Hrp2-Iws1/Spt6-Setd2蛋白复合体调控转录延伸及其偶联的RNA加工的表观遗传学机制

Transcription elongation, together with co-transcriptional RNA processing and histone modification, is becoming increasingly recognized as a critical step of gene expression regulation. Although traditional biochemical studies have identified a number of elongation factors in vitro, the temporal and spatial specificity of these proteins in the regulation of target gene expression in vivo remain largely unknown. Iws1/Spt6 transcription elongation complex associates with the Ser2-phosphorylated C-terminal domain (CTD) of RNA polymerase II (Pol II), stimulates transcription elongation, facilitates pre-mRNA alternative splicing, promotes mRNA export, recruits Setd2 histone methyltransferase and is required for the H3K36me3 across the gene transcribed region. To better understand the molecular mechanisms of their action, we used multiple dimension protein identification technology (MudPIT) to identify Iws1 interacting proteins. We found HDGF related protein 2 (Hrp2) as a new Iws1 interacting protein. Hrp2 recognizes and binds to H3K36me3 through its N-terminal PWWP domain. Thus, Iws1/Spt6 may function as a bridge to connect the “reader” and “writer” of H3K36me3 and coordinately regulate H3K36me3 distribution. Hrp2 has been reported as a cellular co-factor of HIV-1 integrase and may play roles in HIV-1 integration site selection. To further explore the biological function of Hrp2 protein, we examined the Hrp2 protein expression in different mouse tissues. Hrp2 is highly expressed in skeletal muscle and may undergo posttranslational modification. Hrp2 has also been reported to be highly phosphorylated when the C2C12 myoblast cells differentiate into myotube cells. Furthermore, we found that knockdown of Hrp2 or Iws1 significantly suppresses myogenin protein expression and myogenesis. Collectively, these data suggest that Hrp2 and Iws1 may play tissue specific roles in skeletal myogenesis. The central objective of this project is to elucidate the molecular mechanisms by which Hrp2-Iws1/Spt6-Setd2 protein complex regulates myogenesis, activates tissue specific target genes and coordinates transcription elongation, RNA processing and histone modification. To accomplish this goal, we will address the following key questions regarding the Hrp2-Iws1/spt6-Setd2 multi-functional protein complex: (1) How does it assemble in vitro and in vivo? (2) How does it bind to and regulate H3K36me3 level? (3) What role does it play in myogenesis? (4) What are the direct target genes (5) How does it regulate the target gene expression? These studies will not only have a fundamental impact on our understanding on the regulation of myogenesis and target gene expression by the Hrp2-Iws1/spt6-Setd2 complex, but also will be instrumental for an understanding of the mechanisms and functional interactions behind transcription elongation, RNA processing and histone modification.

转录延伸及其偶联的RNA加工和组蛋白修饰在基因表达过程中发挥重要的调控作用。Iws1/Spt6复合体调节转录延伸速率和RNA可变剪接，并招募组蛋白甲基化酶Setd2调节组蛋白H3K36的三甲基化。通过蛋白质组学研究，我们发现H3K36me3识别蛋白Hrp2是新的Iws1相互作用蛋白。Iws1/Spt6可能发挥桥梁作用，偶联H3K36me3的甲基化酶和识别蛋白，精确调节H3K36me3水平。Hrp2特异性地高表达于成年小鼠的肌肉组织，并在成肌细胞分化过程中被磷酸化。基因沉默Iws1或Hrp2抑制成肌细胞分化，提示其调节肌肉发生。拟进行的工作将探讨Hrp2-Iws1/Spt6-Setd2蛋白复合体的组装机制、对组蛋白H3K36me3的识别和修饰、在肌肉发生过程中的作用、调控的靶基因以及调控机制。这一研究将有助于我们更好的理解转录延伸及其偶联的RNA加工的表观遗传学机制以及组织特异性的调控作用。

组蛋白修饰是表观遗传调控的重要组成部分，与基因表达密切相关。转录延伸过程中，多种组蛋白修饰可能发挥调节作用，如H2B K120位点的单泛素化 (H2BK120ub1)、H3K79位点的二甲基化(H3K79me2) 和H3K36位点的三甲基化 (H3K36me3) 等，而H3K36me3 与转录延伸的关系最为密切。组蛋白修饰的生物学功能多是通过识别该修饰的识别蛋白 (reader) 得以实现，比如H3K36me3可以被含有PWWP结构域的蛋白分子结合。我们利用C2C12成肌细胞模型，通过siRNA筛选的方式，鉴定出多个与成肌分化和肌肉发生密切相关的表观调控分子。其中H3K36me3识别蛋白HRP2 (hepatoma-derived growth factor-related protein 2) 敲低后显著抑制肌肉发生。通过蛋白质组学以及一系列生化分析，我们发现HRP2可以通过直接结合BAF染色质重塑复合体中的DPF3a亚单位，进而结合BAF复合体。我们制备了Hrp2基因敲除小鼠，为后续的功能学分析提供了合适的研究模型。接下来通过结合ChIP-sequencing、RNA-sequencing和ATAC-sequencing等技术手段，我们探讨了HRP2-DPF3a-BAF复合体调控轴在肌肉发生过程中的调控模式以及调控的靶基因。通过基因敲除小鼠模型，我们确定了HRP2在体内调控肌肉分化的功能以及在肌肉退行性病变进展中的作用。蛋白结构分析的结果表明，HRP2蛋白的IBD结构域存在不规则区域，提示其在体内、体外可以发生相分离，我们的实验结果证实了这一推测。HRP2的相分离特性可以促进分子间相互作用，进而将多个重要的转录调控分子聚集到染色质的特定区域，促进基因表达，从而调控肌肉的发生和分化过程。这一工作建立了在转录延伸过程中的相变调控、组蛋白修饰与染色质重塑三者之间的互作模式，进一步的体内、体外功能学实验也将有助于我们更好地理解相变调控的生物学功能和在疾病发生发展中的作用。