Replacement Of Histones Research Articles

Chromodomain Protein CDYL Acts as a Crotonyl-CoA Hydratase to Regulate Histone Crotonylation and Spermatogenesis

Lysine crotonylation (Kcr) is a newly identified histone modification that is associated with active transcription in mammalian cells. Here we report that the chromodomain Y-like transcription corepressor CDYL negatively regulates histone Kcr by acting as a crotonyl-CoA hydratase to convert crotonyl-CoA to β-hydroxybutyryl-CoA. We showed that the negative regulation of histone Kcr by CDYL is intrinsically linked to its transcription repression activity and functionally implemented in the reactivation of sex chromosome-linked genes in round spermatids and genome-wide histone replacement in elongating spermatids. Significantly, Cdyl transgenic mice manifest dysregulation of histone Kcr and reduction of male fertility with a decreased epididymal sperm count and sperm cell motility. Our study uncovers a biochemical pathway in the regulation of histone Kcr and implicates CDYL-regulated histone Kcr in spermatogenesis, adding to the understanding of the physiology of male reproduction and the mechanism of the spermatogenic failure in AZFc (Azoospermia Factor c)-deleted infertile men.

CFP1 Regulates Histone H3K4 Trimethylation and Developmental Potential in Mouse Oocytes

Trimethylation of histone H3 at lysine-4 (H3K4me3) isassociated with eukaryotic gene promoters andpoises their transcriptional activation during development. To examine the invivo function of H3K4me3 in the absence of DNA replication, we deleted CXXC finger protein 1 (CFP1), the DNA-binding subunit of the SETD1 histone H3K4 methyltransferase, in developing oocytes. We find that CFP1 isrequired for H3K4me3 accumulation and the deposition of histone variants onto chromatin during oocyte maturation. Decreased H3K4me3 in oocytes caused global downregulation of transcription activity. Oocytes lacking CFP1 failed to complete maturation and were unable to gain developmental competence after fertilization, due to defects in cytoplasmic lattice formation, meiotic division, and maternal-zygotic transition. Our study highlights the importance of H3K4me3 in continuous histone replacement for transcriptional regulation, chromatin remodeling, and normal developmental progression in a non-replicative system.

Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation.

Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development.

DNA Replication Is Required for Circadian Clock Function by Regulating Rhythmic Nucleosome Composition

Although the coupling between circadian and cell cycles allows circadian clocks to gate cell division and DNA replication in many organisms, circadian clocks were thought to function independently of cell cycle. Here, we show that DNA replication is required for circadian clock function in Neurospora. Genetic and pharmacological inhibition of DNA replication abolished both overt and molecular rhythmicities by repressing frequency (frq) gene transcription. DNA replication is essential for the rhythmic changes of nucleosome composition at the frq promoter. The FACT complex, known to be involved in histone disassembly/reassembly, is required for clock function and is recruited to the frq promoter in a replication-dependent manner to promote replacement of histone H2A.Z by H2A. Finally, deletion of H2A.Z uncoupled the dependence of the circadian clock on DNA replication. Together, these results establish circadian clock and cell cycle as interdependent coupled oscillators and identify DNA replication as a critical process in the circadian mechanism.

Post-meiotic DNA double-strand breaks occur in Tetrahymena, and require Topoisomerase II and Spo11.

Based on observations of markers for DNA lesions, such as phosphorylated histone H2AX (γH2AX) and open DNA ends, it has been suggested that post-meiotic DNA double-strand breaks (PM-DSBs) enable chromatin remodeling during animal spermiogenesis. However, the existence of PM-DSBs is unconfirmed, and the mechanism responsible for their formation is unclear. Here, we report the first direct observation of programmed PM-DSBs via the electrophoretic separation of DSB-generated DNA fragments in the ciliate Tetrahymena thermophila. These PM-DSBs are accompanied by switching from a heterochromatic to euchromatic chromatin structure in the haploid pronucleus. Both a topoisomerase II paralog with exclusive pronuclear expression and Spo11 are prerequisites for PM-DSB induction. Reduced PM-DSB induction blocks euchromatin formation, characterized by histone H3K56 acetylation, leading to a failure in gametic nuclei production. We propose that PM-DSBs are responsible for histone replacement during the reprogramming of generative to undifferentiated progeny nuclei.

TSSK6 is required for γH2AX formation and the histone-to-protamine transition during spermiogenesis

Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX (also known as H2AFX) phosphorylation at Ser139 (γH2AX), and replacement of histones by protamines. Previously, we have reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with γH2AX formation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks γH2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6-knockout sperm contained increased levels of histones H3 and H4, and protamine 2 precursor and intermediate(s) indicative of a defective histone-to-protamine transition. These results demonstrate that TSSK6 is required for γH2AX formation during spermiogenesis, and also link γH2AX to the histone-to-protamine transition and male fertility.

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 asTPs 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 thatultimately shift to the fully compacted genomeofthe mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should berevisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.

Histone gene replacement reveals a post-transcriptional role for H3K36 in maintaining metazoan transcriptome fidelity.

Histone H3 lysine 36 methylation (H3K36me) is thought to participate in a host of co-transcriptional regulatory events. To study the function of this residue independent from the enzymes that modify it, we used a 'histone replacement' system in Drosophila to generate a non-modifiable H3K36 lysine-to-arginine (H3K36R) mutant. We observed global dysregulation of mRNA levels in H3K36R animals that correlates with the incidence of H3K36me3. Similar to previous studies, we found that mutation of H3K36 also resulted in H4 hyperacetylation. However, neither cryptic transcription initiation, nor alternative pre-mRNA splicing, contributed to the observed changes in expression, in contrast with previously reported roles for H3K36me. Interestingly, knockdown of the RNA surveillance nuclease, Xrn1, and members of the CCR4-Not deadenylase complex, restored mRNA levels for a class of downregulated, H3K36me3-rich genes. We propose a post-transcriptional role for modification of replication-dependent H3K36 in the control of metazoan gene expression.

The histone H2A isoform Hist2h2ac is a novel regulator of proliferation and epithelial–mesenchymal transition in mammary epithelial and in breast cancer cells

Proliferation and differentiation are controlled through chromatin remodelling. Therefore, there is an enormous biological significance and clinical value in understanding how specific signalling pathways are affected by histone replacement in the nucleosome. In this work, mass spectrometry was used to screen HC11 mammary epithelial cells for changes in histone levels throughout cell differentiation. The canonical histone isoform Histone H2A type 2-C (Hist2h2ac) was found only in undifferentiated/proliferating cells. Hist2h2ac mRNA was induced by EGF, specifically in the CD24+/CD29hi/DC44hi cell subpopulation. Hist2h2ac mRNA was increased by MEK1/2 or PI3-K activation in HC11 and EpH4 mammary epithelial cells, and in MC4-L2 and T47-D breast cancer cells. Hist2h2ac silencing inhibited EGF-induced Zeb-1 expression and E-cadherin down-regulation, and this effect was reverted by Hist2h2ac re-expression. Notably, silencing of Hist2h2ac increased EGFR, ERBB2, and ERK1/2 activation but did not allow EGF-induced proliferation. HIST2H2AC was expressed in all breast cancer molecular subtypes and found altered in 17% breast cancers, being 16.8% of the cases related to HIST2H2AC gene amplification and/or mRNA upregulation. In summary, this is the first study that identifies a canonical histone isoform -Hist2h2ac-downstream of the EGFR pathway, regulating oncogenic signalling and thereby contributing to deregulation of target genes.

Investigating the Histone Replacement Pathway in Sperm Using TPM

DNA is folded in drastically different ways in somatic cells and sperm cells. In somatic cells, DNA is wrapped around histone proteins and remains accessible for transcription and replication. However, in sperm cells, DNA is dramatically condensed into a toroid structure when the histones are replaced by protamine proteins. Here, we investigate the process of histone replacement by protamine using tethered particle motion (TPM). TPM measurements are ideal for looking at DNA folding since measurements are taken at zero force.

Resolving Heart Regeneration by Replacement Histone Profiling

Chromatin regulation is a principal mechanism governing animal development, yet it is unclear to what extent structural changes in chromatin underlie tissue regeneration. Non-mammalian vertebrates such as zebrafish activate cardiomyocyte (CM) division after tissue damage to regenerate lost heart muscle. Here, we generated transgenic zebrafish expressing a biotinylatable H3.3 histone variant in CMs and derived cell-type-specific profiles of histone replacement. We identified an emerging program of putativeenhancers that revise H3.3 occupancy during regeneration, overlaid upon a genome-wide reduction of H3.3 from promoters. In transgenic reporter lines, H3.3-enriched elements directed gene expression in subpopulations of CMs. Other elements increased H3.3 enrichment and displayed enhancer activity in settings of injury- and/or Neuregulin1-elicited CM proliferation. Dozens of consensus sequence motifs containing predicted transcription factor binding sites were enriched in genomic regions with regeneration-responsive H3.3 occupancy. Thus, cell-type-specific regulatory programs of tissue regeneration can be revealed by genome-wide H3.3 profiling.

Investigation of the human H3.3B (H3F3B) gene expression as a novel marker in patients with colorectal cancer.

H3.3 histone is a replacement histone subtype that is express in entire cell cycle phases and overexpress in transcriptionally active regions, promoter regions, and intergenic or intragenic regulatory elements. This histone encoded by two genes termed H3.3A (H3F3A) and H3.3B (H3F3B). Mutations of these two genes lead to some human cancers such as chondroblastoma, osteosarcoma, and epithelial ovarian cancer. The aims of this study were to quantitatively examine the expression of H3.3B gene in colorectal cancer (CRC) and to correlate their expression level with demographics and clinicopathological characteristics. We investigated H3.3B gene expression in CRC by relative quantitative real-time polymerase chain reaction (real-time PCR) technique for the first time. For this purpose, total RNA extracted, then cDNA synthesized and H3.3B gene expression was evaluated with specific primers by real-time PCR in tumoral tissues and adjacent normal tissues of 36 patients with CRC, then statistical analysis was performed using SPSS software. The results of this study indicated that H3.3B gene significantly overexpressed in tumoral tissue than adjacent normal tissue. Furthermore, statistical analysis represented the significant correlation between the H3.3B gene expression and some of the clinicopathological characteristics. Our study showed that H3.3B gene expression changes can be useful as a probable prognosis biomarker in the early stages of CRC before it metastasized.

Histone Variants and Composition in the Developing Brain: Should MeCP2 Care?

Specific compositional chromatin features distinguish brain/neuronal chromatin from that of other tissues and are critical to this organ and cell type development and neuroplasticity. These features include a significant turnover of the major constitutive chromosomal proteins, including the (canonical) replication-dependent histones, the replication-independent replacement histone variants, as well as the chromatin associated transcriptional regulator MeCP2 (methyl CpG binding protein 2). Alterations of histones and MeCP2 have already been implicated in many brain disorders. Despite the relevance of histone variants to chromatin structure and function, only recently has some exciting literature started to re-emerge that directly relates them to neuron plasticity and cognition. However, the amount of information available on the functional role of these histones is still very limited. The purpose of this review is to focus attention to this important group of chromatin proteins, which, in the brain, possess overlapping structural and functional roles with the highly abundant presence of MeCP2. There is an imperative need to understand how all these proteins communicate with each other, and future research will hopefully provide us with answers.

TSSK6 is required for γH2AX formation and the histone-to-protamine transition during spermiogenesis.

Spermiogenesis includes transcriptional silencing, chromatin condensation and extensive morphological changes as spermatids transform into sperm. Chromatin condensation involves histone hyperacetylation, transitory DNA breaks, histone H2AX (also known as H2AFX) phosphorylation at Ser139 (γH2AX), and replacement of histones by protamines. Previously, we have reported that the spermatid protein kinase TSSK6 is essential for fertility in mice, but its specific role in spermiogenesis is unknown. Here, we show that TSSK6 expression is spatiotemporally coincident with γH2AX formation in the nuclei of developing mouse spermatids. RNA-sequencing analysis demonstrates that genetic ablation of Tssk6 does not impact gene expression or silencing in spermatids. However, loss of TSSK6 blocks γH2AX formation, even though the timing and level of the transient DNA breaks is unaltered. Further, Tssk6-knockout sperm contained increased levels of histones H3 and H4, and protamine 2 precursor and intermediate(s) indicative of a defective histone-to-protamine transition. These results demonstrate that TSSK6 is required for γH2AX formation during spermiogenesis, and also link γH2AX to the histone-to-protamine transition and male fertility.

Epigenetics Evolution and Replacement Histones: Evolutionary Changes at Drosophila H2AvD

The evolutionary changes in the Drosophila H2A and H2AvD genes, which encode histones, were analyzed using the sequences of 12 Drosophila sp. for understanding the evolution of histone replacement and epigenetics. The Ball gene, coding for a histone threonine kinase, was located head-to-head with the H2AvD gene in seven Drosophila sp. A strongly conserved DNA sequence was also found in the region upstream of the H2AvD gene; this sequence is most likely a transcriptional signal, because the sequence was also conserved in four other Drosophila sp. that did not have an upstream Ball gene. The SPARC gene, coding for a calcium-binding domain, was located tail-to-tail in the region downstream of the H2AvD gene in 11 Drosophila sp. studied. A moderately conserved DNA sequence was found in the H2AvD gene region at the splicing site in the first intron. Different codon usages for the H2A and H2AvD genes were found for 11 of 17 amino acids, and codon usages characteristic of replacement histones (H2AvD, H4r, H3.3A and H3.3B) were found for amino acids. Codon usage was considerably different at several histone modification sites in the H2A gene. These results suggested that unlike the H3.3 and H4r genes, not only post-transcriptional control, but also transcriptional control played a role in the H2AvD gene. In addition to post-transcriptional controls, such as splicing and translation, the development of a control system for transcription must have occurred during the evolution of histone replacement and epigenetic systems.

Analysis of the histone cluster in Senegalese sole (Solea senegalensis): evidence for a divergent evolution of two canonical histone clusters.

The Senegalese sole (Solea senegalensis) is commercially very important and a priority species for aquaculture product diversification. The main histone cluster was identified within two BAC clones. However, two replacement histones (H1.0 and H3.3) were found in another BAC clone. Different types of canonical histones H2A and H2B were found within the same species for the first time. Phylogenetic analysis demonstrated that the different types of H1, H2A, and H2B histones were all more similar to each other than to canonical histones from other species. The canonical histone H3 of S. senegalensis differs from subtypes H3.1 and H3.2 in humans at the site of residue 96, where a serine is found instead of an alanine. This same polymorphism has been found only in Danio rerio. The karyotype of S. senegalensis comprises 21 pairs of chromosomes, distributed in 3 metacentric pairs, 2 submetacentric pairs, 4 subtelocentric pairs, and 12 acrocentric pairs. The two BAC clones that contain the clusters of canonical histones were both mapped on the largest metacentric pair, and mFISH analysis confirmed the co-location with the dmrt1 gene in that pair. Three chromosome markers have been identified which, in addition to those previously described, account for 18 chromosome pairs in S. senegalensis.

Histone variants on the move: substrates for chromatin dynamics.

Most histones are assembled into nucleosomes behind the replication fork to package newly synthesized DNA. By contrast, histone variants, which are encoded by separate genes, are typically incorporated throughout the cell cycle. Histone variants can profoundly change chromatin properties, which in turn affect DNA replication and repair, transcription, and chromosome packaging and segregation. Recent advances in the study of histone replacement have elucidated the dynamic processes by which particular histone variants become substrates of histone chaperones, ATP-dependent chromatin remodellers and histone-modifying enzymes. Here, we review histone variant dynamics and the effects of replacing DNA synthesis-coupled histones with their replication-independent variants on the chromatin landscape.

127 ABUNDANCE OF mRNA FOR HISTONE VARIANTS, HISTONE, AND DNA MODIFICATION ENZYMES IN BOVINE IN VIVO OOCYTES AND PRE-IMPLANTATION EMBRYOS

During early embryogenesis, chromatin composition and structure undergo dramatic changes due to replacement of canonical histones by histone variants, post-translational modifications of histones, and changes in DNA methylation. These dynamics of chromatin play important roles in the regulation of gene expression and development of embryonic cells. Our goal here is to describe the above-mentioned changes using recently established transcriptome profiles of bovine in vivo-produced oocytes and pre-implantation embryos (Jiang et al. 2014 BMC Genomics, 15, 1). Ten multiparous Holstein cows were synchronized and superovulated. Artificial insemination was conducted at 12 and 24 h post-standing heat using semen from bulls of proven fertility. In vivo-matured oocytes and 2- to 16-cell stage embryos were collected at 30 h, and 2 to 4 days after oestrus by oviducal flushing. Early morulae, compact morulae, and blastocysts were collected by non-surgical uterine flushing on days 5, 6, and 7 after oestrus. Single-cell deep sequencing libraries were prepared from oocytes/embryos (2 samples/stage) using a SOLiDTM Total RNA-seq Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions and sequenced on a 5500xl Genetic Analyzer. The reproducibility of the preparation and sequencing methods were indicated by high Pearson correlation efficiencies between the replicates. Sequencing reads were normalized to transcripts per million as final results after trimming and mapping of the reads. We found that 8, 8, 7, 13, 10, 2, and 2 out of the 14, 52, 22, 31, 23, 4, and 3 annotated histone variants, histone methyl-tranferases, histone demethylases, histone acetyl-tranferases, histone deacetylases, DNA methyl-transferases, and DNA demethylases, respectively, were highly abundant (mean transformed transcripts per kilobase million > 50) in at least one of the pre-implantation development stages studied. Among histone variants with high mRNA abundance, H1FOO, H3F3A, and H3F3B were highly stored in oocytes, whereas other variants such as H2AFJ, H2AFV, H2AFX, H2AFY, H2AFZ, and CENPA were largely transcribed after the embryonic genome activation. H3F3A and H3F3B, however, were maintained at relatively high levels throughout pre-implantation development. Additionally, the mRNA for histone acetyl-transferases, TADA2A and TADA1; histone deacetylase, HDAC1 and HDAC3; histone methyl-transferases, EED and PRMT5; histone demethylase, KDM1A, were more abundant than others. It was also found that oocytes stored a large amount of DNA methyl-transferase, DNMT1, which degraded gradually after fertilization. Overall, in vivo-produced oocytes and early embryos contained more mRNA for histone-modifying enzymes than those for DNA modification. Taken together, our results suggest that although there are widely recognised and dramatic changes in embryonic DNA methylation through both active and passive mechanisms, the pre-implantation embryos may be more engaged in modifying histones than DNA.

Unlocking sperm chromatin at fertilization requires a dedicated egg thioredoxin in Drosophila.

In most animals, the extreme compaction of sperm DNA is achieved after the massive replacement of histones with sperm nuclear basic proteins (SNBPs), such as protamines. In some species, the ultracompact sperm chromatin is stabilized by a network of disulfide bonds connecting cysteine residues present in SNBPs. Studies in mammals have established that the reduction of these disulfide crosslinks at fertilization is required for sperm nuclear decondensation and the formation of the male pronucleus. Here, we show that the Drosophila maternal thioredoxin Deadhead (DHD) is specifically required to unlock sperm chromatin at fertilization. In dhd mutant eggs, the sperm nucleus fails to decondense and the replacement of SNBPs with maternally-provided histones is severely delayed, thus preventing the participation of paternal chromosomes in embryo development. We demonstrate that DHD localizes to the sperm nucleus to reduce its disulfide targets and is then rapidly degraded after fertilization.

The Drosophila chromosomal protein Mst77F is processed to generate an essential component of mature sperm chromatin.

In most animals, the bulk of sperm DNA is packaged with sperm nuclear basic proteins (SNBPs), a diverse group of highly basic chromosomal proteins notably comprising mammalian protamines. The replacement of histones with SNBPs during spermiogenesis allows sperm DNA to reach an extreme level of compaction, but little is known about how SNBPs actually function in vivo. Mst77F is a Drosophila SNBP with unique DNA condensation properties in vitro, but its role during spermiogenesis remains unclear. Here, we show that Mst77F is required for the compaction of sperm DNA and the production of mature sperm, through its cooperation with protamine-like proteins Mst35Ba/b. We demonstrate that Mst77F is incorporated in spermatid chromatin as a precursor protein, which is subsequently processed through the proteolysis of its N-terminus. The cleavage of Mst77F is very similar to the processing of protamine P2 during human spermiogenesis and notably leaves the cysteine residues in the mature protein intact, suggesting that they participate in the formation of disulfide cross-links. Despite the rapid evolution of SNBPs, sperm chromatin condensation thus involves remarkably convergent mechanisms in distantly related animals.