Dissecting the Functions of Macroh2A1 in Transcriptional Regulation and the DNA Damage Response

Abstract

Chromatin is an essential regulatory platform for transcription and DNA damage repair. Chromatin structure is regulated by covalent post translational modifications (PTMs) of histones, ATP-dependent remodeling and incorporation of specialized histone variants to create functionally distinct chromatin environments. The histone variant macroH2A has distinct roles in regulating chromatin structure and function. MacroH2A is composed of a histone-like region attached to a carboxyl-terminal macrodomain by a flexible linker. The macrodomain emerges from the nucleosome allowing macroH2A to organize the same amount of DNA as canonical H2A. Three highly homologous isoforms of macroH2A have been identified in humans. Only the macrodomain of macroH2A1.1 contains a unique exon that allows it to bind to NAD+-derived ligands, such as poly(ADPribose) (PAR). MacroH2A1 regulates transcription and DNA damage responses, in part, through its ability to bind PAR.

In normal cells, macroH2A1 colocalizes with two functionally distinct types of chromatin, facultative heterochromatin marked by H3K27me3 and transcriptionally active chromatin marked by H2B acetylation. In cancer cells, macroH2A1 continues to colocalize with H3K27me3 but is notably absent from H2B acetylated chromatin. Furthermore, the majority of macroH2A1-regulated genes, including components of the senescence-associated secretory phenotype (SASP), reside in H2B-acetylated chromatin resulting in the loss of an important macroH2A1-mediatated tumor suppressive gene regulatory program. Overall, this led us to hypothesize that distinct mechanisms govern macroH2A1 incorporation into these two types of chromatin. Using macroH2A1 mutants, we demonstrate that the carboxyl-terminal region of the histone-like domain of macroH2A1 is required for accurate deposition into H2B-acetylated chromatin distinguishing this process from incorporation into H3K27me3-containing chromatin in which multiple features of macroH2A1 are sufficient for targeting. Furthermore, we determined that H2B K20 acetylation is a key post-translational modification (PTM) for targeting macroH2A1 deposition into transcriptionally active chromatin. The loss of macroH2A1 from H2Bacetylated chromatin, either by mutating macroH2A1 itself or removing the H2B K20ac chromatin cue for macroH2A1 deposition, impeded the upregulation of SASP gene expression, highlighting their critical role in regulating macroH2A1 function. Our findings have allowed us to definitively establish that macroH2A1's local regulation of an important transcriptional program, the senescence-associated secretory phenotype (SASP), requires its accurate genomic localization.

In addition to transcription, PAR chains function as a scaffold to recruit DNA repair factors. Poly(ADP-ribose) polymerase-1 (PARP1) is activated upon sensing DNA damage can either promote cell survival or death. To accumulate the PAR chains required to recruit repair factors, the rate of PAR chain synthesis must exceed the rate of PAR turnover which catalyzed by constitutively active PARG and ARH3. If the rate of PAR synthesis is too high, due to unstable PAR chains and PARP1 overactivation, NAD+ depletion can result, leading to necrotic cell death. We show that macroH2A1.1 binds to the ends of PAR chains leading to their sustained accumulation which preserves cellular NAD+ levels and consequentially promotes cell survival. As a histone variant, macroH2A1 is part of the local chromatin regulatory platform for transcription. Through its ability to stabilize PAR chains, macroH2A1 regulates the kinetics of PAR accumulation and serves as a critical support structure for the PAR scaffold built upon chromatin itself. MacroH2A is not only an important platform for transcriptional regulation but mediates aspects of the DNA damage response as well.