Nuclear histone deacetylases are not required for global histone deacetylation during meiotic maturation in porcine oocytes

Histone acetylation plays an important role in the regulation of chromatin structure and gene function. In mammalian oocytes, histones H3 and H4 are highly acetylated during the germinal vesicle (GV) stage, and global histone deacetylation takes place via a histone deacetylase (HDAC)-dependent mechanism after GV breakdown (GVBD). The presence of HDACs in the GVs of mammalian oocytes in spite of the high acetylation states of nuclear histones indicates that the HDACs in the nucleus are inactive but become activated after GVBD. However, the fluctuation pattern, the localization of HDAC activity during meiotic maturation and, moreover, the responsibility of nuclear HDACs for global histone deacetylation are still unknown. Here, we demonstrated using porcine oocytes that total HDAC activity was maintained throughout meiotic maturation, and high HDAC activity was observed in both the nucleus and the cytoplasm at the GV stage. The experiments with valproic acid (VPA), a specific class I HDAC inhibitor, revealed that the HDACs in GVs were class I, and those in the cytoplasm were other than class I. Interestingly, VPA had no effect on global histone deacetylation after GVBD, indicating that nuclear HDACs were not required for global histone deacetylation. To confirm this possibility, we removed the nuclei from immature oocytes, injected somatic cell nuclei into the enucleated oocytes, and showed that injected somatic cell nuclei were dramatically deacetylated after nuclear envelope breakdown. These results revealed that nuclear contents, including class I HDACs, are not required for the global histone deacetylation during meiosis, and that cytoplasmic HDACs other than class I are responsible for this process.     

Inhibition of histone deacetylases by Trichostatin A leads to a HoxB4-independent increase of hematopoietic progenitor/stem cell frequencies as a result of selective survival

BackgroundDNA and chromatin modifications are critical mediators in the establishment and maintenance of cell type-specific gene expression patterns that constitute cellular identities. One type of modification, the acetylation and deacetylation of histones, occurs reversibly on lysine ε-NH₃⁺ groups of core histones via histone acetyl transferases (HAT) and histone deacetylases (HDAC). Hyperacetylated histones are associated with active chromatin domains, whereas hypoacetylated histones are enriched in non-transcribed loci.MethodsWe analyzed global histone H4 acetylation and HDAC activity levels in mature lineage marker-positive (Lin⁺) and progenitor lineage marker-negative (Lin^?) hematopoietic cells from murine bone marrow (BM). In addition, we studied the effects of HDAC inhibition on hematopoietic progenitor/stem cell (HPSC) frequencies, cell survival, differentiation and HoxB4 dependence.ResultsWe observed that Lin^? and Lin⁺ cells do not differ in global histone H4 acetylation but in HDAC activity levels. Further, we saw that augmented histone acetylation achieved by transient Trichostatin A (TSA) treatment increased the frequency of cells with HPSC immunophenotype and function in the heterogeneous pool of BM cells. Induction of histone hyperacetylation in differentiated BM cells was detrimental, as evidenced by preferential death of mature BM cells upon HDAC inhibition. Finally, TSA treatment of BM cells from HoxB4^?/? mice revealed that the HDAC inhibitor-mediated increase in HPSC frequencies was independent of HoxB4.ConclusionsOverall, these data indicate the potential of chromatin modifications for the regulation of HPSC. Chromatin-modifying agents may provide potential strategies for ex vivo expansion of HPSC.     

Cell shape regulates global histone acetylation in human mammary epithelial cells

Extracellular matrix (ECM) regulates cell morphology and gene expression in vivo; these relationships are maintained in three-dimensional (3D) cultures of mammary epithelial cells. In the presence of laminin-rich ECM (lrECM), mammary epithelial cells round up and undergo global histone deacetylation, a process critical for their functional differentiation. However, it remains unclear whether lrECM-dependent cell rounding and global histone deacetylation are indeed part of a common physical-biochemical pathway. Using 3D cultures as well as nonadhesive and micropatterned substrata, here we showed that the cell 'rounding' caused by lrECM was sufficient to induce deacetylation of histones H3 and H4 in the absence of biochemical cues. Microarray and confocal analysis demonstrated that this deacetylation in 3D culture is associated with a global increase in chromatin condensation and a reduction in gene expression. Whereas cells cultured on plastic substrata formed prominent stress fibers, cells grown in 3D lrECM or on micropatterns lacked these structures. Disruption of the actin cytoskeleton with cytochalasin D phenocopied the lrECM-induced cell rounding and histone deacetylation. These results reveal a novel link between ECM-controlled cell shape and chromatin structure and suggest that this link is mediated by changes in the actin cytoskeleton.     

HDAC inhibition is associated to valproic acid induction of early megakaryocytic markers

Valproic acid (VPA), a histone deacetylase inhibitor, causes differentiation in different cell lines and in a cell-specific manner; yet, its effect on megakaryocytic (MK) differentiation has not been studied. We evaluated whether VPA induces MK differentiation in a UT-7 cell line through histone acetylation in the GpIIIa gene region and activation of the ERK pathway. UT-7 cells, derived from megakaryoblastic leukemia, were treated with VPA at various concentrations, and the expression of differentiation markers as well as the gene expression profile was assessed. Flow cytometry, immunoblot analysis, and RT-PCR demonstrated that VPA induced the expression of the early MK markers GpIIIa (CD61) and GpIIb/IIIa (CD41) in a dose-dependent manner. The VPA-treated cells showed hyperacetylation of the histones H3 and H4; in particular, histone acetylation was found to have been associated with CD61 expression, in that the GpIIIa promoter showed H4 hyperacetylation, as demonstrated by the chromatin immunoprecipitation assay. Furthermore, activation of the ERK pathway was involved in VPA-mediated CD61/CD41 expression and in cell adhesion, as demonstrated by using the MEK/ERK inhibitor U0126. In conclusion, the capacity of VPA to commit UT-7 cells to MK differentiation is mediated by its inhibitory action on HDAC and the long-lived activation of ERK1/2.     

Metformin Attenuates Cognitive Impairments in Hypoxia–Ischemia Neonatal Rats via Improving Remyelination

Perinatal hypoxia–ischemia (H/I) causes brain injury and myelination damage. Finding efficient methods to restore myelination is critical for the recovery of brain impairments. By applying an H/I rat model, we demonstrate that metformin (Met) treatment significantly ameliorates the loss of locomotor activity and cognition of H/I rat in the Morris water maze and open field task tests. After administration of Met to H/I rat, the proliferation of Olig2+ oligodendrocyte progenitor cells and the expression of myelin basic protein are obviously increased in the corpus callosum. Additionally, the myelin sheaths are more compact and the impairments are evidently attenuated. These data indicate that Met is beneficial for the amelioration of H/I-induced myelination and behavior deficits.     

Combination of valproic acid and ATRA restores RARβ2 expression and induces differentiation in cervical cancer through the PI3K/Akt pathway

Epigenetic silencing of the tumor suppressor gene, RARβ2, through histone deacetylation has been established as an important process of cervical carcinogenesis. This pivotal role has led to the suggestion that a combination of retinoids selective for RARβ2 with histone deacetylase (HDAC) inhibitors may have therapeutic potential. Valproic acid (VPA), a HDAC inhibitor, has a critical role in the regulation of gene expression through histone acetylation and causes transformed cells to undergo growth arrest, differentiation, and apoptosis. Therefore, we hypothesized that the combination of VPA and ATRA could restore RARβ2 expression, thus resulting in enhanced anti-neoplastic activity in cervical cancer. Here, we show that VPA combined with ATRA led to hyperacetylation of histone H3 and a significant alteration of gene expression in cervical cancer cells, including RARβ2 gene expression, which was upregulated 50- to 90-fold. The combination therapy effectively inhibited the growth of cervical cancer cells more than the single agent treatment both in vitro and in vivo. The additive effects were associated with a significant upregulation of p21(CIP1) and p53 as well as a pronounced decrease in p-Stat3. Furthermore, the combined treatment led to cell cycle arrest predominantly at the G1 phase, and it preferentially induced cell differentiation rather than apoptosis in cervical cancer cells. The differentiation program was determined by the presence of E-cadherinmediated adhesion and activation of the PI3K/Akt pathway. Taken together, these results provide new insight into the mechanisms of enhanced antitumor activity of the HDAC inhibitor and ATRA regimen, thus offering a new therapeutic strategy for cervical cancer patients.     

Bromodomain‐dependent stage‐specific male genome programming by Brdt

Male germ cell differentiation is a highly regulated multistep process initiated by the commitment of progenitor cells into meiosis and characterized by major chromatin reorganizations in haploid spermatids. We report here that a single member of the double bromodomain BET factors, Brdt, is a master regulator of both meiotic divisions and post‐meiotic genome repackaging. Upon its activation at the onset of meiosis, Brdt drives and determines the developmental timing of a testis‐specific gene expression program. In meiotic and post‐meiotic cells, Brdt initiates a genuine histone acetylation‐guided programming of the genome by activating essential genes and repressing a ‘progenitor cells’ gene expression program. At post‐meiotic stages, a global chromatin hyperacetylation gives the signal for Brdt's first bromodomain to direct the genome‐wide replacement of histones by transition proteins. Brdt is therefore a unique and essential regulator of male germ cell differentiation, which, by using various domains in a developmentally controlled manner, first drives a specific spermatogenic gene expression program, and later controls the tight packaging of the male genome.     

The activity of 2'',3''-cyclic nucleotide 3''-phosphohydrolase in the corpus callosum, subcortical white matter, and spinal cord in infants dying from sudden infant death syndrome

Abstract: The activity of 2',3'-cyclic nucleotide 3'-phos-phohydrolase (CNPase) has been determined in corpus callosum, subcortical white matter, and spinal cord of infants whose death was attributed to the sudden infant death syndrome (SIDS), and compared with enzyme activity in other cases in which the cause of death was not associated with respiratory distress. In nearly half the SIDS cases, CNPase activity and oligodendroglial cell numbers were reduced before the onset of myelination, but only in the corpus callosum. In other SIDS cases, enzyme activity and cell numbers were the same as in non-SIDS cases. If the expression of CNPase activity reflects glioblast differentiation to oligodendrocytes with myelinating potential, then this transformation is abnormal in certain SIDS cases, as also evidenced in cases of prolonged neonatal respiratory insufficiency and gives rise to a subsequent deficit of myelin in the corpus callosum.     

Acetylation of histones in nucleosomes

Summary A short review is given on the biochemistry of histone acetylation. Sites of acetylation in nucleosomal histones and enzymes involved in acetylation and deacetylation are discussed. Studies relating to the influence of these modifications on the structure of nucleosomes and chromatin are especially emphasized in this article.     

Changes in histone acetylation during mouse oocyte meiosis

We examined global changes in the acetylation of histones in mouse oocytes during meiosis. Immunocytochemistry with specific antibodies against various acetylated lysine residues on histones H3 and H4 showed that acetylation of all the lysines decreased to undetectable or negligible levels in the oocytes during meiosis, whereas most of these lysines were acetylated during mitosis in preimplantation embryos and somatic cells. When the somatic cell nuclei were transferred into enucleated oocytes, the acetylation of lysines decreased markedly. This type of deacetylation was inhibited by trichostatin A, a specific inhibitor of histone deacetylase (HDAC), thereby indicating that HDAC is able to deacetylate histones during meiosis but not during mitosis. Meiosis-specific deacetylation may be a consequence of the accessibility of HDAC1 to the chromosome, because HDAC1 colocalized with the chromosome during meiosis but not during mitosis. As histone acetylation is thought to play a role in propagating the gene expression pattern to the descendent generation during mitosis, and the gene expression pattern of differentiated oocytes is reprogrammed during meiosis to allow the initiation of a new program by totipotent zygotes of the next generation, our results suggest that the oocyte cytoplasm initializes a program of gene expression by deacetylating histones.     

Development of live-cell imaging probes for monitoring histone modifications

The combination of histone posttranslational modifications occurring in nucleosomal histones determines the epigenetic code. Histone modifications such as acetylation are dynamically controlled in response to a variety of signals during the cell cycle and differentiation, but they are paradoxically maintained through cell division to impart tissue specific gene expression patterns to progeny. The dynamics of histone modifications in living cells are poorly understood, because of the lack of experimental tools to monitor them in a real-time fashion. Recently, FRET-based imaging probes for histone H4 acetylation have been developed, which enabled monitoring of changes in histone acetylation during the cell cycle and drug treatment. Further development of this type of fluorescent probes for other modifications will make it possible to visualize complicated epigenetic regulation in living cells.