Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment

To ensure genome stability, cells have evolved a robust defense mechanism to detect, signal, and repair damaged DNA that is generated by exogenous stressors such as ionizing radiation, endogenous stressors such as free radicals, or normal physiological processes such as DNA replication. Homologous recombination (HR) repair is a critical pathway of repairing DNA double strand breaks, and it plays an essential role in maintaining genomic integrity. Previous studies have shown that BRIT1, also known as MCPH1, is a key regulator of HR repair. Here, we report that chromodomain helicase DNA-binding protein 4 (CHD4) is a novel BRIT1 binding partner that regulates the HR repair process. The BRCA1 C-terminal domains of BRIT1 are required for its interaction with CHD4. Depletion of CHD4 and overexpression of the ATPase-dead form of CHD4 impairs the recruitment of BRIT1 to the DNA damage lesions. As a functional consequence, CHD4 deficiency sensitizes cells to double strand break-inducing agents, reduces the recruitment of HR repair factor BRCA1, and impairs HR repair efficiency. We further demonstrate that CHD4-depleted cells are more sensitive to poly(ADP-ribose) polymerase inhibitor treatment. In response to DNA damage induced by poly(ADP-ribose) polymerase inhibitors, CHD4 deficiency impairs the recruitment of DNA repair proteins BRIT1, BRCA1, and replication protein A at early steps of HR repair. Taken together, our findings identify an important role of CHD4 in controlling HR repair to maintain genome stability and establish the potential therapeutic implications of targeting CHD4 deficiency in tumors.     

Charge-based interaction conserved within histone H3 lysine 4 (H3K4) methyltransferase complexes is needed for protein stability, histone methylation, and gene expression

Histone H3 lysine 4 (H3K4) methyltransferases are conserved from yeast to humans, assemble in multisubunit complexes, and are needed to regulate gene expression. The yeast H3K4 methyltransferase complex, Set1 complex or complex of proteins associated with Set1 (COMPASS), consists of Set1 and conserved Set1-associated proteins: Swd1, Swd2, Swd3, Spp1, Bre2, Sdc1, and Shg1. The removal of the WD40 domain-containing subunits Swd1 and Swd3 leads to a loss of Set1 protein and consequently a complete loss of H3K4 methylation. However, until now, how these WD40 domain-containing proteins interact with Set1 and contribute to the stability of Set1 and H3K4 methylation has not been determined. In this study, we identified small basic and acidic patches that mediate protein interactions between the C terminus of Swd1 and the nSET domain of Set1. Absence of either the basic or acidic patches of Set1 and Swd1, respectively, disrupts the interaction between Set1 and Swd1, diminishes Set1 protein levels, and abolishes H3K4 methylation. Moreover, these basic and acidic patches are also important for cell growth, telomere silencing, and gene expression. We also show that the basic and acidic patches of Set1 and Swd1 are conserved in their human counterparts SET1A/B and RBBP5, respectively, and are needed for the protein interaction between SET1A and RBBP5. Therefore, this charge-based interaction is likely important for maintaining the protein stability of the human SET1A/B methyltransferase complexes so that proper H3K4 methylation, cell growth, and gene expression can also occur in mammals.     

Regulation of glycolytic enzyme phosphoglycerate mutase-1 by Sirt1 protein-mediated deacetylation

Emerging proteomic evidence suggests that acetylation of metabolic enzymes is a prevalent post-translational modification. In a few recent reports, acetylation down-regulated activity of specific enzymes in fatty acid oxidation, urea cycle, electron transport, and anti-oxidant pathways. Here, we reveal that the glycolytic enzyme phosphoglycerate mutase-1 (PGAM1) is negatively regulated by Sirt1, a member of the NAD(+)-dependent protein deacetylases. Acetylated PGAM1 displays enhanced activity, although Sirt1-mediated deacetylation reduces activity. Acetylation sites mapped to the C-terminal "cap," a region previously known to affect catalytic efficiency. Overexpression of a constitutively active variant (acetylated mimic) of PGAM1 stimulated flux through glycolysis. Under glucose restriction, Sirt1 levels dramatically increased, leading to PGAM1 deacetylation and attenuated activity. Previously, Sirt1 has been implicated in the adaptation from glucose to fat burning. This study (i) demonstrates that protein acetylation can stimulate metabolic enzymes, (ii) provides biochemical evidence that glycolysis is modulated by reversible acetylation, and (iii) demonstrates that PGAM1 deacetylation and activity are directly controlled by Sirt1.     

Identification of PR-SET7 and EZH2 selective inhibitors inducing cell death in human leukemia U937 cells

Chemical manipulations undertaken on some bis(bromo- and dibromo-phenol) compounds previously reported by us as wide-spectrum epigenetic inhibitors let us to identify bis (bromo- and dibromo-methoxyphenyl) derivatives highly selective for PR-SET7 and EZH2 (compounds 4, 5, 9, and 10). Western blot analyses were carried out in U937 cells to determine the effects of such compounds on the methyl marks related to the tested enzymes (H3K4me1, H3K9me2, H4H20me1, and H3K27me3). The 1,5-bis(3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one 4 (EC₅₀ vs EZH2 = 74.9 μM), tested in U937 cells at 50 μM, induced massive cell death and 28% of granulocytic differentiation, highlighting the potential use of EZH2 inhibitors in cancer.     

Dynamics of γH2AX formation and elimination in mammalian cells after X-irradiation

Phosphorylation of the replacement histone H2AX occurs in megabase chromatin domains around DNA double-strand breaks (DSBs), and this modification called γH2AX can be used as an effective marker for DSB repair and DNA damage response. In this study, we examined a bystander effect (BE) in locally irradiated embryonic human fibroblasts. Using fluorescence microscopy, we found that BE could be observed 1 h after X-ray irradiation (IR) and was completely eliminated 24 h after IR. Using immunohistochemistry and immunoblotting, we also studied kinetics of γH2AX formation and elimination in Syrian hamster and mouse tissues after whole body IR of animals. Analysis of hamster tissues at different times after IR at the dose 5 Gy showed that γH2AX-associated fluorescence in heart was decreased slowly with about a half level remaining 24 h after IR; at the same time, in brain, the level of γH2AX was about 3 times increased over the control level, and in liver, γH2AX level decreased to control values. We also report that in mouse heart the level of γH2AX measured by immunoblotting is lower than in brain, kidney and liver at different times after IR at the dose 3 Gy. Our observations indicate that there are significant variations in dynamics of γH2AX formation and elimination between non-proliferating mammalian tissues. These variations in γH2AX dynamics in indicated organs partially correlated with the expression level of the major kinase genes involved in H2AX phosphorylation (ATM and DNA-PK).     

八肋游仆虫大核基因组中组蛋白基因 H4A和H4B的克隆及分析

组蛋白作为核小体的基本组分,是染色质的结构和功能必需的。组蛋白的变体和修饰共同参与染色质修饰及基因的表达调控。真核生物细胞中的5种组蛋白在进化中高度保守,然而纤毛虫的组蛋白H4与其他真核生物相比有较大的差异。本实验应用PCR技术从八肋游仆虫(Euplotes octocarinatus)中获得了2种组蛋白H4基因,分别为H4A和H4B,GenBank登录号为:JN715068和JN715069。序列分析表明,H4A基因开放阅读框324 bp,预测编码107个氨基酸,分子量为11.6 ku,等电点为10.99。而H4B基因编码框384 bp,编码127个氨基酸,分子量为14.4 ku,等电点为9.93。Blast结果显示,H4A序列与其他生物中H4的一致性相对较高,达81%～94%,而H4B的一致性为36%～70%。H4A和H4B的一致性仅为44.7%。实时荧光定量PCR表明,H4A的转录本高于H4B。结果提示:在进化过程中八肋游仆虫可能进化出特殊的组蛋白H4基因,不同的组蛋白H4可能发挥不同的功能。     

The antiepileptic drug valproic Acid restores T cell homeostasis and ameliorates pathogenesis of experimental autoimmune encephalomyelitis

Maintaining a constant number and ratio of immune cells is one critical aspect of the tight regulation of immune homeostasis. Breakdown of this balance will lead to autoimmune diseases such as multiple sclerosis (MS). The antiepileptic drug valproic acid (VPA) was reported to regulate the growth, survival, and differentiation of many cells. However, its function in T cell homeostasis and MS treatment remains unknown. In this study, VPA was found to reduce spinal cord inflammation, demyelination, and disease scores in experimental autoimmune encephalomyelitis, a mouse model of MS. Further study indicated that VPA induces apoptosis in activated T cells and maintains the immune homeostasis. This effect was found to be mainly mediated by the caspase-8/caspase-3 pathway. Interestingly, this phenomenon was also confirmed in T cells from normal human subjects and MS patients. Considering the long history of clinical use and our new findings, we believe VPA might be a safe and effective therapy for autoimmune diseases, such as multiple sclerosis.