Determining gene targets and pathways for primate-specific zinc finger proteins

描述（由申请人提供）：kruppel型锌指（ZNF）基因座是所有人类基因家族中最大的基因之一。在哺乳动物中，大多数ZNF基因属于单一亚型，编码蛋白质，其中dma结合锌指阵列连接到称为KRAB的染色质相互作用结构域，该结构域具有有效的转录抑制活性。虽然某些KRAB-ZNF位点是高度保守的，但持续的片段复制事件在每个哺乳动物谱系中创造了很大程度上独特的基因集。在404个人类KRAB- ZNF基因座中，超过三分之一是灵长类特有的，甚至最近的重复基因也出现了分化，这表明人类选择了具有不同DNA识别位点的新蛋白质。这个单基因家族占所有预测的人类转录因子位点的五分之一，现有数据表明，它在调节对人类健康至关重要的过程中发挥着广泛的作用。然而，由于已知的调控靶点和途径只有少数KRAB-ZNF蛋白，大多数家族成员的功能仍然是一个猜测问题。我们假设这个动态基因家族在塑造人类健康相关生物学方面发挥了重要作用，包括深度保守和灵长类特有的特征。拟议的研究计划旨在通过对25个KRAB-ZNF基因的功能分析来解决这一假设。作为主要焦点，我们将分析25个基因编码的蛋白质，这些基因参与了最近的灵长类特异性复制，重点是最适合实验分析的旁系蛋白质。具体来说，我们将(1)通过操纵人类细胞中的基因表达来确定每个ZNF蛋白所调节的基因和途径；(2)利用染色质免疫沉淀技术确定ZNF蛋白在人染色质中结合的基因组区域；(3)利用生物信息学和实验相结合的方法，鉴定和验证确定每种蛋白质有利识别位点的一致DNA基序。这项研究将首次深入研究这一人类转录抑制因子大家族的功能，使我们能够研究它们在调节人类免疫、生殖、发育、癌症易感性和其他涉及KRAB-ZNF基因的过程中的作用。我们生成的数据将为预测其他家族成员的功能以及评估KRAB-ZNF基因的获得、丢失、突变和失调对人类健康的潜在影响提供新的指导。

DESCRIPTION (provided by applicant): Kruppel-type zinc finger (ZNF) loci comprise one of the largest of all human gene families. In mammals, the majority of ZNF genes are of a single subtype, encoding proteins in which DMA-binding zinc finger arrays are attached to a chromatin-interacting domain, called KRAB, that confers a potent transcriptional repressor activity. Although certain KRAB-ZNF loci are highly conserved, ongoing segmental duplication events have created largely unique gene sets in each mammalian lineage. More than one-third of the 404 human KRAB- ZNF loci are primate-specific, and even the most recent duplicate genes have diverged in ways that indicate a selection for novel proteins with distinct DNA recognition sites. This single gene family comprises one-fifth of all predicted human transcription factor loci, and available data suggest broad roles in regulating processes that are critical to human health. However, since regulatory targets and pathways are known for only a handful of KRAB-ZNF proteins the functions of most family members remain a matter of conjecture. We hypothesize that this dynamic gene family has played a significant role in shaping human health-related biology, including both deeply conserved and primate-specific traits. The proposed research program is designed to address this hypothesis through functional analysis of 25 KRAB-ZNF genes. As a primary focus we will analyze proteins encoded by 25 genes involved in the most recent primate-specific duplications with emphasis on paralogous proteins that are most amenable to experimental analysis. Specifically, we will (1) determine genes and pathways that are regulated by each ZNF protein by manipulating gene expression in human cells; (2) define genomic regions to which the ZNF proteins bind in human chromatin using chromatin immunoprecipitation techniques; and (3) identify and validate consensus DNA motifs defining favored recognition sites for each protein using combined bioinformatics and experimental approaches. This study will provide a first in-depth look at functions for this large family of human transcriptional repressors, permitting us to examine their roles in regulating in human immunity, reproduction, development, cancer susceptibility and other processes in which KRAB-ZNF genes have been implicated. The data we generate will provide new guidelines to predict the functions of additional family members and to assess the potential impact of the gain, loss, mutation and dsyregulation of KRAB-ZNF genes on human health.