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

描述（由申请人提供）：含KRAB结构域的锌指蛋白（KRAB- znfs）是哺乳动物中一个大的，但特征不明显的转录调节因子家族。KRAB- znfs被认为是通过KRAB盒介导的与KAP1辅阻遏因子复合物的相互作用发挥转录阻遏因子的功能。尽管有大量的生化证据，但已知的KRAB-ZNFs靶基因很少，这些调节因子的生理作用基本上仍然未知。编码锌指蛋白的基因通常位于几个不同的染色体上。这些群集被认为是由串联复制产生的，在哺乳动物物种中数量要多得多。最近的系统发育分析表明，人类第19染色体上的KRAB ZNF基因簇与小鼠第7染色体上的KRAB ZNF基因簇高度相关；似乎一个小鼠基因Zfp61在19号染色体上产生了一个由10个人类基因组成的集群。这些基因中的每一个都含有一个KRAB转录抑制域，并具有不同数量的C2H2锌指DNA结合域。这种KRAB-ZNFs的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。我认为人类特异性KRAB-ZNF基因的进化对谱系特异性转录网络有重大影响。具体来说，KRAB-ZNF基因簇C2H2区域内的差异导致10种KRAB-ZNF转录抑制因子的DNA结合活性不同。因此，这些单独的因子能够调节10个不同的基因亚群，否则在小鼠中仅由单个转录抑制因子控制。我将表达10个人类基因和一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）分析它们的靶标。

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.