Structural insights into p300 regulation and acetylation-dependent genome organisation.

Histone modifications are deposited by chromatin modifying enzymes and read out by proteins that recognize the modified state. BRD4-NUT is an oncogenic fusion protein of the acetyl lysine reader BRD4 that binds to the acetylase p300 and enables formation of long-range intra- and interchromosomal interactions. We here examine how acetylation reading and writing enable formation of such interactions. We show that NUT contains an acidic transcriptional activation domain that binds to the TAZ2 domain of p300. We use NMR to investigate the structure of the complex and found that the TAZ2 domain has an autoinhibitory role for p300. NUT-TAZ2 interaction or mutations found in cancer that interfere with autoinhibition by TAZ2 allosterically activate p300. p300 activation results in a self-organizing, acetylation-dependent feed-forward reaction that enables long-range interactions by bromodomain multivalent acetyl-lysine binding. We discuss the implications for chromatin organisation, gene regulation and dysregulation in disease.

RNA-Guided Genomic Localization of H2A.L.2 Histone Variant.

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.

Transcription factor dimerization activates the p300 acetyltransferase.

The transcriptional co-activator p300 is a histone acetyltransferase (HAT) that is typically recruited to transcriptional enhancers and regulates gene expression by acetylating chromatin. Here we show that the activation of p300 directly depends on the activation and oligomerization status of transcription factor ligands. Using two model transcription factors, IRF3 and STAT1, we demonstrate that transcription factor dimerization enables the trans-autoacetylation of p300 in a highly conserved and intrinsically disordered autoinhibitory lysine-rich loop, resulting in p300 activation. We describe a crystal structure of p300 in which the autoinhibitory loop invades the active site of a neighbouring HAT domain, revealing a snapshot of a trans-autoacetylation reaction intermediate. Substrate access to the active site involves the rearrangement of an autoinhibitory RING domain. Our data explain how cellular signalling and the activation and dimerization of transcription factors control the activation of p300, and therefore explain why gene transcription is associated with chromatin acetylation.

Characterization of Post-Meiotic Male Germ Cell Genome Organizational States.

Dramatic and unique genome reorganizations accompany the differentiation of haploid male germ cells, characterized by a gradual loss of the vast majority of histones leading to a final tight compaction of the genome by protamines. Despite being essential for procreation and the life cycle, the mechanisms driving the transformation of nucleosomes into nucleoprotamines remain poorly understood. To address this issue, our laboratory has developed a number of specific approaches, ranging from the purification of spermatogenic cells at specific stages, the analysis of chromatin transitional states, the functional characterization of histone variants, histone-replacing proteins and their chaperones. This chapter will detail all related relevant techniques with a particular emphasis on methods allowing the functional studies of histone variants and the genome organizational states associated with the studied histones in spermatogenic cells undergoing histone-to-protamine exchange.

Histone variants: essential actors in male genome programming.

Prior to its transmission to the offspring, the male genome has to be tightly compacted. A genome-scale histone eviction and the subsequent repackaging of DNA by protamines (Prms) direct this essential genome condensation step. The requirement for male germ cells to undergo such a dramatic and unique genome reorganization explains why these cells express the largest number of histone variants, including many testis-specific ones. Indeed, an open chromatin, nucleosome instability and a facilitated process of histone disassembly are direct consequences of the presence of these histone variants in the chromatin of male germ cells. These histone-induced changes in chromatin first control a stage-specific gene expression program and then directly mediate the histone-to-Prm transition process. This review aims at summarizing and discussing a series of recent functional studies of male germ cell histone variants with a focus on their impact on the process of histone eviction and male genome compaction.