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Zinc finger methyl-CpG binding proteins: Bimodal regulators of cellular transcription

锌指甲基-CpG 结合蛋白：细胞转录的双峰调节因子

基本信息

批准号：
1715370
负责人：
Bethany Buck-Koehntop
金额：
$ 62.43万
依托单位：
University of Utah
依托单位国家：
美国
项目类别：
Continuing Grant
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1715370&HistoricalAwards=false
关键词：
Zinc finger methyl CpG binding

项目摘要

The primary goal of this research is to understand the mechanisms by which gene expression is regulated within the cell. DNA is the long-term storage unit for genetic information, providing instructions for cellular and life functions. Access to different genetic regions is partially regulated by reversible chemical changes to localized regions of the DNA, defined as epigenetic modifications. This project evaluates the role of a small family of proteins, the ZBTB family, that are important in making these epigenetic modifications. The broader impacts for this project include two major aspects: First, the training of graduate students in an interdisciplinary research program that utilizes a broad spectrum of techniques to identify and characterize the three ZBTB. Second, this project includes the development of biochemical and DNA-focused hands-on and inquiry-based activities at The Leonardo, a museum in downtown Salt Lake City. In addition to the broader public, The Leonardo accommodates a significant number of students from underrepresented and underserved groups from Salt Lake Valley schools. It is well recognized that the interest of a child in science is best instilled at an early and impressionable age, and it is expected that development and implementation of hands-on activities will have a long-term significant impact on the state's diversity growth in STEM areas.DNA methylation is one form of epigenetic modification. While insight into how the DNA methylation mark is established and removed has been gained, there is only minimal knowledge for the mechanisms by which methyl-CpG binding proteins (MBPs),read DNA methylation marks and translate this information into a specific transcriptional outcome, particularly for the ZBTB MBPs. An understanding for how the ZBTB MBPs modulate transcription is complicated by the fact that they exhibit bimodal DNA recognition, targeting both methylated and sequence-specific non-methylated DNA sites, which affords them the capability of functioning as both transcriptional repressors and activators. Additionally, each of the ZBTB MBPs can interact with multiple co-repressor/-activator protein complexes at target gene sites. Thus, an understanding for how each of these proteins elicits selectivity for their binding partners is central to discerning their role in transcriptional regulation. To investigate this, a combined in vitro biophysical and in cell approach will be utilized to delineate the mechanisms by which this family of MBPs recognize their DNA and proteins targets. The overall goals are to structurally characterize ZBTB protein:DNA interactions to gain mechanistic insight into how each protein preferentially selects DNA targets; to utilize function-specific mutations to probe the molecular basis for bimodal DNA recognition; and to define protein interactomes for each ZBTB MBP. The outcomes of this research will provide an essential foundation for the larger goal of discerning the full mechanisms by which each member of this under studied family of ZBTB MBPs elicits selective gene targeting, recruits specific protein complexes to the target site and alters transcription to provide a particular cellular function.

这项研究的主要目标是了解基因表达在细胞内调节的机制。DNA是遗传信息的长期存储单位，为细胞和生命功能提供指令。进入不同的遗传区域部分地受到DNA局部区域的可逆化学变化的调节，定义为表观遗传修饰。该项目评估了一个小的蛋白质家族ZBTB家族的作用，该家族在进行这些表观遗传修饰中非常重要。该项目更广泛的影响包括两个主要方面：第一，在跨学科研究计划中培训研究生，该计划利用广泛的技术来识别和表征三种ZBTB。第二，该项目包括在湖城中心的列奥纳多博物馆开展以生物化学和DNA为重点的实践和探究活动。除了更广泛的公众，列奥纳多容纳了来自盐湖山谷学校的代表性不足和服务不足群体的大量学生。众所周知，孩子对科学的兴趣最好在早期和易受影响的年龄灌输，预计实践活动的开发和实施将对该州STEM领域的多样性增长产生长期的重大影响。DNA甲基化是表观遗传修饰的一种形式。虽然已经深入了解了DNA甲基化标记是如何建立和去除的，但对于甲基-CpG结合蛋白（MBP）读取DNA甲基化标记并将此信息翻译成特定转录结果的机制，特别是对于ZBTB MBP，只有很少的知识。ZBTB MBP如何调节转录的理解是复杂的，因为它们表现出双峰DNA识别，靶向甲基化和序列特异性非甲基化DNA位点，这使它们能够同时作为转录抑制因子和激活因子发挥作用。此外，每个ZBTB MBP可以与靶基因位点处的多个共阻遏物/激活蛋白复合物相互作用。因此，了解这些蛋白质中的每一种如何激发其结合伴侣的选择性对于辨别它们在转录调控中的作用至关重要。为了研究这一点，结合在体外生物物理和细胞的方法将被用来描绘的机制，这个家庭的MBPs识别其DNA和蛋白质的目标。总体目标是从结构上表征ZBTB蛋白：DNA相互作用，以获得对每个蛋白如何优先选择DNA靶点的机制性洞察;利用功能特异性突变来探测双峰DNA识别的分子基础;并为每个ZBTB MBP定义蛋白质相互作用组。这项研究的结果将为更大的目标提供必要的基础，即识别ZBTB MBP家族的每个成员通过其进行选择性基因靶向，招募特定的蛋白质复合物到靶位点并改变转录以提供特定的细胞功能的完整机制。