Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells.

Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.

Chromatin-to-nucleoprotamine transition is controlled by the histone H2B variant TH2B.

The conversion of male germ cell chromatin to a nucleoprotamine structure is fundamental to the life cycle, yet the underlying molecular details remain obscure. Here we show that an essential step is the genome-wide incorporation of TH2B, a histone H2B variant of hitherto unknown function. Using mouse models in which TH2B is depleted or C-terminally modified, we show that TH2B directs the final transformation of dissociating nucleosomes into protamine-packed structures. Depletion of TH2B induces compensatory mechanisms that permit histone removal by up-regulating H2B and programming nucleosome instability through targeted histone modifications, including lysine crotonylation and arginine methylation. Furthermore, after fertilization, TH2B reassembles onto the male genome during protamine-to-histone exchange. Thus, TH2B is a unique histone variant that plays a key role in the histone-to-protamine packing of the male genome and guides genome-wide chromatin transitions that both precede and follow transmission of the male genome to the egg.

Chemical Screen Identifies Diverse and Novel Histone Deacetylase Inhibitors as Repressors of NUT Function: Implications for NUT Carcinoma Pathogenesis and Treatment.

NUT carcinoma (NC), characterized most commonly by the BRD4-NUTM1 fusion, is a rare, aggressive variant of squamous carcinoma with no effective treatment. BRD4-NUT drives growth and maintains the poorly differentiated state of NC by activating pro-growth genes such as MYC, through the formation of massive, hyperacetylated, superenhancer-like domains termed megadomains. BRD4-NUT-mediated hyperacetylation of chromatin is facilitated by the chromatin-targeting tandem bromodomains of BRD4, combined with NUT, which recruits the histone acetyltransferase, p300. Here, we developed a high-throughput small-molecule screen to identify inhibitors of transcriptional activation by NUT. In this dCAS9-based GFP-reporter assay, the strongest hits were diverse histone deacetylase (HDAC) inhibitors. Two structurally unrelated HDAC inhibitors, panobinostat and the novel compound, IRBM6, both repressed growth and induced differentiation of NC cells in proportion to their inhibition of NUT transcriptional activity. These two compounds repressed transcription of megadomain-associated oncogenic genes, such as MYC and SOX2, while upregulating pro-differentiation, non-megadomain-associated genes, including JUN, FOS, and key cell-cycle regulators, such as CDKN1A. The transcriptional changes correlate with depletion of BRD4-NUT from megadomains, and redistribution of the p300/CBP-associated chromatin acetylation mark, H3K27ac, away from megadomains toward regular enhancer regions previously populated by H3K27ac. In NC xenograft models, we demonstrated that suppression of tumor growth by panobinostat was comparable with that of bromodomain inhibition, and when combined they improved both survival and growth suppression. IMPLICATIONS: The findings provide mechanistic and preclinical rationale for the use of HDAC inhibitors, alone or combined with other agents, in the treatment of NUT carcinoma.

"Z4" Complex Member Fusions in NUT Carcinoma: Implications for a Novel Oncogenic Mechanism.

Nuclear protein in testis (NUT) carcinoma (NC) is a rare, distinctly aggressive subtype of squamous carcinoma defined by the presence of NUT-fusion oncogenes resulting from chromosomal translocation. In most cases, the NUT gene (NUTM1) is fused to bromodomain containing 4 (BRD4) forming the BRD4-NUT oncogene. Here, a novel fusion partner to NUT was discovered using next-generation sequencing and FISH from a young patient with an undifferentiated malignant round cell tumor. Interestingly, the NUT fusion identified involved ZNF592, a zinc finger containing protein, which was previously identified as a component of the BRD4-NUT complex. In BRD4-NUT-expressing NC cells, wild-type ZNF592 and other associated "Z4" complex proteins, including ZNF532 and ZMYND8, colocalize with BRD4-NUT in characteristic nuclear foci. Furthermore, ectopic expression of BRD4-NUT in a non-NC cell line induces sequestration of Z4 factors to BRD4-NUT foci. Finally, the data demonstrate the specific dependency of NC cells on Z4 modules, ZNF532 and ZNF592. IMPLICATIONS: This study establishes the oncogenic role of Z4 factors in NC, offering potential new targeted therapeutic strategies in this incurable cancer.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/12/1826/F1.large.jpg.

Nut Directs p300-Dependent, Genome-Wide H4 Hyperacetylation in Male Germ Cells.

Nuclear protein in testis (Nut) is a universal oncogenic driver in the highly aggressive NUT midline carcinoma, whose physiological function in male germ cells has been unclear. Here we show that expression of Nut is normally restricted to post-meiotic spermatogenic cells, where its presence triggers p300-dependent genome-wide histone H4 hyperacetylation, which is essential for the completion of histone-to-protamine exchange. Accordingly, the inactivation of Nut induces male sterility with spermatogenesis arrest at the histone-removal stage. Nut uses p300 and/or CBP to enhance acetylation of H4 at both K5 and K8, providing binding sites for the first bromodomain of Brdt, the testis-specific member of the BET family, which subsequently mediates genome-wide histone removal. Altogether, our data reveal the detailed molecular basis of the global histone hyperacetylation wave, which occurs before the final compaction of the male genome.

Novel O-GlcNAcylation on Ser(40) of canonical H2A isoforms specific to viviparity.

We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser(40) (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser(40) and the other five have Ala(40). The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser(40) isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala(40) isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser(40) isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser(40) and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.

Exogenous Expression of Human Protamine 1 (hPrm1) Remodels Fibroblast Nuclei into Spermatid-like Structures.

Protamines confer a compact structure to the genome of male gametes. Here, we find that somatic cells can be remodeled by transient expression of protamine 1 (Prm1). Ectopically expressed Prm1 forms scattered foci in the nuclei of fibroblasts, which coalescence into spermatid-like structures, concomitant with a loss of histones and a reprogramming barrier, H3 lysine 9 methylation. Protaminized nuclei injected into enucleated oocytes efficiently underwent protamine to maternal histone TH2B exchange and developed into normal blastocyst stage embryos in vitro. Altogether, our findings present a model to study male-specific chromatin remodeling, which can be exploited for the improvement of somatic cell nuclear transfer.

Genome-scale acetylation-dependent histone eviction during spermatogenesis.

A genome-wide histone hyperacetylation is known to occur in the absence of transcription in haploid male germ cells, spermatids, before and during the global histone eviction and their replacement by non-histone DNA-packaging proteins. Although the occurrence of this histone hyperacetylation has been correlated with histone removal for a long time, the underlying mechanisms have remained largely obscure. Important recent discoveries have not only shed light on how histone acetylation could drive a subsequent transformation in genome organization but also revealed that the associated nucleosome dismantlement is a multi-step process, requiring the contribution of histone variants, critical destabilizing histone modifications and chromatin readers, including Brdt, working together to achieve the full packaging of the male genome, indispensable for the propagation of life.