Role of Linker Histone H1 in Epigenetic Gene Regulation Project Summary Epigenetic mechanisms are crucial for chromatin structure reprogramming and gene expression during mammalian development and cell differentiation. H1 linker histones are major chromatin structural proteins that associate with nucleosome core particles and play a key role in mediating higher order chromatin structure folding. However, the role of H1 in epigenetic gene regulation has not been explored. Previously we showed, by inactivating 3 H1 linker histone genes simultaneously, that the total amount of H1 is essential for mammalian development. We derived triple H1 null embryonic stem cell lines and showed that H1 is particularly important in controlling several imprinted gene expression in these cells. We also found that DNA methylation level at the imprinting control regions of these two affected loci is decreased with H1 reduction, thus suggesting a new link between H1 and DNA methylation. We now propose to address the underlying mechanisms of this novel epigenetic link and to investigate additional H1 regulated epigenetic events in embryonic stem cells and in mouse models. Specifically, we will: 1) Test whether H1 histones regulate establishment or maintenance of specific DNA methylation patterns through recruitment of DNA methyltransferases;2) Identify additional critical H1 regulated epigenetic events and characterize the sequence and functional dependence of such events that are required for proper control of gene expression;3) Study the in vivo regulatory role of H1 in epigenetic gene regulation during embryogenesis and gametogenesis. Findings from these studies will advance our knowledge of epigenetic gene regulation mechanisms. Relevance: Aberrant epigenetic gene regulation has been responsible for many human diseases, such as cancer and imprinted gene diseases. Understanding the epigenetic gene regulation mechanisms will provide critical clues as to how the epigenetic information can be experimentally reprogrammed for therapeutic purposes.