactions occurring at silencers between Sir1, Sir4, ORC, and Rap1. An interesting consequence of the comparative Sir4 ChIP-Seq experiments was the generation of a high-resolution picture of the architecture of silent chromatin in yeast. The unexpected non-uniform distributions of Sir4 protein across HML and HMR bring into question the standard "spreading" model for yeast silent chromatin formation, and will fuel future experiments to determine how Sir-based chromatin structures determine gene silencing and the epigenetic inheritance of gene expression states. I describe the novel ChIP-Seq picture of Sir protein association with silenced loci in Appendix A. Finally, in addition to these specific biological insights, my comparative genetic studies provide guidelines for using the genetic variation between S. bayanus and S. cerevisiae as a tool to learn more about conserved genetic circuits and gene regulation mechanisms in general. Two substantial advances in evolutionary genetic techniques are presented in Chapters 3 and 4, which involve the use of yeast hybrids. First, I show that the genetic facility of S. cerevisiae/S. bayanus hybrids can be used to tease apart interspecies genetic variation of functional consequence that resides in cis-regulatory DNA elements from that in trans-acting transcriptional regulatory proteins. Second, in the case of silencing, the very act of re-introducing genetic factors that have been independently evolving for millions of years leads to unexpected, emergent phenotypes in the hybrids that can be used to understand the silencing mechanism itself. Lessons from my work should inform principles of comparative genetics using organisms closely related to classical "model organism" species such as S. cerevisiae