KyoT3, an isoform of murine FHL1, associates with the transcription factor RBP-J and represses the RBP-J-mediated transactivation

Previously, we have shown that KyoT2, an isoform of the four and a half LIM domain protein 1 (FHL1), modulates Notch signaling via repressing RBP-J-mediated transactivation. In this study, we investigated the effect of another isoform of FHL1, KyoT3, on transactivation of a RBP-J-dependent promoter. We found that KyoT3 was expressed widely in a variety of tissues. By constructing EGFP fusion proteins, we showed that KyoT3 locates preferentially in nucleus. KyoT3 interacted with RBP-J, as shown by co-immunoprecipitation assays. Moreover, we demonstrated by a reporter assay that KyoT3 repressed transactivation of a RBP-J-dependent promoter, which was activated by both the Notch intracellular domain and Epstein-Barr virus nuclear antigen 2, an EB virus-encoded oncoprotein. These results suggest a multi-elemental control of the Notch signaling pathway, which is critical for cell differentiation in development.     

The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate

Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression. The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins. Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate. This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily. However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone. These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.     

组蛋白乙酰转移酶PCAF在原核细胞的表达及其生物学活性检测

p300/CBP相关因子（p300/CBPassociated factor,PCAF）是真核细胞内一种重要的组蛋白乙酰转移酶,它主要通过催化核心组蛋白的乙酰化,促进特定基因的转录,参与细胞内多种生物学过程。国内目前尚没有制备出具有生物学活性的组蛋白乙酰转移酶PCAF的报道。为此, PCAF全长cDNA被克隆入原核表达载体pGEX-5X-1,通过对诱导条件进行优化,实现了PCAF在大肠杆菌BL21（DE3）菌株中的高效可溶性表达并进行了亲和纯化。利用体外乙酰转移酶活性分析实验,检测到所表达的GST-PCAF融合蛋白能够使组蛋白H3发生乙酰化。这种具有生物学活性的PCAF蛋白的成功制备为进一步研究PCAF的转录调控功能以及它与其它蛋白间的相互作用奠定了基础。     

小鼠心脏发育中组蛋白乙酰化酶GCN5和PCAF的时空表达特征

目的研究组蛋白乙酰化酶氨合成通用控制蛋白5（general control nonderepressible-5 GCN5）和p300/CREB结合蛋白相关因子（p300/CREB binding protein-associated factor PCAF）在小鼠心脏发育过程中的时空表达规律。方法选取胎龄7.5-18d、出生1d和3月成年昆明小鼠正常心脏,利用免疫组织化学法和RT-PCR或Western blot技术半定量检测GCN5和PCAF在小鼠心脏发育过程中的时空表达变化。结果1.GCN5在E7.5-E9.5心脏原基中不表达;E10.5后心内膜垫及室间隔膜部和房室瓣相对强表达,心肌组织弱表达。GCN5 mRNA在E10.5-E11.5出现高峰表达,E12.5-E15.5表达水平下降,E16.5至成年鼠期表达极低。2.PCAF在E7.5-E9.5心脏原基中广泛弱表达;E10.5后心肌膜较强表达,心内膜、房室瓣和小梁网较弱表达,室间隔肌部弱表达,室间隔膜部极弱表达;PCAF蛋白在E10.5已有相对高表达,E11.5达到表达高峰,此后表达逐渐降低,在E13.5-E15.5和E16.5-生后1d仔鼠期出现两个表达平台期,成年鼠期心脏中表达最低。结论GCN5和PCAF在发育心脏中存在不同表达分布和变化规律,表明二者均参与了心脏的发生发育过程,且二者可能与心脏的特定结构发育密切相关。     

RUNX3 directly interacts with intracellular domain of Notch1 and suppresses Notch signaling in hepatocellular carcinoma cells

RUNX3 takes a strong suppressive effect in many tumors including hepatocellular carcinoma (HCC). HES-1, a downstream target of Notch signaling, is shown to be decreased in human HCC cell line SMMC7721 with RUNX3 gene transfection. Since Notch signaling is oncogenic in HCC, RUNX3 might exert its inhibitory effect in HCC partly through the suppression on Notch signaling. To investigate the possible mechanism of the down-regulation of HES-1 by RUNX3, we performed Western blot and reporter assay and found that RUNX3 suppressed intracellular domain of Notch1 (ICN1)-mediated transactivation of Notch signaling while it did not alter the expression of ICN1 and recombination signal binding protein-Jκ (RBP-J) in SMMC7721 cells. Besides, confocal microscopy, co-immunoprecipitation and GST pull-down assays showed that RUNX3 could co-localize with ICN1 and RBP-J, forming a complex with these two molecules in nucleus of SMMC7721 cells by its direct interaction with ICN1. Furthermore, RUNX3 was recruited to RBP-J recognition motif of HES-1 promoter, which was identified by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Taken together, these findings indicate that RUNX3 suppresses Notch signaling in HCC SMMC7721 cells by its interaction with ICN1 and thus recruitment to the RBP-J recognition motif of downstream genes of Notch signaling.     

Histone Deacetylase 3 Regulates Cyclin A Stability

PCAF and GCN5 acetylate cyclin A at specific lysine residues targeting it for degradation at mitosis. We report here that histone deacetylase 3 (HDAC3) directly interacts with and deacetylates cyclin A. HDAC3 interacts with a domain included in the first 171 aa of cyclin A, a region involved in the regulation of its stability. In cells, overexpression of HDAC3 reduced cyclin A acetylation whereas the knocking down of HDAC3 increased its acetylation. Moreover, reduction of HDAC3 levels induced a decrease of cyclin A that can be reversed by proteasome inhibitors. These results indicate that HDAC3 is able to regulate cyclin A degradation during mitosis via proteasome. Interestingly, HDAC3 is abruptly degraded at mitosis also via proteasome thus facilitating cyclin A acetylation by PCAF/GCN5, which will target cyclin A for degradation. Because cyclin A is crucial for S phase progression and mitosis entry, the knock down of HDAC3 affects cell cycle progression specifically at both, S phase and G₂/M transition. In summary we propose here that HDAC3 regulates cyclin A stability by counteracting the action of the acetylases PCAF/GCN5.