Our long term goal is to understand the functional roles and the factors that regulate the developmental appearance of several novel chromosomal proteins that are common features of mammalian spermatogenesis. During the pachytene stage of meiosis a set of novel variants for all the common histones except H4 replace to varying degrees their somatic-type counterparts. Perhaps the most unusual testis histone variant is H1t, whose primary structure differs from standard H1 proteins throughout its length. Because H1 is the histone responsible for determining the compaction of nucleosomal DNA, H1t may well impart an unusual structure to late meiotic and postmeiotic chromosomes. Later, in spermatids with condensing nuclei, the histones are replaced by a set of transition proteins (TP1-4). The major transition proteins (TP1,2) remain in the nucleus for approximately 2 days but are then removed, leaving protamines as the major basic proteins in the mature sperm nucleus. Presumably the unusual chromosomal proteins of spermatogenesis play some role ensuring that meiotic events, genomic imprinting, and chromosome organization in the sperm nucleus occur appropriately for the male gamete's part in the development of the embryo. Information about the functions and regulation of these proteins could lead to better assessment of some types of male infertility, the identification of causes of chromosomal abnormalities, of failures of early development, and a better understanding of chromosomal structure and gene regulation in general. Experiments are planned to dissect the nucleotide sequences that impart specialized expression to the H1t gene. Deleted versions of the natural gene and of H1t promoter- reporter fusions will be tested for appropriate expression in transgenic mice and somatic cell lines. In vitro assays of cell free transcription, gel retardation, and footprinting will identify specific promoter regions important to regulated expression as well as protein factors that bind to them. At least one such factor will be cloned. To understand the functional role of H1t, targeted gene disruptions will be made in mouse ES cells that will be used to produce chimeric mice that can be bred to generate animals that lack H1t. The human H1t gene will be isolated and sequenced to permit its chromosomal localization. The TP1 promoter region will be characterized to see if it is responsive to cAMP, as is suggested by preliminary binding studies with nuclear protein extracts.
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