9304394 Poccia The long term goal of this work is to understand the inactivation/activation of the sperm nucleus that occurs during spermatogenesis and following fertilization. Successful genetic reactivation of the dormant sperm nucleus by egg cytoplasm is essential to successful biparental embryonic development. Male pronuclear reactivation is preceded by decondensation of chromatin and alteration of DNA associated basic proteins. Dr. Poccia will test the abilities of two sperm specific histones of the sea urchin (Sp H1 and Sp H2B) to determine first order structure of sperm chromatin and its alteration following fertilization. His model predicts that phosphorylation of "SPKK" sites in the N-terminal domains of these molecules controls average nucleosomal repeat length and accessibility of linker DNA to enzymatic digestion. These properties have been previously correlated in vivo, but whether N-terminal phosphorylation plays a role in determining repeat lengths or sensitivity to MNase digestion is a major untested postulate. Novel features of his experiments include the use of identical histone variants which differ in degree of phosphorylation, employment of N- and C-terminal peptide as well as intact molecules to assess the contribution of those domains, and evaluation of the contributions of H2B as well as H1. Relevance of "SPKK" phosphorylation extends beyond Sp histones. Similar sites occur in most H1's and in several nucleic acid packaging proteins and regulators, some of which are phosphorylated in a cell cycle dependent manner. Moreover, positioning of nucleosomes and alteration of histone-DNA binding affinities is critically important in replication and transcriptional control mechanisms. ***
