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

描述(申请人提供)：Krupbel型锌指(ZNF)基因座是人类最大的基因家族之一。在哺乳动物中，大多数ZNF基因都是单一的亚型，编码的蛋白质中，DMA结合的锌指阵列连接到一个名为KRAB的染色质相互作用结构域，该结构域赋予了强大的转录抑制活性。虽然某些KRAB-ZNF基因座是高度保守的，但正在进行的节段性复制事件已经在每个哺乳动物谱系中创造了基本上独特的基因集。在人类的404个KRAB-ZNF基因座中，超过三分之一是灵长类特有的，即使是最新的重复基因也出现了分歧，表明选择了具有不同DNA识别位点的新蛋白质。这个单基因家族包括所有预测的人类转录因子基因座的五分之一，现有数据表明，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，由于只知道少数KRAB-ZNF蛋白的调控靶点和调控途径，大多数家族成员的功能仍然是一个猜测的问题。我们假设，这个动态的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，包括高度保守的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说。作为主要焦点，我们将分析最近灵长类特定复制中涉及的25个基因编码的蛋白质，重点放在最适合实验分析的平行蛋白质上。具体地说，我们将(1)通过操纵基因在人类细胞中的表达来确定每种ZNF蛋白调控的基因和途径；(2)使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；以及(3)使用生物信息学和实验相结合的方法识别和验证定义每种蛋白质青睐识别位点的共识DNA基序。这项研究将首次深入了解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、癌症易感性和其他与KRAB-ZNF基因有关的过程中的作用。我们生成的数据将为预测额外家庭成员的功能和评估KRAB-ZNF基因的获得、丢失、突变和缺失调节对人类健康的潜在影响提供新的指导方针。

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.