Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division.     

MAT2A promotes porcine adipogenesis by mediating H3K27me3 at Wnt10b locus and repressing Wnt/β-catenin signaling

Methionine adenosyltransferase (MAT) is a critical biological enzyme and that can catalyze L-met and ATP to form S-adenosylmethionine (SAM), which is acted as a biological methyl donor in transmethylation reactions involving histone methylation. However, the regulatory effect of methionine adenosyltransferase2A (MAT2A) and its associated methyltransferase activity on adipogenesis is still unclear. In this study, we investigate the effect of MAT2A on adipogenesis and its potential mechanism on histone methylation during porcine preadipocyte differentiation. We demonstrated that overexpression of MAT2A promoted lipid accumulation and significantly up-regulated the levels of adipogenic marker genes including PPARγ, SREBP-1c, and aP2. Whereas, knockdown of MAT2A or inhibition MATII enzyme activity inhibited lipid accumulation and down-regulated the expression of the above-mentioned genes. Mechanistic studies revealed that MAT2A interacted with histone-lysine N-methyltransferase Ezh2 and was recruited to Wnt10b promoter to repress its expression by promoting H3K27 methylation. Additionally, MAT2A interacted with MafK protein and was recruited to MARE element at Wnt10b gene. The catalytic activity of MAT2A as well as its interacting factor-MAT2B, was required for Wnt10b repression and supplying SAM for methyltransferases. Moreover, MAT2A suppressed Wnt10b expression and further inhibited Wnt/β-catenin signaling to promote adipogenesis.     

Interactions of HP1 Bound to H3K9me3 Dinucleosome by Molecular Simulations and Biochemical Assays

Heterochromatin protein 1 (HP1), associated with heterochromatin formation, recognizes an epigenetically repressive marker, trimethylated lysine 9 in histone H3 (H3K9me3), and generally contributes to long-term silencing. How HP1 induces heterochromatin is not fully understood. Recent experiments suggested that not one, but two nucleosomes provide a platform for this recognition. Integrating previous and new biochemical assays with computational modeling, we provide near-atomic structural models for HP1 binding to the dinucleosomes. We found that the dimeric HP1α tends to bind two H3K9me3s that are in adjacent nucleosomes, thus bridging two nucleosomes. We identified, to our knowledge, a novel DNA binding motif in the hinge region that is specific to HP1α and is essential for recognizing the H3K9me3 sites of two nucleosomes. An HP1 isoform, HP1γ, does not easily bridge two nucleosomes in extended conformations because of the absence of the above binding motif and its shorter hinge region. We propose a molecular mechanism for chromatin structural changes caused by HP1.     

Msx1重新分布Ezh2和H3K27me3到细胞核核周具有细胞类型特异性

细胞核内部的空间排布并不是随机的,而是高度动态且具细胞特异性的。在发育过程中,序列特异性的转录调控因子和表观遗传修饰因子的协同作用及其核定位与基因的表达调控密切相关。我们前期研究发现,在成肌细胞中,同源异型框蛋白Msx1通过重新分布Ezh2复合物和抑制标记H3K27me3到细胞核核周来调控靶标基因的表达,从而抑制成肌细胞的分化。这种机制是成肌细胞所特有的,还是Msx1在抑制非成肌细胞分化时也会重新分布Ezh2复合物和转录抑制性标记H3K27me3到细胞核核周,目前还不清楚。在发育过程中,Msx1可以抑制乳腺上皮细胞HC11和成骨细胞BMP2T3的分化,因此我们选取了这两种非成肌细胞做进一步探究。我们发现,在这两种非肌肉细胞中,Msx1虽也富集在细胞核核周,却并不能重新分布Ezh2和转录抑制性标记H3K27me3到细胞核核周。我们的研究表明Msx1重新分布Ezh2和抑制性标记H3K27me3到细胞核核周具有细胞类型特异性。提示我们,Msx1可能是通过不同的分子机制来调控不同类型细胞的分化。     

肿瘤干细胞中受H3K9me2调控的干性基因的筛选

为了分析比较甲基转移酶G9a和组蛋白H3K9me2修饰在胶质瘤干细胞与非干细胞中存在的差异,筛选出维持胶质瘤干细胞干性的相关基因。通过G9a抑制剂促进U87细胞成球和过表达G9a促进U87细胞分化的方法,培养了成球的胶质瘤干细胞和贴壁的非干细胞,这两种细胞的CD133表达差异明显。再利用H3K9me2抗体通过Ch IP-seq技术比较H3K9me2修饰在干细胞组与非干细胞组中的差异,在存在差异的基因中,对TSS±2 000 bp范围内的基因进行了GO分析,并随机选出10个转录因子进行QPCR验证,结果与Ch IP-seq实验基本一致。     

The PR-Set7 binding domain of Riz1 is required for the H4K20me1-H3K9me1 trans-tail ‘histone code’ and Riz1 tumor suppressor function

PR-Set7/Set8/KMT5a is the sole histone H4 lysine 20 monomethyltransferase (H4K20me1) in metazoans and is essential for proper cell division and genomic stability. We unexpectedly discovered that normal cellular levels of monomethylated histone H3 lysine 9 (H3K9me1) were also dependent on PR-Set7, but independent of its catalytic activity. This observation suggested that PR-Set7 interacts with an H3K9 monomethyltransferase to establish the previously reported H4K20me1-H3K9me1 trans-tail ‘histone code’. Here we show that PR-Set7 specifically and directly binds the C-terminus of the Riz1/PRDM2/KMT8 tumor suppressor and demonstrate that the N-terminal PR/SET domain of Riz1 preferentially monomethylates H3K9. The PR-Set7 binding domain was required for Riz1 nuclear localization and maintenance of the H4K20me1-H3K9me1 trans-tail ‘histone code’. Although Riz1 can function as a repressor, Riz1/H3K9me1 was dispensable for the repression of genes regulated by PR-Set7/H4K20me1. Frameshift mutations resulting in a truncated Riz1 incapable of binding PR-Set7 occur frequently in various aggressive cancers. In these cancer cells, expression of wild-type Riz1 restored tumor suppression by decreasing proliferation and increasing apoptosis. These phenotypes were not observed in cells expressing either the Riz1 PR/SET domain or PR-Set7 binding domain indicating that Riz1 methyltransferase activity and PR-Set7 binding domain are both essential for Riz1 tumor suppressor function.     

Clinical differences between patients with MODY-3, MODY-2 and type 2 diabetes mellitus with I27L polymorphism in the HNF1α gene

ObjectiveThe aim of our study was to describe and evaluate the clinical and metabolic characteristics of patients with MODY-3, MODY-2 or type 2 diabetes who presented I27L polymorphism in the HNF1α gene.MethodsThe study included 31 previously diagnosed subjects under follow-up for MODY-3 (10 subjects from 5 families), MODY-2 (15 subjects from 9 families), or type 2 diabetes (6 subjects) with I27L polymorphism in the HNF1α gene. The demographic, clinical, metabolic, and genetic characteristics of all patients were analyzed.ResultsNo differences were observed in distribution according to sex, age of onset, or form of diagnosis. All patients with MODY-2 or MODY-3 had a family history of diabetes. In contrast, 33.3% of patients with type 2 diabetes mellitus and I27L polymorphism in the HNF1α gene had no family history of diabetes (p < 0.05). No differences were observed in body mass index, prevalence of hypertension, or microvascular or macrovascular complications. Drug therapy was required by 100% of MODY-3 patients, but not required by 100% of MODY-2 patients or 16.7% of patients with type 2 diabetes mellitus and I27L polymorphism in the HNF1α gene (p < 0.05).ConclusionsOccasional difficulties may be encountered when classifying patients with MODY-2, MODY-3 or type 2 diabetes of atypical characteristics, in this case patients who present I27L polymorphism in the HNF1α gene.     

The Weak Complex between RhoGAP Protein ARHGAP22 and Signal Regulatory Protein 14-3-3 Has 1∶2 Stoichiometry and a Single Peptide Binding Mode

ARHGAP22 is a RhoGAP protein comprising an N-terminal PH domain, a RhoGAP domain and a C-terminal coiled-coil domain. It has recently been identified as an Akt substrate that binds 14-3-3 proteins in response to treatment with growth factors involved in cell migration. We used a range of biophysical techniques to investigate the weak interaction between 14-3-3 and a truncated form of ARHGAP22 lacking the coiled-coil domain. This weak interaction could be stabilized by chemical cross-linking which we used to show that: a monomer of ARHGAP22 binds a dimer of 14-3-3; the ARHGAP22 PH domain is required for the 14-3-3 interaction; the RhoGAP domain is unlikely to participate in the interaction; Ser16 is the more important of two predicted 14-3-3 binding sites; and, phosphorylation of Ser16 may not be necessary for 14-3-3 interaction under the conditions we used. Small angle X-ray scattering and cross-link information were used to generate solution structures of the isolated proteins and of the cross-linked ARHGAP22:14-3-3 complex, showing that no major rearrangement occurs in either protein upon binding, and supporting a role for the PH domain and N-terminal peptide of ARHGAP22 in the 14-3-3 interaction. Small-angle X-ray scattering measurements of mixtures of ARHGAP22 and 14-3-3 were used to establish that the affinity of the interaction is ～30 μM.