[Spermiogenesis: histone acetylation triggers male genome reprogramming]. / Spermiogenèse: l'acétylation des histones déclenche la reprogrammation du génome mâle.

During their post-meiotic maturation, male germ cells undergo an extensive reorganization of their genome, during which histones become globally hyperacetylated, are then removed and progressively replaced by transition proteins and finally by protamines. The latter are known to tightly associate with DNA in the mature sperm cell. Although this is a highly conserved and fundamental biological process, which is a necessary prerequisite for the transmission of the male genome to the next generation, its molecular basis remains mostly unknown. We have identified several key factors involved in this process, and their detailed functional study has enabled us to propose the first model describing molecular mechanisms involved in post-meiotic male genome reprogramming. One of them, Bromodomain Testis Specific (BRDT), has been the focus of particular attention since it possesses the unique ability to specifically induce a dramatic compaction of acetylated chromatin. Interestingly, a mutation was found homozygous in infertile men which, according to our structural and functional studies, disrupts the function of the protein. A combination of molecular structural and genetic approaches has led to a comprehensive understanding of new major actors involved in the male genome reprogramming and transmission.

HDAC6, at the crossroads between cytoskeleton and cell signaling by acetylation and ubiquitination.

Histone deacetylase 6 (HDAC6) is a unique enzyme with specific structural and functional features. It is actively or stably maintained in the cytoplasm and is the only member, within the histone deacetylase family, that harbors a full duplication of its deacetylase homology region followed by a specific ubiquitin-binding domain at the C-terminus end. Accordingly, this deacetylase functions at the heart of a cellular regulatory mechanism capable of coordinating various cellular functions largely relying on the microtubule network. Moreover, HDAC6 action as a regulator of the HSP90 chaperone activity adds to the multifunctionality of the protein, and allows us to propose a critical role for HDAC6 in mediating and coordinating various cellular events in response to different stressful stimuli.

Functional significance of histone deacetylase diversity.

Nucleocytoplasmic shuttling of histone deacetylases is emerging as a major step in determining the composition, and hence the activity, of the corresponding nuclear regulatory complexes. This shuttling process is one of the distinctive characteristics of these enzymes, themselves belonging to structurally and functionally different classes. Considering the specific features of each class of deacetylases, it is possible to determine how each member can contribute to particular cellular functions.

Identity crisis in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) shares many hallmarks with cancer. Cancer cells acquire their hallmarks by a pathological Darwinian evolution process built on the so-called cancer cell "identity crisis." Here we demonstrate that PAH shares the most striking features of the cancer identity crisis: the ectopic expression of normally silent tissue-specific genes.

p14ARF promotes RB accumulation through inhibition of its Tip60-dependent acetylation.

p14ARF is a tumour suppressor which plays a critical role in p53-dependent or -independent cell growth control. Several studies have recently provided evidence that p14ARF can also interfere either directly or indirectly with some components of the RB signalling pathway to mediate its antiproliferative activity. The aim of this study was to explore the existence of direct relationships between p14ARF and RB proteins. We show that p14ARF promotes the accumulation of a hypoacetylated RB protein, when it is upregulated in a model of stable-inducible clones or physiologically induced following cell exposure to cytotoxic agents. Looking for the mechanisms involved in this process, we demonstrate that the histone acetyl transferase Tip60 directly interacts with RB and stimulates its degradation by the proteasome through acetylation of its C-terminus. Furthermore, and consistent with p14ARF-induced RB accumulation, we provide evidence that p14ARF prevents Tip60-mediated RB acetylation, therefore precluding its proteasomal degradation. Overall, our results identify a novel mechanism by which p14ARF controls the RB pathway to trigger its antiproliferative function.

Higher concentrations of histone macroH2A in the Barr body are correlated with higher nucleosome density.

Histone macroH2A, which is a subtype of histone H2A, possesses a histone H2A-like portion fused to a relatively long non-histone portion. MacroH2A has been shown to associate preferentially with the inactive X chromosome [1]. To investigate the specificity of this association, the nuclear distribution of macroH2A was compared with that of regular core histones. In normal human female fibroblasts, all anti-histone antibodies that were tested (including anti-macroH2A antibody) preferentially labeled the inactive X chromosome. Moreover, when expressed as green fluorescent protein (GFP) fusions, both histone H2A and macroH2A were concentrated in the Barr body. These data clearly show the presence of a higher density of nucleosomes in the inactive X chromosome. Accordingly, the specificity of the macroH2A association with the inactive X chromosome should be reconsidered. While investigating the role of macroH2A, we found that the proximity of the non-histone region of macroH2A to a promoter could lead to a specific repression of transcription, suggesting that the incorporation of macroH2A into chromatin might help to establish the stable pattern of gene expression in differentiated cells.

Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm.

The intracellular localization, and thereby the function, of a number of key regulator proteins tagged with a short leucine-rich motif (the nuclear export signal or NES) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus [1]. A common characteristic of these regulators is their transient action in the nucleus during either a specific phase of the cell cycle or in response to specific signals [1]. Here, we show that a particular member of the class II histone-deacetylases mHDA2/mHDAC6 [2] belongs to this family of cellular regulators that are present predominantly in the cytoplasm, but are also capable of shuttling between the nucleus and the cytoplasm. A very potent NES present at the amino terminus of mHDAC6 was found to play an essential role in this shuttling process. The sub-cellular localization of mHDAC6 appeared to be controlled by specific signals, since the arrest of cell proliferation was found to be associated with the translocation of a fraction of the protein into the nucleus. Data presented here suggest that mHDAC6 might be the first member of a functionally distinct class of deacetylases, responsible for activities not shared by other known histone deacetylases.

Involvement of retinoblastoma protein and HBP1 in histone H1(0) gene expression.

The histone H1(0)-encoding gene is expressed in vertebrates in differentiating cells during the arrest of proliferation. In the H1(0) promoter, a specific regulatory element, which we named the H4 box, exhibits features which implicate a role in mediating H1(0) gene expression in response to both differentiation and cell cycle control signals. For instance, within the linker histone gene family, the H4 box is found only in the promoters of differentiation-associated subtypes, suggesting that it is specifically involved in differentiation-dependent expression of these genes. In addition, an element nearly identical to the H4 box is conserved in the promoters of histone H4-encoding genes and is known to be involved in their cell cycle-dependent expression. The transcription factors interacting with the H1(0) H4 box were therefore expected to link differentiation-dependent expression of H1(0) to the cell cycle control machinery. The aim of this work was to identify such transcription factors and to obtain information concerning the regulatory pathway involved. Interestingly, our cloning strategy led to the isolation of a retinoblastoma protein (RB) partner known as HBP1. HBP1, a high-mobility group box transcription factor, interacted specifically with the H1(0) H4 box and moreover was expressed in a differentiation-dependent manner. We also showed that the HBP1-encoding gene is able to produce different forms of HBP1. Finally, we demonstrated that both HBP1 and RB were involved in the activation of H1(0) gene expression. We therefore propose that HBP1 mediates a link between the cell cycle control machinery and cell differentiation signals. Through modulating the expression of specific chromatin-associated proteins such as histone H1(0), HBP1 plays a vital role in chromatin remodeling events during the arrest of cell proliferation in differentiating cells.

Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways.

The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins. Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3). Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6. By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them. All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination. The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Histone acetylation-mediated chromatin compaction during mouse spermatogenesis.

One of the most dramatic chromatin remodelling events takes place during mammalian spermatogenesis involving massive incorporation of somatic and testis-specific histone variants, as well as generalized histone modifications before their replacement by new DNA packaging proteins. Our data suggest that the induced histone acetylation occurring after meiosis may direct the first steps of genome compaction. Indeed, a double bromodomain-containing protein expressed in postmeiotic cells, Brdt, shows the extraordinary capacity to specifically condense acetylated chromatin in vivo and in vitro. In elongating spermatids, Brdt widely co-localizes with acetylated histones before accumulating in condensed chromatin domains. These domains preferentially maintain their acetylation status until late spermatogenesis. Based on these data, we propose that Brdt mediates a general histone acetylation-induced chromatin compaction and also maintains differential acetylation of specific regions, and is therefore involved in organizing the spermatozoon's genome.

[Epigenetics of the sperm cell]. / Epigénétique du spermatozoïde.

In addition to genetic information, the spermatozoon carries another type of information, named epigenetic, which is not associated with variations of the DNA sequence. In somatic cells, it is now generally admitted that epigenetic information is not only regulated by DNA methylation but also involves modifications of the genome structure, or epigenome. During male germ cell maturation, the epigenome is globally re-organized, since most histones, which are associated to DNA in somatic cells, are removed and replaced by sperm specific nuclear proteins, the protamines, responsible for the tight compaction of the sperm DNA. However, a small proportion of histones, and probably other proteins, are retained within the sperm nucleus, and the structure of the sperm genome is actually heterogeneous. This heterogeneity of the sperm epigenome could support an epigenetic information, transmitted to the embryo, which could be crucial for its development. Although it is nowadays possible to appreciate the global structure of the sperm genome, the precise constitution of the sperm epigenome remains unknown. In particular, very recent data suggest that specific regions of the genome could be associated with particular proteins and define specific structures. This structural partitioning of the sperm genome could convey important epigenetic information, crucial for the embryo development.

Regulation of histone H1(0) accumulation during induced differentiation of murine erythroleukemia cells.

Histone H1(0) is one of the potential candidates that may contribute to the onset and stabilization of a genetic program during induced differentiation of murine erythroleukemia cells. In an attempt to understand better the role of H1(0) in this process we have tried to determine at which level the regulation of its induced accumulation occurs. Protein H1(0) was found to increase by a factor of 3 while its mRNA increased by a factor of 14, due to activation of gene transcription. As shown by H1(0) half-life measurements, the difference between the actual amount of H1(0) and that expected from the amount of mRNA was not due to increased turnover of the protein. Fractionation of the translational apparatus at several times during induction, revealed that H1(0) mRNA was efficiently transferred to the high molecular weight polysomes. The rate of synthesis of H1(0) was also increased by a factor of 4. Taken together, these results suggest the existence of a strong control at the translational level, which regulates H1(0) accumulation.

Early events in murine erythroleukemia cells induced to differentiate. Accumulation and gene expression of the transformation-associated cellular protein p53.

Oncogenes may play a crucial role in the genetic program of cellular differentiation; even, probably, at a very early stage in this program, which can be described as pre-commitment. We have investigated the variation in, and the control level of, the accumulation of the transformation-associated cellular protein p53 in murine erythroleukemia cells induced to differentiate by hexamethylene bisacetamide. Using flow cytofluorimetry after double staining of the cells, we have found that p53 decreased from two hours after the input of the inducer, to reach a basal level of about 30% of the starting value. The stability of the protein was found to be affected neither by the inducer nor by the position of the cells in the cell cycle. Looking for the regulation mechanism of the p53 decay, we found that the mRNA started to decrease as early as half an hour after the hexamethylene bisacetamide was put in the culture medium, and that the transcription rate of the gene itself could not account for the observed down-regulation of the mRNA, suggesting a post-transcriptional control for the mRNA accumulation. This control did not require the de-novo synthesis of a protein component, as shown by cycloheximide experiments, but seemed to be governed by the induced synthesis of an RNA molecule. Hypothetical models for such a regulation process are discussed in the light of recent reports on the metabolism of mRNA.

Differential remodeling of the HIV-1 nucleosome upon transcription activators and SWI/SNF complex binding.

Here we have examined HIV-1 nucleosome remodeling upon the binding of transcription factors and the SWI/SNF complex using a novel approach. The approach combines UV laser protein-DNA crosslinking, electrophoretic mobility-shift analysis and DNase I protection analysis with immunochemical techniques. It was found that single activator-bound HIV-1 nucleosomes exhibit very weak perturbation in histone NH(2) tail-DNA interactions. However, the simultaneous binding of the transcription activators Sp1, NF-kB1, LEF-1 and USF synergistically increased the release of histone NH(2) tails from nucleosomal DNA. In contrast, the binding of SWI/SNF complex to HIV-1 nucleosome disrupted structured histone domain-DNA contacts, but not histone NH(2) tail-DNA interactions. Stable remodeled nucleosomes, (obtained after detachment of SWI/SNF), displayed identical structural alterations with those bound to SWI/SNF. These results demonstrate a different in vitro remodeling of the HIV-1 nucleosome upon the binding of multiple transcription activators and of SWI/SNF complex.

Developmentally regulated chromatin acetylation and histone H1(0) accumulation.

There exists a close relationship between core histone acetylation and the induced expression of the histone H1(0) gene. We took advantage of this fact to evaluate the influence of chromatin hyperacetylation on the developmentally regulated expression of this specific gene. In this study, the in situ immunodetection approach has been used to analyze both the acetylated histone H4 isoforms and histone H1(0) accumulation during early Xenopus laevis development. We have chosen two stages of development, gastrula stage, when H1(0) is not expressed and not inducible by butyrate treatment, and stage 27 when H1(0) is not expressed but is inducible by butyrate. At stage 27 of development, the early induced accumulation of histone H1(0) under butyrate treatment, occurs mainly in tissues that express the protein normally during later development. These experiments suggest that histone acetylation may be part of a pathway which, in a specific set of cells, keeps H1(0) and probably a series of specific genes, competent for transcription, but cell-specific factors are involved in the induced expression of these genes.

Regulated hyperacetylation of core histones during mouse spermatogenesis: involvement of histone deacetylases.

Here we report a detailed analysis of waves of histone acetylation that occurs throughout spermatogenesis in mouse. Our data showed that spermatogonia and preleptotene spermatocytes contained acetylated core histones H2A, H2B and H4, whereas no acetylated histones were observed throughout meiosis in leptotene or pachytene spermatocytes. Histones remained unacetylated in most round spermatids. Acetylated forms of H2A and H2B, H3 and H4 reappeared in step 9 to 11 elongating spermatids, and disappeared later in condensing spermatids. The spatial distribution pattern of acetylated H4 within the spermatids nuclei, analyzed in 3D by immunofluorescence combined with confocal microscopy, showed a spatial sequence of events tightly associated with chromatin condensation. In order to gain an insight into mechanisms controlling histone hyperacetylation during spermiogenesis, we treated spermatogenic cells with a histone deacetylase inhibitor, trichostatin A (TSA), which showed a spectacular increase of histone acetylation in round spermatids. This observation suggests that deacetylases are responsible for maintaining a deacetylated state of histones in these cells. TSA treatment could not induce histone acetylation in condensing spermatids, suggesting that acetylated core histones are replaced by transition proteins without being previously deacetylated. Moreover, our data showed a dramatic decrease in histone deacetylases in condensing spermatids. Therefore, the regulation of histone deacetylase activity/concentration appears to play a major role in controling histone hyperacetylation and probably histone replacement during spermiogenesis.

Histone H1 diversity: bridging regulatory signals to linker histone function.

Genes encoding linker histone variants have evolved to link their expression to signals controlling the proliferative capacities of cells, i.e. cycling and growth-arrested cells express distinct and specific H1 subtypes. In metazoan, these variants show a tripartite structure, with considerably divergent sequences in their amino and carboxyl terminus domains. The aim of this review is to show how specific regulatory signals control the expression of an individual H1 and to discuss the functional significance of the two variables associated with a linker histone: its primary sequence and the timing of its expression.

Developmental regulation and butyrate-inducible transcription of the Xenopus histone H1(0) promoter.

We have isolated genomic clones of the Xenopus laevis histone H1(0) promoter and identified regulatory elements mediating the transcriptional regulation of the H1(0) gene. Expression of H1(0) is associated with the terminal differentiation of many cell types. During X. laevis development, H1(0) mRNA is present in the oocyte and egg, but remains at low levels during embryogenesis until hatching. After this time, mRNA levels accumulate dramatically correlating with the differentiation of many tissue types, e.g., liver and skin. Accumulation of H1(0) mRNA can be induced at earlier development stages by treating embryos with butyrate. The enhanced transcription of H1(0) in adult somatic cells, as well as the butyrate inducibility of the gene, have been investigated using transfection of adult X. laevis A6 somatic cells. We have defined specific protein-nucleic acid interactions with three cis-acting elements. Two previously defined gene regulatory elements: the H1 box, normally involved in the regulation of the H1 gene, and the H4TF2 site, normally involved in the regulation of the H4 gene, appear to have novel roles in determining differentiation-specific H1(0) expression. These two elements act together with a new distal cis-acting element in order to sustain high levels of basal transcription and to potentiate transcription following butyrate treatment.