新功能、新界面、新体验,扫描即可下载生物谷APP!
首页 » BIOON报道 » NSMB:特殊蛋白复合物可在小鼠胚胎干细胞中实现特殊基因的激活组蛋白标记

NSMB:特殊蛋白复合物可在小鼠胚胎干细胞中实现特殊基因的激活组蛋白标记

来源:生物谷 2013-08-12 23:01

2013年8月12日 讯 /生物谷BIOON/ --10年前我们觉得基因表达看似很简单,无非就是被开启或者被关闭,2006年一项突破性研究揭示了小鼠胚胎干细胞中的调节基因,具有很多和活性基因以及被抑制基因相关的特性标记;而这些所谓的活性基因及被抑制的基因被认为在机体发育和分化过程中具有重要作用。

近日,刊登在国际杂志Nature Structural & Molecular Biology上的一篇研究论文中,来自斯托瓦斯医学研究所的研究人员通过研究鉴别出了一种蛋白复合物,其可以在小鼠胚胎干细胞中的特殊基因中实现激活组蛋白标记的功能,但是如果缺乏了该蛋白复合物却对发育中基因的激活并无多大影响,这就暗示研究者需要进行更多关于胚胎甚至是癌细胞的组蛋白修饰模式等相关研究。

(a) The COMPASS family of H3K4 methylases in Drosophila (three members; left) and mammals (six members; right). Subunits common to all COMPASS family members in eukaryotes fromyeast to human are shown in green; complex-specific subunits are shown in blue, orange and purple. Magenta indicates Host Cell Factor (HCF1), a substochiometric component found in some branches of the COMPASS family. (b) Mll2 and Mll3 mRNA levels after RNAi-mediated knockdown in mouse embryonic stem cells with shRNAs targeting GFP (shGFP), Mll3 (shMll3) or Mll2 (shMll2). Expression determined by quantitative reverse-transcription PCR is shown relative to β-actin (Actb). Bars indicate range of values for two technical replicates, representative of three biological-replicate experiments. (c) ChIP-seq track file examples of H3K4me3 at mouse Hox gene clusters. Red and black bars above the tracks indicate bivalent and nonbivalent regions, respectively. C.p.m., counts per million mapped reads. H3K27me3 data from Mikkelsen et al.4 are shown for comparison (purple). (d) ChIP-seq track examples of bivalent and nonbivalent chromatin in control and Mll2-shRNA–treated cells. Bivalently marked genes such as Prr18, Brachyury (T ), Vgll4 and Syn2 are shown with red bars above the tracks as in (c).

2001年研究者首次对一种名为COMPASS的酵母蛋白特性进行了描述,其可以以特殊方式对组蛋白进行甲基化修饰;随后研究者又在哺乳动物体内发现了6种类似COMPASS的蛋白:两种SET蛋白和四种MLL蛋白。

研究人员表示,名为PRC2的酶复合体可以实现H3K27me3的抑制性标记,H3K27me3是组蛋白H3残基的一种甲基化修饰。为了鉴别出哪种COMPASS家族成员参与了H3K27me3的抑制过程,研究者以Mll2作为效应因子,Mll2缺陷的细胞可以表现出H3K27me3缺失的情况,并不是所有的基因,只是其在启动子位置的时候才可以调节基因的表达,比如Hox基因。

当研究者研究Mll2缺失的小鼠胚胎干细胞的行为时,发现细胞可以继续展现其自我更新的特性,这就表明允许干细胞多样化的基因并不会因Mll2的缺失而受到干扰。而当培养基中含有诱导成熟的因子时,Mll2缺失的小鼠胚胎干细胞在基因表达上并没有任何异常,实际上展现二价组蛋白标记的Hox基因的表达在Mll2缺陷细胞中的表达就跟在非突变细胞中表达一样。研究者表示,接收分化信号的Mll2缺陷小鼠胚胎干细胞,尽管其缺失了H3K4me3的标记区域,但其可以一直激活需要成熟的基因的表达,这就为理解多能干细胞的发育提供了一定的基础。

(a) H3K4me3 occupancy change in Mll2- and Mll3-depleted mouse embryonic stem cells for all H3K4me3-enriched genes. Left, ChIP-seq enrichment profiles for ±5 kb around the TSS of all H3K4me3-enriched genes. Bivalent genes are shown as a separate group from the H3K4me3-only modified genes. Right, H3K4me3 occupancy log2 fold change after depletion of Mll2 (shMll2/shGFP) or Mll3 (shMll3/shGFP), measured ±5 kb around the TSS (indicated by arrowheads). (b) Percentage of bivalent and nonbivalent genes with H3K4me3 occupancy loss. The percentages are shown at four levels: total H3K4me3-enriched genes, genes with >50% decrease of H3K4me3, genes with >75% decrease and genes with >87.5% decrease. Numbers shown are the total number of genes for each level. H3K27me3 data in a and b are from Wamstad et al.20. (c) Average gene occupancy plots of H3K4me3 in shGFP (blue), shMll2 (red) and shMll3 (green) knockdown embryonic stem cells. Top and bottom panels show nonbivalent and bivalent genes, respectively. Plots are averaged from both H3K4me3 ChIP-seq biological replicates. (d) Gene ontology (GO) analysis of genes with H3K4me3 loss after Mll2 knockdown. Benjamini-corrected P values as reported by DAVID21, 22 are shown.

这项研究也重点对肿瘤的发生进行了研究,由于引发肿瘤的癌症干细胞也会在某些基因上表现出二价的组蛋白标记特性,而且癌症干细胞对于化疗表现出一定的耐受性,这就使得成功去除癌干细胞变得非常困难,研究者的研究工作为揭示癌症干细胞形成肿瘤的分子机制以及为开发特定疗法提供了希望和思路,相关研究成果由美国国家癌症研究所等机构提供资助。(生物谷Bioon.com)

Figure 3: Loss of bivalency has little effect on transcriptional induction kinetics in response to retinoic acid.

(a) Expression analysis of mouse V6.5 embryonic stem cells infected with GFP-shRNA (shGFP) or Mll2-shRNA (shMll2) lentivirus and induced to differentiate with 2 μM retinoic acid for 6 h (RA 6) or 12 h (RA 12). Heat maps of expression and H3K4me3 occupancy are shown. Genes with expression increased by 6 h and 12 h of RA treatment are sorted by the wild-type occupancy of H3K4me3 and separated as bivalent or not bivalent. Left, expression heat map in control and Mll2-shRNA–treated cells. r.p.k.m., reads per kilobase per million mapped reads. Middle, log2 fold changes for expression after Mll2 knockdown. Induction defects would appear as an increase in green in the 6 and 12 h time points. Right, log2 fold change of H3K4me3 occupancy. (b) Scatter plots of log2 fold changes in expression after RA induction at 6 h (top) or 12 h (bottom). Each dot represents a gene that was induced by 6 h. Correlation coefficients are calculated in the log2 scale. (c) Gene expression track examples before and after RA induction. (d) Induction kinetics at one and three hours of RA treatment. Bars represent the range of values for two technical replicates, representative of three biological replicate experiments. A second Mll2-shRNA with an alternate sequence is denoted shMll2_2.

The Mll2 branch of the COMPASS family regulates bivalent promoters in mouse embryonic stem cells

Deqing Hu, Alexander S Garruss, Xin Gao, Marc A Morgan, Malcolm Cook, Edwin R Smith & Ali Shilatifard

Promoters of many developmentally regulated genes, in the embryonic stem cell state, have a bivalent mark of H3K27me3 and H3K4me3, proposed to confer precise temporal activation upon differentiation. Although Polycomb repressive complex 2 is known to implement H3K27 trimethylation, the COMPASS family member responsible for H3K4me3 at bivalently marked promoters was previously unknown. Here, we identify Mll2 (KMT2b) as the enzyme catalyzing H3K4 trimethylation at bivalentlymarked promoters in embryonic stem cells. Although H3K4me3 at bivalent genes is proposed to prime future activation, we detected no substantial defect in rapid transcriptional induction after retinoic acid treatment in Mll2-depleted cells. Our identification of the Mll2 complex as the COMPASS family member responsible for H3K4me3 marking bivalent promoters provides an opportunity to reevaluate and experimentally test models for the function of bivalency in the embryonic stem cell state and in differentiation.

温馨提示:87%用户都在生物谷APP上阅读,扫描立刻下载! 天天精彩!


相关标签

最新会议 培训班 期刊库