Barbara J. Meyer - US grants

DESCRIPTION (provided by applicant): A major challenge in biomedical research is to understand the functional interplay between gene regulation, chromosome structure, chromatin modifications, and human disease. X-chromosome dosage compensation (DC) in the nematode C. elegans is exemplary for such analysis for the following reasons: DC is mediated by a condensin complex, a chromosome restructuring complex essential for chromosome segregation; DC is essential for proper gene expression and viability because it distinguishes X chromosome from autosomes to control hundreds of genes on X simultaneously; and X-chromosome-specific chromatin modifications are essential for DC. The dosage compensation complex (DCC) binds selectively to X chromosomes of XX embryos to reduce transcription by half, thereby equalizing X-chromosome transcription between XX and XO embryos. Our recent experiments indicate that the DCC imposes a unique spatial organization onto the X chromosomes of XX embryos by binding to its highest affinity recruitment sites on X (rex sites). Mutations that disrupt DCC binding disrupt that X-specific conformation. Proposed experiments extend this analysis and explore the relationship between chromosome structure and gene expression by using our highly efficient genome editing strategies to delete and insert rex sites on X and autosomes and then assess the consequent changes in chromosome conformation and gene expression. Our study is unique in providing a robust example of a major change in chromosome structure imposed by a specific complex, on a specific chromosome, through high- affinity binding to its targets. We have also shown recently that a DCC subunit has a demethylase activity that is responsible for the selective enrichment of H4K20me1 on X chromosomes of XX embryos upon DCC binding. Selective mutation of the catalytic residues abrogates H4K20me1 enrichment and disrupts dosage compensation. These highly specific mutations allow us to test, in an unusually precise way, the role of histone modifications in X-chromosome structure and gene expression. H4K20me1 is also enriched on the mammalian inactive X chromosome, but the role of this enrichment in transcriptional silencing is not known, nor is a selective reagent available to test its role. Changes in histone lysine methylation states are a common occurrence during tumor formation, and strong correlation now exists between an increase in activity of histone demethylases and tumor progression. Hence, our studies of a chromosome-specific demethylase enzyme may become directly relevant to human health. Lastly, we have gained an evolutionary perspective on the X-chromosome DNA sequences that recruit DCC complexes to X chromosomes of highly diverged nematode species. Surpisingly, while the DCC complexes are conserved, the cis-acting motifs are highly diverged. More typically, the target site specificity of conserved regulatory proteins that control multiple celluar processes by targeting hundreds of sites is far more evolutionarily constrained. Hence, the divergence in DCC binding specificity provides an unusual opportunity to understand the path for a concerted change in hundreds of target sites.

Our long-term goals are two-fold, (1) to understand, in genetic and molecular terms, the basis for the male/hermaphrodite decision in the nematode C. elegans and (2) to understand the mechanism of X chromosome dosage compensation at the genetic and molecular levels. The primary sex-determining signal in C. elegans is the X/A ratio; 2X/2A animals are hermaphrodite, and IX/2A animals are male. How the primary sex-determining signal is assessed and subsequently translated into the choice of either the hermaphrodite or male mode of sexual development re- mains a mystery, but is a primary focus of this grant. We have acquired significant insight into understanding the basis of the male/hermaphrodite decision by demonstrating that the sex determination and dosage compensation processes share common early steps prior to their divergence into two separate pathways. The hermaphrodite mode is coordinately controlled by at least two genes, sdc-I and sdc-2; the male mode is controlled by xol-l. The sdc genes appear to play a role in either assessing the X/A ratio or transmitting this signal to both the sex determination and dosage compensation pathways. Genetic interactions suggest that xol-l is the earliest acting gene in the known hierarchy that controls the male/hermaphrodite decision and is likely to be the gene nearest to the primary sex-determining signal. A fourth gene, dpy,-29, is also involved in controlling both processes, and illustrates a paradoxical feedback between dosage compensation disruptions and sex determination. Further insight into the male/hermaphrodite decision will be sought by: (1) Identification and analysis of additional genes central to both sex determination and dosage compensation. (2) A mosaic analysis of sdc-2, xol-l and dpy-29 to determine when the sex determination decision is made and whether it is a cell autonomous process or whether it involves factors extrinsic to the cell, such as diffusible products or cell-cell interactions. (3) An extensive molecular analysis of the genes that play a central role in the male/hermaphrodite decision, sdc-1, sdc-2, xol-l and dpy-29. The molecular analysis will include discerning how these genes are regulated in response to the X/A signal, (or how they help assess the X/A ratio), as well as how they regulate each other and the downstream sex determination and dosage compensation genes. The molecular characterization of these early-acting components of the sex determination decision will be the first molecular analysis of genes that control both processes in this organism. Further insight into the mechanism of X chromosome dosage compensation will be sought by: (1) Identification and analysis of additional dosage compensation genes. (2) Molecular characterization of two genes critical to the process, dpy-27 and dpy-28. This represents the first molecular characterization of dosage compensation genes in this organism. Elucidating the mechanism of sex determination will provide fundamental insight into developmental processes that go awry in human diseases.

[unreadable] DESCRIPTION (provided by applicant): The enclosed proposal represents a competitive renewal of an established graduate training program in Genetics at UC Berkeley. We request funds to support 17 graduate trainees in the areas of Developmental Genetics and Genomics, Cell & Systems Genetics, and Population Genetics and Evolution. Students enter this inter-disciplinary training program after gaining admission into one of four Departments on campus. Two of the Departments, Molecular and Cell Biology (MCB) and Integrative Biology (IB), are located within the College of Letters and Sciences. A third Department, Plant and Microbial Biology (PMB), is located in the College of Natural Resources. Students can also enter the training program through the School of Public Health (SPH). Students are selected into the program based on an interest in one of the broadly defined areas of genetics that are sponsored by this training grant. They are also selected based on merit and future promise as independent investigators. Despite their diverse backgrounds, the training program includes a number of mechanisms to ensure that all of the graduate students obtain rigorous training in genetics. Once they enter the program, students are free to select any one of 41 different faculty mentors located in the four aforementioned performance sites. These faculty employ a variety of genetic and genomic methods to study a broad spectrum of problems in cell, developmental, and evolutionary biology, including metazoan and plant patterning, embryogenesis, sex determination, cell determination and differentiation, morphogenesis, and organogenesis. The study of cellular processes in the areas of signal transduction, DNA replication, transcription, cell trafficking and the cytoskeleton, transcription, and DNA replication are the focus of many faculty. Other faculty use genetic approaches to study evolutionary processes such as the evolution of chordates and patterning in invertebrates. In the first year of the program, trainees complete advanced lecture, laboratory, and seminar courses. Students in PMB and MCB are also required to complete three 10-week laboratory rotations with potential dissertation mentors. All students commence their dissertation research by the beginning of the second year. They are also required to complete an oral qualifying exam that is administered by four faculty from at least two different departments within the training program. All students are required to complete at least two semesters of teaching as graduate instructors during the second and third years of the training program. In addition to formal course offerings in the different areas of genetics, students are expected to participate in a variety of seminar programs, joint lab meetings, journal clubs, and retreats that are sponsored by the Genetics Training Program. [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Male infertility is a long-standing enigma of significant medical concern. The integrity of sperm chromatin is a clinical indicator of male fertility and in vitro fertilization potential: chromosome aneuploidy and DNA decondensation or damage are correlated with reproductive failure. Identifying conserved proteins important for sperm chromatin structure and packaging can reveal universal causes of infertility. Here we combine proteomics, cytology and functional analysis in Caenorhabditis elegans to identify spermatogenic chromatin-associated proteins that are important for fertility. Our strategy employed multiple steps: purification of chromatin from comparable meiotic cell types, namely those undergoing spermatogenesis or oogenesis;proteomic analysis by multidimensional protein identification technology (MudPIT) of factors that co-purify with chromatin;prioritization of sperm proteins based on abundance;and subtraction of common proteins to eliminate general chromatin and meiotic factors. Our approach reduced 1,099 proteins co-purified with spermatogenic chromatin, currently the most extensive catalogue, to 132 proteins for functional analysis. Reduction of gene function through RNA interference coupled with protein localization studies revealed conserved spermatogenesis-specific proteins vital for DNA compaction, chromosome segregation, and fertility. Unexpected roles in spermatogenesis were also detected for factors involved in other processes. Our strategy to find fertility factors conserved from C. elegans to mammals achieved its goal: of mouse gene knockouts corresponding to nematode proteins, 37% (7/19) cause male sterility. Our list therefore provides significant opportunity to identify causes of male infertility and targets for male contraceptives.

PROJECT SUMMARY Studies are proposed to dissect one of the fundamental, binary development decisions that most metazoans make: their sex. The nematode C. elegans determines sex with remarkable precision by tallying X- chromosome number relative to the sets of autosomes (X:A signal): ratios of 1X:2A (0.5) and 2X:3A (0.67) signal male fate, while ratios of 3X:4A (0.75) and 2X:2A (1.0) signal hermaphrodite fate. We have discovered much about the nature and action of the X:A signal and its direct target, a master sex-determination-switch gene that also controls X-chromosome dosage compensation. However, a fundamental question remains: how is the signal interpreted reproducibly in an all or none manner to elicit fertile male or hermaphrodite development, never intersexual development? We pioneer new methods using machine learning neural networks to address this question with single-molecule and single-cell resolution. We also propose to dissect the functional interplay between chromatin modification and chromosome structure in regulating gene expression over vast chromosomal territories. X-chromosome dosage compensation in C. elegans is exemplary for this analysis: we found recently that dosage-compensated X chromosomes have (i) elevated levels of modified histone H4K20me1 compared to autosomes and (ii) a unique three-dimensional architecture. Both are imposed by the dosage compensation complex (DCC). Loss of H4K20me1 disrupts 3D architecture and elevates X gene expression. In the nematode DCC, one subunit is an H4K20me2 demethylase and five subunits are homologs of condensin subunits, which compact and resolve mitotic and meiotic chromosomes. All DCC subunits are recruited specifically to hermaphrodite X chromosomes by an XX-specific subunit that triggers binding to cis- acting regulatory elements on X (rex) to reduce gene expression by half. The DCC remodels the structure of X into topologically associating domains (TADs) using its highest affinity rex sites to establish domain boundaries. Despite this knowledge, important questions underlying the mechanisms of dosage compensation remain. What DCC subunits recognize the X-enriched motifs in rex sites to bind X directly? How does the DCC regulate RNA polymerase II to repress gene expression? What mechanisms underlie H4K20me1's control of chromosome structure, and how does DCC-mediated higher-order structure affect gene expression? Our findings should have broad implications, because (i) condensin complexes control chromosome structure from bacteria to man, (ii) H4K20me1 is enriched on the inactive X of female mammals, (iii) demethylases are linked to tumor progression, and (iv) the H4K20me2 demethylase modulates nematode growth, metabolism, and entry into the quiescent dauer state. Lastly, we will exploit our unexpected finding that rex sites have diverged across Caenorhabditis species separated by 30 MYR, retaining no functional overlap despite strong conservation of the core DCC machinery. This divergence provides an unusual opportunity to study the path for a concerted co- evolutionary change in hundreds of target sites across X chromosomes and the protein complexes that bind them.