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 much of its length. Because H1 is the histone class responsible for determining the compaction of nucleosomal DNA, H1t may well impart an unusual structure to late meiotic and postmeiotic chromosomes. Later, in condensing spermatids, most or all of the histones are replaced with sperm-specific chromosomal proteins, so that these testis-specific histone variants are scarce or absent in spermatozoa. Presumably the unusual histone variants of spermatogenesis play some role ensuring that meiotic events, spermatid development, and chromosome organization in the sperm occur appropriately. Information about the function and regulation of these proteins, and H1t in particular, could lead to better assessment of some forms of male infertility, the identification of causes of chromosomal abnormalities and failures of early development and a better understanding of chromosomal structure and gene regulation in general. Experiments are planned to assess the functional importance of H1t by using targeted gene disruption in embryonic stem cells to make an H1t knockout mouse. If spermatogenesis is affected, the same technique will be used to substitute a somatic H1 for H1t and thus determine whether H1t's unique structure is important. To understand the transcriptional mechanisms that account for the complete silence of the H1t gene in somatic cells but its expression in late spermatocytes, we will clone and characterize a testis-specific binding factor for a conserved palindrome in the Hlt promoter. It is expected that eventual study of the promoter for this apparent transcription factor will lead to better understanding of how the process of spermatogenesis is driven forward. the H1t gene is repressed in rodent cell lines by a G/C-rich region just downstream of its TATA box. We will attempt to identify the factor(s) responsible for repression. Transgenic mice will be used to analyse the effect of mutations in the conserved Hlt palindrome and the repressor element downstream of the TATA box.
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