Saher S. Hammoud, Ph.D. - US grants

Summary: The epigenome consists of several types of chemical modifications on either histone proteins or DNA. In somatic cells, these modifications are maintained across mitotic divisions, whereas in the germline and embryo, the epigenome is reset to ensure proper development and to prevent the trans-generational inheritance of acquired epigenetic information. Despite the two waves of genome-wide reprogramming during mammalian development, the inheritance of epigenetic information has been observed in various organisms, including humans. These findings raise a fundamental question: is epigenetic information in gametes transmitted from generation to generation and, if so, what are the consequences of trans-generational epigenetic inheritance? The epigenetic contribution from the paternal genome is considered limited since the sperm genome is largely depleted of histones. The small amounts of histones retained in sperm were believed to be remnants of incomplete histone-to-protamine exchange. However, my studies revealed that key developmental gene promoters in sperm are DNA hypomethylated and are enriched for both active and repressive histone modifications: a ?poised? chromatin state presumed to be specific to a totipotent/pluripotent cell. However, our findings demonstrated that competency for totipotency is already embedded in sperm chromatin, and possibly inherited through the paternal lineage, therefore, challenging prior notions and expanding the breadth of paternal contribution to the developing embryo beyond the DNA sequence. Furthermore, subsequent studies have shown that the epigenetic modifications retained in sperm chromatin are distinct from those present in the oocyte. Whether these retained parent-of-origin epigenetic marks (apart from imprinted genes) are instructive for early embryonic development or the first cell fate determination events remains unknown. This proposal aims to address two fundamental questions in biology: 1) What is the biological function and significance of retained histones and their attendant modifications in sperm, and 2) Is the preservation of the parent-of-origin epigenetic marks (apart from imprinted genes) important for early development and the first cell fate determination events or are these marks merely a remnant of the paternal or maternal origin of the genomes? To address these questions and others, we propose to develop exciting, cutting edge cell biology and molecular genetic tools that will enable the visualization, tracking, and temporal control of paternal histones in the developing embryo. Through these studies we hope to uncover whether the modified nucleosomes retained in sperm chromatin are inherited and instructive for development, therefore, providing a molecular mechanism for transgenerational inheritance, a notion supported by observation, yet without a clear molecular explanation.

Abstract Spermatogenesis is a well-organized and tightly regulated biological process composed of four distinct biological activities: 1) Continuous stem cell self-renewal and the generation of transit amplifying progenitor spermatogonia 2) the formation of fully differentiated, mitotic spermatogonia from the progenitors, 3) meiosis, and 4) the orderly and coordinated differentiation of haploid cells into spermatozoa (spermiogenesis). The transition between these developmental states is precisely timed, and is dependent upon both germ cell intrinsic and extrinsic factors secreted by supporting cells (e.g. Sertoli, Leydig, Myoid and Immune cells). In the past, the spatial organization of these cells across the seminiferous tubule has been studied histologically, and their contribution to spermatogenesis has been explored through the purification of predefined cell types, followed by gene expression profiling of the cell populations. However, these approaches rely on known markers to define cell population. Furthermore, past genomic profiling methods reveal average properties of cell populations ? not individual cells. Hence, the existing view of spermatogenesis fails to: 1) describe the full extent of inter-cellular heterogeneity 2) identify rare cell populations e.g., transitional states, or 3) integrate and reconcile variation in gene expression of a cell depending on its specific stage of the cycle of the seminiferous epithelium. To overcome these limitations, we will use the newly available single-cell RNA-sequencing technology to create an unbiased cellular catalog of the adult testis. However, single cell RNA-seq, as currently performed, requires the dissociation of complex tissues and the loss of spatial information. Therefore, to begin addressing and resolving the complexity of the seminiferous tubule, we will integrate gene expression profiles from individual cells with their spatial location using single molecule RNA fluorescent in-situ hybridization (smFISH). To demonstrate the power of combining single-cell sequencing and smFISH we will begin with the development of a high-resolution map of Sertoli cells (SC), across the seminiferous tubule? a cell type known to exhibit stage-specific transcriptional dynamics. The smFISH on the SC populations will resolve the distribution of individual subtypes of SC and potentially highlight their changes during different stages of the seminiferous epithelium cycle. In short, this proposal will provide a systematic analysis of germ cells and the somatic environment in the mouse testis. We will provide a complete inventory of major cell types, mRNA markers, and will specifically map subtypes of Sertoli cells to known stages of the seminiferous epithelium at an unprecedented resolution. All genomic data and spatial maps will be shared with the community as a resource essential for understanding the structural and functional diversity of germ cell development, helping to answer new mechanistic questions regarding the regulation and signaling processes that guide this important developmental process. The information gained from these studies will provide a foundation for future efforts to drive in vitro gametogenesis or restore germ cell in vivo.

Ancillary reproductive health projects to existing large and/or longitudinal studies Sperm and egg cells carry genetic and epigenetic information from parents to offspring, serving as a link between the past, present and future of a species. Unlike oocytes and somatic cells, which package their DNA with histones, the DNA of mature sperm is bound by protamines, highly basic and rapidly evolving proteins that are essential for the compaction of paternal chromatin. This differential packaging traces back >500 million years, but the biological and functional significance of protamine protein packaging remains poorly understood. Here we identified a functional role for protamine proteins in coordinating proper embryonic development. This proposal aims to develop additional molecular genetic tools to dissect the role of protamines in fertility, early development, and evolution. Together, the findings presented here will increase our molecular understanding of these ancient, yet rapidly evolving proteins and may overturn the long-held dogma of their presumed function.