Functions of Mammalian H1 Linker Histones in Gene Regulation and Chromatin Activity

The goals of this proposal are to elucidate the functions of mammalian H1 linker histones in gene regulation and chromatin activity during the cell cycle and to reveal functional differences amongst the 11 variants that constitute the mammalian H1 gene family. Along with the four core histones (H2A, H2B, H3 and H4) that assemble DNA into nucleosomes, the H1 linker histones play key roles in organizing the structure of the chromatin fiber. In comparison to the progress in understanding the roles of core histones, our knowledge about the functions of linker histones in chromatin biology is quite limited. Much of our knowledge about H1’s roles is derived from in vitro studies, whereas our understanding of its functions in vivo remains very incomplete. We propose to help fill this gap by elucidating the functions, mechanisms of action and variant-specific activities of mammalian H1 linker histones in gene regulation and chromatin activity in vivo. Our strategy for studying the in vivo functions of mammalian H1’s has been to generate and characterize mouse embryonic stem cells and mice that are partially depleted of H1 by inactivation of several H1 genes. By analyzing these H1-depleted models, we showed that H1 is essential for mammalian development, plays unexpected roles in epigenetic regulation, and is very important for the integrity and silencing of heterochromatin. Furthermore, our studies with Drosophila H1 showed that it is required for late replication of heterochromatin during endoreplication of salivary gland polytene chromosomes. Recently, we also discovered that, in addition to their important functions in heterochromatin, mammalian H1’s also play important roles in euchromatin where they inhibit deposition of active gene body chromatin marks (methylation of H3K79, H3K36 and H3K4) and affect Pol II transcription elongation and alternative mRNA splicing outcomes. To gain a deeper understanding of the mechanisms by which mammalian H1’s participate in gene regulation and chromatin activities, we propose to pursue the following specific aims: (1) Understand the functions of H1 in RNA Pol II transcription, mRNA splicing and gene body histone H3 lysine methylation; (2) Determine the roles of H1 in late S phase replication timing of heterochromatin and its reorganization during the mitosis to G1 phase transition; (3) Identify functional differences amongst H1 variants and the contributions of their N- and C-terminal domains and post-translational modifications. The successful completion of this project will provide important insights into the ways in which this major constituent of chromatin regulates the structure and activity of mammalian chromosomes.