Transcription cofactors in differentiation

Harvard Medical School, Boston MA

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Abstract

DESCRIPTION (provided by applicant): Histone N-terminal tails undergo multiple post-translational modifications including methylation, which significantly impact chromatin structure and transcription. Our main interest is to understand histone methylation regulation, a modification that was considered irreversible until our discovery of the first histone demethylase, LSD1. Recent studies implicated LSD1 in differentiation, heterochromatin biology and tumorigenesis. However, the LSD1 biology and in vivo mechanism of action remain incompletely understood. Loss of the C. elegans LSD1 homolog SPR-5 has recently been reported to result in progressive sterility (W. Kelly, per. comm.). In the past funding period, we pursued this further and uncovered an important link between LSD1 and C. elegans germline maintenance based on the findings that 1) spr-5 mutants display increased, p53-dependent germ cell apoptosis, suggesting activation of a DNA damage checkpoint;2) RAD-51 foci levels are elevated during meiotic prophase in spr-5 mutants suggesting that progression of double-strand break repair is impaired. Thus, a focus of this application is to understand the mechanisms by which SPR-5 regulates germline maintenance. We will determine the demethylase activity of SPR-5 and its related proteins T08D10.2 and AMX-1 in vitro and in vivo, and assess the role of the enzymatic activity in germline maintenance (Aim 1). We will explore the mechanisms by which SPR-5 regulates germline maintenance by considering two alternative but not mutually exclusive models (Aim 2): 1) SPR-5 exerts a direct effect on chromatin structure impacting DNA damage susceptibility and repair. 2) SPR-5 exerts its function indirectly via controlling transcription of DNA damage response genes. We will also investigate the progressive infertility observed in spr-5 mutants, which is reminiscent of defects in telomere maintenance (Aim 2). Lastly, we will identify protein networks that regulate SPR-5 germline functions through protein complex purification and investigate the genetic relationship between SPR-5 and other histone modifying enzymes by genetic suppressor/enhancer screens (Aim 3). Collectively, this application will provide significant new insights into the poorly understood role of chromatin and chromatin modifying enzymes in germ cell maintenance. Findings will also shed new light on molecular mechanisms that control germ cell functions and will impact on our understanding of analogous processes in higher eukaryotes.