Sequence-specific DNA-binding of evolutionary divergent KRAB-zinc finger proteins

描述(申请人提供)：KRAB结构域包含锌指蛋白(KRAB-ZNFs)是哺乳动物中一个大的但特征不佳的转录调控家族。KRAB-ZNF被认为是通过KRAB盒介导的与KAP1辅阻遏子复合体的相互作用而发挥转录抑制因子的功能。尽管有大量的生化证据，但已知的KRAB-ZNF的靶基因很少，这些调控因子的生理作用基本上还不清楚。编码锌指蛋白的基因通常分布在几条不同的染色体上。这些簇被认为是由串联复制产生的，在哺乳动物物种中存在的数量要多得多。最近的系统发育分析表明，人类19号染色体上的KRAB ZNF基因簇与小鼠7号染色体上的KRAB ZNF基因簇高度相关；似乎单个小鼠基因Zfp61在19号染色体上形成了10个人类基因簇。每个基因都包含一个KRAB转录抑制因子结构域，并具有不同数量的C2H2锌指DNA结合域。KRAB-ZNF的这种谱系特异性复制可能对定义物种特异性转录网络具有重要意义，并可能对发育具有重要意义。我认为人类特定的KRAB-ZNF基因的进化对谱系特异的转录网络有重大影响。具体地说，KRAB-ZNF基因簇的C2H2区域内的差异导致了10个KRAB-ZNF转录抑制因子的不同DNA结合活性。因此，这些单独的因子能够调节10个不同的基因亚集，否则这些基因只能由小鼠中的一个转录抑制因子控制。我将表达10个人类基因和1个小鼠基因的标记版本，并使用体内染色质免疫沉淀和高通量测序(CHIP-SEQ)来分析它们的靶标。 公共卫生相关性：这项建议旨在研究一组特征不佳的锌指蛋白，这些蛋白可能参与调节组织或物种特定的转录网络。癌症已经被证明含有与本研究中的那些在结构上相关的锌指蛋白的拷贝数和表达水平改变。最终，这项研究将增加社区对KRAB-ZNF基因的获得、丢失和错误调节对人类健康的潜在影响的了解。

DESCRIPTION (provided by applicant): KRAB domain-containing zinc finger proteins (KRAB-ZNFs) are a large, but poorly characterized family of transcriptional regulators in mammals. KRAB-ZNFs are thought to function as transcriptional repressors through a KRAB box-mediated interaction with the KAP1 corepressor complex. Despite much biochemical evidence, there are very few known target genes of KRAB-ZNFs and the physiological roles of these regulators remain essentially unknown. The genes encoding zinc finger proteins are typically found in clusters located on several different chromosomes. These clusters are thought to have arisen from tandem duplications and are present in much higher numbers in mammalian species. Recent phylogenetic analysis suggests that the KRAB ZNF gene clusters on human chromosome 19 and mouse chromosome 7 are highly related; it appears that a single mouse gene Zfp61 has given rise to a cluster of 10 human genes on chromosome 19. Each of these genes contains a KRAB transcriptional repressor domain and has varying numbers of C2H2 zinc finger DNA binding domains. Such lineage specific duplications of KRAB-ZNFs may have important implications for defining species-specific transcriptional networks and may have significant implications on development. I propose that the evolution of human specific KRAB-ZNF genes has a significant impact on lineage-specific transcriptional networks. Specifically, the divergence within the C2H2 regions of the cluster of KRAB-ZNF genes leads to diverse DNA binding activities of the 10 KRAB-ZNF transcriptional repressors. These separate factors are therefore capable of regulating 10 distinct subsets of genes that are otherwise only controlled by a single transcription repressor in mouse. I will express tagged versions of the 10 human genes and one mouse gene and analyze their targets using an in vivo chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). PUBLIC HEALTH RELEVANCE: This proposal aims to study a group of poorly characterized zinc finger proteins, which are likely to be involved in regulating tissue- or species-specific transcriptional networks. Cancers have been shown to contain altered copy numbers and expression levels of zinc finger proteins structurally related to those in this study. Ultimately, this research will add to the community's understanding of the potential impact of the gain, loss, and misregulation of KRAB-ZNF genes on human health.