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

这项研究的主要目标是了解细胞内基因表达的调节机制。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。对不同遗传区域的访问受到DNA局部区域可逆化学变化的部分调节，这种变化被定义为表观遗传修饰。这个项目评估了一个小的蛋白质家族ZBTB家族的作用，ZBTB家族在进行这些表观遗传修饰方面很重要。该项目的更广泛影响包括两个主要方面：第一，对研究生进行跨学科研究计划的培训，该计划利用广泛的技术来识别和描述三个ZBTB。其次，该项目包括在盐湖城市中心的莱昂纳多博物馆开展以生化和DNA为重点的动手和基于探究的活动。除了更广泛的公众，莱昂纳多还容纳了相当数量的学生，他们来自盐湖谷学校的代表不足和服务不足的群体。众所周知，儿童对科学的兴趣最好在早期和易受影响的年龄灌输，预计动手活动的发展和实施将对国家在STEM地区的多样性增长产生长期重大影响。DNA甲基化是表观遗传修饰的一种形式。虽然人们已经深入了解DNA甲基化标记是如何建立和移除的，但对于甲基-CpG结合蛋白(Mbps)、读取DNA甲基化标记并将这些信息转化为特定转录结果，特别是ZBTB Mbps的机制，人们知之甚少。对于ZBTB MBPS如何调控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的非甲基化DNA位点，这使它们能够同时作为转录抑制物和激活剂发挥作用。此外，每个ZBTB Mbps都可以与靶基因位点上的多个共抑制/激活蛋白复合体相互作用。因此，了解这些蛋白质中的每一种如何引起对其结合伙伴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，将利用体外生物物理和细胞内相结合的方法来描述这个Mbps家族识别其DNA和蛋白质靶标的机制。总体目标是确定ZBTB蛋白质的结构特征：DNA相互作用以获得对每个蛋白质如何优先选择DNA靶标的机械性洞察；利用功能特定的突变来探索双峰DNA识别的分子基础；以及定义每个ZBTB MBP的蛋白质相互作用。这项研究的结果将为更大的目标提供必要的基础，即了解这个被研究的ZBTB Mbps家族的每个成员通过什么机制来诱导选择性的基因靶向，将特定的蛋白质复合体招募到目标位置，并改变转录来提供特定的细胞功能。

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.