The goals of this project are to advance our knowledge about the functions of H1 linker histones and to understand the functional significance of the diversity present in this family of chromatin proteins. H1 linker histones play a key role in the structure of chromatin and thereby affect gene expression as well as other processes requiring access to DNA. Most of our knowledge about the functions of H1 histones has been derived from in vitro experiments. Our approach is to analyze the functions of H1 histones in vivo in mice. The mouse (and other mammalian) H1 histones consist of at least 8 subtypes that differ considerably, both in their primary sequences and in their expression during development and tissue differentiation. These subtypes offer an additional, potential level of regulation of chromatin function. Our strategy for studying the function of linker histones has been to generate and characterize mice in which one or more H1 genes has been inactivated by gene targeting. We have generated a large repertoire of mouse strains consisting of 6 single H1 null mice and several compound null strains and cultured cell lines. We have used these mutants to show that, unlike in lower organisms, H1 histones are essential for mammalian development. Some of the mutants have been analyzed for their effects on gene expression. The results show that loss of individual subtypes as well as reduction in total amount of H1 causes changes in expression of specific genes. We now propose to use our unique set of mouse strains to: (1) study the mechanisms by which individual H1 subtypes and H1 stoichiometry affect gene transcription and regulation in vivo. We will compare the chromatin structure in the vicinity of specific genes in wild-type and mutant H1-depleted cells and mice; and (2) study the role of H1 linker histones in the structure, composition and post-translational modifications of chromatin in vivo. We also propose to develop new cell lines with extremely low amounts or completely lacking H1 histones. We also propose to perform an in vivo test of specific H1 subtype function by a gene replacement strategy in mice. There is evidence that H1 histones are downstream targets of cyclin-dependent kinases regulated by the retinoblastoma protein. Thus H1 histones may be key transducers of information between cell cycle regulators and chromatin structure, gene expression and other activities of the genome in normal and malignant cells.
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