Roles of histone H3K9 methyltransferases during Drosophila spermatogenesis

Epigenetic regulation of gene expression by covalent modification of histones is important for germ line cell development. In mammals, histone H3 lysine 9 (H3K9)-specific histone methyltransferases (HMTases), such as G9a, SETDB1, and SUV39H, play critical roles, but the contribution of H3K9-specific HMTases in Drosophila remains to be clarified, especially in male sperm. Here, we performed immunocytochemical analyses with a specific antibody to dG9a, Drosophila G9a ortholog, and demonstrated localization in the cytoplasm from the growth to elongation stages of spermatogenesis. In the subsequent early canoe stage, strong dG9a signals were detected exclusively in nuclei, suggesting a regulatory role. However, mono-, di-, and trimethylated H3K9 signals were not extensively decreased in a homozygous dG9a null mutant throughout these stages. In contrast, mono- and trimethylated H3K9 signals were extensively decreased in a heterozygous DmSetdb1 mutant during spermatogenesis, and similar reduction in monomethylated H3K9 signals was observed in a homozygous Su(var)3–9 mutant. Therefore, DmSETDB1 is likely to be mainly responsible for mono- and trimethylation of H3K9 and SU(VAR)3–9 for monomethylation of H3K9 during spermatogenesis. However, the reduced methylation of H3K9 in premeiotic spermatocytes did not influence X–Y chromosome disjunction in male meiosis, suggesting that it may not be critical for spermatogenesis in Drosophila.     

Effect of pitavastatin against expression of S100β protein in the gerbil hippocampus after transient cerebral ischaemia

Aim and methods: We investigated the immunohistochemical alterations of S100β‐, S100‐, glial fibrillary acidic protein (GFAP)‐ and isolectin B₄‐positive cells in the hippocampus after 5 min of transient cerebral ischaemia in gerbils. We also examined the effect of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase inhibitor pitavastatin against neuronal damage in the hippocampal CA1 sector after ischaemia. Results: Severe neuronal damage was observed in the hippocampal CA1 pyramidal neurons from 5 days after ischaemia. GFAP‐positive cells increased gradually in the hippocampus from 5 days after ischaemia. Five and 14 days after ischaemia, significant increases in the number of GFAP‐positive cells and isolectin B₄‐positive cells were observed in the hippocampal CA1 and CA3 sector. Mild increases in the number of S100 and S100β‐positive cells were observed in the hippocampal CA1 sector from 1 h to 2 days after ischaemia. Thereafter, S100β‐positive cells increased in the hippocampal CA1 sector after ischaemia, whereas S100‐positive cells decreased in this region. In our double‐labelled immunostainings, S100 and S100β immunoreactivity was found in GFAP‐positive astrocytes, but not in isolectin B₄‐positive microglia. Pharmacological study showed that HMG‐CoA reductase inhibitor, pitavastatin, can protect against the hippocampal CA1 neuronal damage after ischaemia. This drug also prevented increases in the number of GFAP‐positive astrocytes, isolectin B₄‐positive microglia, S100‐positive astrocytes and S100β‐positive astrocytes after ischaemia. Conclusion: The present study demonstrates that pitavastatin can decrease the neuronal damage of hippocampal CA1 sector after ischaemia. This beneficial effect may be, at least in part, mediated by inhibiting the expression of astrocytic activation in the hippocampus at the acute phase after ischaemia. Thus the modulation of astrocytic activation may offer a novel therapeutic strategy of ischaemic brain damage.     

Ubiquitination-dependent and -independent repression of target genes by SETDB1 reveal a context-dependent role for its methyltransferase activity during adipogenesis

The lysine methyltransferase SETDB1, an enzyme responsible for methylation of histone H3 at lysine 9, plays a key role in H3K9 tri-methylation-dependent silencing of endogenous retroviruses and developmental genes. Recent studies have shown that ubiquitination of human SETDB1 complements its catalytic activity and the silencing of endogenous retroviruses in human embryonic stem cells. However, it is not known whether SETDB1 ubiquitination is essential for its other major role in epigenetic silencing of developmental gene programs. We previously showed that SETDB1 contributes to the formation of H3K4/H3K9me3 bivalent chromatin domains that keep adipogenic Cebpa and Pparg genes in a poised state for activation and restricts the differentiation potential of pre-adipocytes. Here, we show that ubiquitin-resistant K885A mutant of SETDB1 represses adipogenic genes and inhibits pre-adipocyte differentiation similar to wild-type SETDB1. We show this was due to a compensation mechanism for H3K9me3 chromatin modifications on the Cebpa locus by other H3K9 methyltransferases Suv39H1 and Suv39H2. In contrast, the K885A mutant did not repress other SETDB1 target genes such as Tril and Gas6 suggesting SETDB1 represses its target genes by two mechanisms; one that requires its ubiquitination and another that still requires SETDB1 but not its enzyme activity.     

C3H1型锌指结合蛋白36介导的星形胶质细胞活化在肌萎缩侧索硬化症运动神经元退变中的作用

目的 探讨C3H1型锌指结合蛋白36（ZFP36L1）介导的星形胶质细胞活化在肌萎缩侧索硬化症（ALS）运动神经元退变中的作用。 方法 以铜锌超氧化物歧化酶1（SOD1)-G93A转基因小鼠作为动物模型,同窝野生型小鼠作为对照（突变型及野生型小鼠各时间点分别取13只小鼠）;Real-time PCR、Western blotting检测小鼠发病早期、中期及晚期脊髓内ZFP36L1 mRNA及蛋白变化,免疫荧光染色检测ZFP36L1在脊髓内的表达及分布;出生后 1～2 d新生小鼠15只,建立SOD1-G93A突变型原代星形胶质细胞模型,Real-time PCR、Western blotting检测星形胶质细胞内ZFP36L1 mRNA及蛋白水平的变化,si-ZFP36L1转染SOD1-G93A突变型原代星形胶质细胞,Western blotting检测转染效率,Western blotting及ELISA检测转染后星形胶质细胞分泌炎性因子肿瘤坏死因子α（TNF-α）、白细胞介素18（IL-18）变化;沉默SOD1-G93A突变型原代星形胶质细胞内ZFP36L1后,与SOD1-G93A突变型NSC34细胞共培养,通过5’-乙炔基-2’-脱氧尿苷（EdU）实验和观察增殖细胞核抗原（PCNA）的水平明确ZFP36L1对NSC34细胞增殖的影响,通过TUNEL实验及观察剪切Caspase-3（cleaved-Caspase-3）的水平明确ZFP36L1对NSC34细胞凋亡的影响,以转染空白小干扰RNA（siRNA）作为对照组。 结果 与野生型小鼠相比,ZFP36L1在SOD1-G93A转基因小鼠脊髓组织内mRNA及蛋白水平均下调,在野生型小鼠脊髓组织内,ZFP36L1主要与β-微管蛋白Ⅲ（β-tubulin Ⅲ）阳性的神经元共表达,而SOD1-G93A突变型小鼠的脊髓组织内,ZFP36L1在神经元内表达减弱,与胶质纤维酸性蛋白（GFAP）标记的星形胶质细胞共表达明显增加;SOD1-G93A突变型原代星形胶质细胞内ZFP36L1表达增加,si-ZFP36L1能明显降低SOD1-G93A突变型原代星形胶质细胞内ZFP36L1水平;沉默ZFP36L1后星形胶质细胞分泌炎性因子 TNF-α、 IL-18明显降低。此外,沉默ZFP36L1后,SOD1-G93A突变型原代星形胶质细胞能显著增强NSC34细胞增殖活性,抑制NSC34细胞凋亡。 结论 在ALS发病过程中,星形胶质细胞被激活,ZFP36L1通过星形胶质细胞分泌的炎性因子促进了ALS运动神经元的退变。     

Mouse ZAR1‐like (XM_359149) colocalizes with mRNA processing components and its dominant‐negative mutant caused two‐cell‐stage embryonic arrest

Maternal effect genes and encoding proteins are necessary for nuclear reprogramming and zygotic genome activation. However, the mechanisms that mediate these functions are largely unknown. Here we identified XM_359149, a Zar1‐like gene that is predominantly expressed in oocytes and zygotes, which we designated Zar1‐like (Zar1l). ZAR1L‐EGFP formed multiple cytoplasmic foci in late two‐cell‐stage embryos. Expression of the ZAR1L C‐terminus induced two‐cell‐stage embryonic arrest, accompanied with abnormal methylation of histone H3K4me2/3 and H3K9me2/3, and marked down‐regulation of a group of chromatin modification factors including Dppa2, Dppa4, and Piwil2. When ectopically expressed in somatic cells, ZAR1L colocalized with P‐body components including EIF2C1(AGO1), EIF2C2(AGO2), DDX6 and LSM14A, and germline‐specific chromatoid body components including PIWIL1, PIWIL2, and LIN28. ZAR1L colocalized with ZAR1 and interacted with human LIN28. Our data suggest that ZAR1L and ZAR1 may comprise a novel family of processing‐body/chromatoid‐body components that potentially function as RNA regulators in early embryos. Developmental Dynamics 239:407–424, 2010. © 2009 Wiley‐Liss, Inc.     

H3K27me3 demethylase UTX regulates the differentiation of a subset of bipolar cells in the mouse retina

Di‐ and trimethylation of lysine 27 on histone 3 (H3K27me2/3) is a critical gene repression mechanism. We previously showed that down‐regulation of the H3K27 demethylase, Jumonji domain‐containing protein 3 (JMJD3), resulted in a reduced number of protein kinase C (PKC)α‐positive rod ON‐bipolar cells. In this work, we focused on the role of another H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX), in retinal development. UTX was expressed in the retinal progenitor cells of the embryonic mouse retina and was observed in the inner nuclear layer during late retinal development and in the mature retina. The short hairpin RNA‐mediated knockdown of Utx in a mouse retinal explant led to a reduced number of PKCα‐positive rod ON‐bipolar cells. However, other retinal subtypes were unaffected by this knockdown. Using a retina‐specific knockout of Utx in mice, the in vivo effects of UTX down‐regulation were examined. Again, the number of PKCα‐positive rod ON‐bipolar cells was reduced, and no other apparent phenotypes, including retinal progenitor proliferation, apoptosis or differentiation, were observed. Finally, we examined retina‐specific Utx and Jmjd3 double‐knockout mice and found that although the number of rod ON‐bipolar cells was reduced, no additional effects from the loss of Utx and Jmjd3 were observed. Taken together, our data show that UTX contributes to retinal differentiation in a lineage‐specific manner.     

The Polycomb Group Protein SUZ12 regulates histone H3 lysine 9 methylation and HP1α distribution

Regulation of histone methylation is critical for proper gene expression and chromosome function. Suppressor of Zeste 12 (SUZ12) is a requisite member of the EED/EZH2 histone methyltransferase complexes, and is required for full activity of these complexes in vitro. In mammals and flies, SUZ12/Su(z)12 is necessary for trimethylation of histone H3 on lysine 27 (H3K27me3) on facultative heterochromatin. However, Su(z)12 is unique among Polycomb Group Proteins in that Su(z)12 mutant flies exhibit gross defects in position effect variegation, suggesting a role for Su(z)12 in constitutive heterochromatin formation. We investigated the role of Suz12 in constitutive heterochromatin and discovered that Suz12 is required for histone H3 lysine 9 tri-methylation (H3K9me3) in differentiated but not undifferentiated mouse embryonic stem cells. Knockdown of SUZ12 in human cells caused a reduction in H3K27me3 and H3K9me3, and altered the distribution of HP1α. In contrast, EZH2 knockdown caused loss of H3K27me3 but not H3K9me3, indicating that SUZ12 regulates H3-K9 methylation in an EZH2-independent fashion. This work uncovers a role for SUZ12 in H3-K9 methylation.     

Hyperpolarized 13C magnetic resonance spectroscopy detects toxin‐induced neuroinflammation in mice

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) ¹³C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1–¹³C] pyruvate has shown potential in assessing neuroinflammation‐linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS‐induced neuroinflammatory changes using HP [1–¹³C] pyruvate and HP ¹³C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1–¹³C] pyruvate and HP ¹³C urea was performed at baseline (day 0) and at days 3 and 7 post‐intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1–¹³C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS‐treated mouse brain, but not in either the contralateral side or saline‐injected animals. HP ¹³C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS‐injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post‐LPS injection. In conclusion, we can detect LPS‐induced changes in the mouse brain using HP ¹³C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP ¹³C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.     

Prenatal dexamethasone exposure in rats results in long‐term epigenetic histone modifications and tumour necrosis factor‐α production decrease

Glucocorticoid (GC) is often given when preterm delivery is expected. This treatment is successful in stimulating the development of the fetal lung. However, reports and related research regarding the prolonged effects of prenatal GC on the development of immunity are very limited. Some data, derived from infants whose mothers were given immunosuppressants during pregnancy for the treatment of autoimmune disorders, suggest that prenatal exposure to GC may have only a limited effect on the development of the immune system. What is unknown is whether the immune modulation effects of prenatal GC might appear at a later childhood stage and beyond. Here we evaluated the immune programming influenced by prenatal GC. Pregnant Sprague‐Dawley rats received dexamethasone (DEX; 0·1 mg/kg/day) or saline at gestational days 14–20. Male offspring were killed at day 7 or day 120 after birth. Spleens were collected for immune study. Of the inflammation mediators, matrix metalloproteinase‐9, tumour necrosis factor‐α (TNF‐α) and granulocyte–macrophage colony‐stimulating factor mRNAs decreased in the prenatal DEX group at an early stage after birth. Upon concanavalin A stimulation, prenatal DEX treatment reduced TNF‐α production, but not interferon‐γ production, by splenocytes at day 120 after birth compared with the vehicle group. Decreased levels of active chromatin signs (acetylation of histone H3 lysines, H3K4me1/3, and H3K36me3) in TNF‐α promoter were compatible with the expressions of TNF‐α. Our results suggest that prenatal DEX has a profound and lasting impact on the developing immune system even to the adult stage. Epigenetic histone modifications regulate TNF‐α expression following prenatal DEX in rats.     

In vivo tracking of histone H3 lysine 9 acetylation in Xenopus laevis during tail regeneration

Xenopus laevis tadpoles can completely regenerate their appendages, such as tail and limbs, and therefore provide a unique model to decipher the molecular mechanisms of organ regeneration in vertebrates. Epigenetic modifications are likely to be involved in this remarkable regeneration capacity, but they remain largely unknown. To examine the involvement of histone modification during organ regeneration, we generated transgenic X. laevis ubiquitously expressing a fluorescent modification‐specific intracellular antibody (Mintbody) that is able to track histone H3 lysine 9 acetylation (H3K9ac) in vivo through nuclear enhanced green fluorescent protein (EGFP) fluorescence. In embryos ubiquitously expressing H3K9ac‐Mintbody, robust fluorescence was observed in the nuclei of somites. Interestingly, H3K9ac‐Mintbody signals predominantly accumulated in nuclei of regenerating notochord at 24 h postamputation following activation of reactive oxygen species (ROS). Moreover, apocynin (APO), an inhibitor of ROS production, attenuated H3K9ac‐Mintbody signals in regenerating notochord. Our results suggest that ROS production is involved in acetylation of H3K9 in regenerating notochord at the onset of tail regeneration. We also show this transgenic Xenopus to be a useful tool to investigate epigenetic modification, not only in organogenesis but also in organ regeneration.     

Epigenome and chromatin structure in human embryonic stem cells undergoing differentiation

Epigenetic histone (H3) modification patterns and the nuclear radial arrangement of select genetic elements were compared in human embryonic stem cells (hESCs) before and after differentiation. H3K9 acetylation, H3K9 trimethylation, and H3K79 monomethylation were reduced at the nuclear periphery of differentiated hESCs. Differentiation coincided with centromere redistribution, as evidenced by perinucleolar accumulation of the centromeric markers CENP‐A and H3K9me3, central repositioning of centromeres 1, 5, 19, and rearrangement of other centromeres at the nuclear periphery. The radial positions of PML, RARα genes, and human chromosomes 10, 12, 15, 17, and 19 remained relatively stable as hESCs differentiated. However, the female inactive H3K27‐trimethylated X chromosome occupied a more peripheral nuclear position in differentiated cells. Thus, pluripotent and differentiated hESCs have distinct nuclear patterns of heterochromatic structures (centromeres and inactive X chromosome) and epigenetic marks (H3K9me3, and H3K27me3), while relatively conserved gene density‐related radial chromatin distributions are already largely established in undifferentiated hES cells. Developmental Dynamics 237:3690–3702, 2008. © 2008 Wiley‐Liss, Inc.     

Identification of prognostic markers in diffuse midline gliomas H3K27M‐mutant

Pediatric diffuse midline gliomas are devastating diseases. Among them, diffuse midline gliomas H3K27M‐mutant are associated with worse prognosis. However, recent studies have highlighted significant differences in clinical behavior and biological alterations within this specific subgroup. In this context, simple markers are needed to refine the prognosis of diffuse midline gliomas H3K27M‐mutant and guide the clinical management of patients. The aims of this study were (i) to describe the molecular, immunohistochemical and, especially, chromosomal features of a cohort of diffuse midline gliomas and (ii) to focus on H3K27M‐mutant tumors to identify new prognostic markers. Patients were retrospectively selected from 2001 to 2017. Tumor samples were analyzed by immunohistochemistry (including H3K27me3, EGFR, c‐MET and p53), next‐generation sequencing and comparative genomic hybridization array. Forty‐nine patients were included in the study. The median age at diagnosis was 9 years, and the median overall survival (OS) was 9.4 months. H3F3A or HIST1H3B mutations were identified in 80% of the samples. Within the H3K27M‐mutant tumors, PDGFRA amplification, loss of 17p and a complex chromosomal profile were significantly associated with worse survival. Three prognostic markers were identified in diffuse midline gliomas H3K27M‐mutant: PDGFRA amplification, loss of 17p and a complex chromosomal profile. These markers are easy to detect in daily practice and should be considered to refine the prognosis of this entity.