Sperm chromatin structure and remodeling during spermiogenesis and fertilization

PROJECT SUMMARY Genetic inheritance material of a eukaryotic organism (DNA) is stored in chromosomes. Understanding of cellular activities in normal development and disease requires the analyses of critical regulatory roles that are played by packaging the genome into a nucleoprotein complex termed chromatin. The structure of chromatin and the regulatory machinery of its metabolism are uniquely important and thus, strongly conserved in evolution. The objective of our work is to understand the relationship between the establishment of chromatin structure in the male gametes (sperm cells) and regulation of their function. We will focus on a systematic study of sperm chromatin structure and factors that mediate its assembly and remodeling during spermiogenesis and egg fertilization in a model organism, fruit fly (Drosophila melanogaster). Sperm chromatin differs in composition and architecture from the normal cell chromatin, in particular by an extraordinary high degree of DNA condensation. It is rigid, enzymatically static and is formed by compaction of the DNA with small cysteine-rich proteins termed protamines (and other sperm nuclear basic proteins, SNBPs). Despite its importance for the life cycle of any sexually reproducing organism, sperm chromatin structure has been poorly studied. To fill this gap, we will perform biochemical, biophysical and structural analyses of in vitro reconstituted Drosophila sperm chromatin. Sperm chromatin replaces that of somatic cells and is assembled/remodeled in multiple steps during sperm differentiation (spermiogenesis). At fertilization, the egg also faces the challenge of remodeling the condensed sperm chromatin into an accessible, transcription- and replication-competent form. We recently discovered cellular machinery that mediates sperm chromatin remodeling (SCR) in vitro and in vivo. Thus, we will study the factors (protamine chaperones and enzymes of the thioredoxin system) that mediate SCR. Protamines in sperm undergo cysteine oxidation and oligomerization via intra- and inter-molecular disulfide bonds. For fertilization success, the disulfides have to be reduced by egg-specific thioredoxins. Thus, we will evaluate small-molecule inhibitors of the thioredoxin system as candidate agents to manipulate fly and mouse fertility (putative contraceptive drugs). The successful completion of this project will lead to a comprehensive biochemical and biological characterization of factors that mediate the assembly and remodeling of metazoan sperm chromatin. More globally, our work will provide insights into the role of chromosome assembly and maintenance in regulation of the cell function and will be applicable to understanding, diagnosis and treatment of human conditions that involve defects in chromatin structure.