Frascati-mediated Mitochondrial Metabolism

无效红细胞生成障碍导致相当大的人类发病率，并占用主要的卫生资源。设计新的治疗策略的一个主要障碍是对介导正常终末红细胞成熟的分子机制的不完全理解。转录因子加塔-1是红细胞生成的主要调节因子。2004年，韦斯和同事发现，加塔-1不仅激活了许多红细胞特异性基因，而且抑制了几乎相同数量的基因。随后的研究表明，激活和抑制的基因都是由加塔-1直接控制的。这导致了该领域两个主要的未回答的问题：（1）加塔-1如何区分激活和抑制基因？（2）加塔-1如何执行这些相反的转录功能？我们的长期目标是进一步阐明这些机制，并应用这些信息更好地了解和治疗人类无效的红细胞生成障碍。作为第一步，我们最近进行了加塔-1 ChIP-seq， 在小鼠红系细胞中进行cDNA微阵列分析以鉴定全基因组直接功能性加塔-1靶基因。这提供了激活的（454）和抑制的（325）加塔-1靶基因及其加塔-1结合顺式调节元件的大数据集。生物信息学分析揭示了区分加塔-1激活基因与抑制基因的候选特征。与其他人最近的工作一致，我们发现SCL复合物的组合占用与基因激活密切相关。然而，复合加塔：E-box（SCL结合）DNA结合基序本身的简单存在并不能完全区分激活和抑制基因。因此，必须要求提供额外信息， 指定加塔-1激活基因。该提议的具体目的是：（1）鉴定与SCL合作以区分激活和抑制的加塔-1靶基因的其他因子;和（2）进一步理解SCL共占据导致加塔-1正转录活性的机制。 基于我们的初步研究，我们假设某些富含GC和CAAT结合的转录因子有助于区分加塔-1激活基因。我们还假设SCL复合物阻断了加塔-1和Polycomb抑制复合物2之间的相互作用，并招募了转录阳性细胞。 伸长调节剂将使用ChIP-seq、基因表达分析、转基因报告基因测定和生物化学技术来测试这些假设。这些研究的预期结果是鉴定参与指定加塔-1基因激活与抑制的新机制。

Disorders of ineffective erythropoiesis cause considerable human morbidity and utilize major health resources. A major barrier to the design of novel treatment strategies is an incomplete understanding of the molecular mechanisms that mediate normal terminal erythroid maturation. The transcription factor GATA-1 is a master regulator of erythropoiesis. In 2004, Weiss and colleagues showed that GATA-1 not only activates many erythroid-specific genes, but also represses nearly an equal number. Subsequent studies showed that both the activated and repressed genes are directly controlled by GATA-1. This has led to two major unanswered questions in the field: (1) how does GATA-1 distinguish between activated and repressed genes? And (2) how does GATA-1 carry out these opposing transcriptional functions? Our long-term obiective is to further elucidate these mechanisms and apply this information to better understand and treat human ineffective erythropoietic disorders. As a first step, we recently performed GATA-1 ChlP-seq and cDNA microarray analysis in murine erythroid cells to identify genome-wide direct functional GATA-1 target genes. This provided a large dataset of activated (454) and repressed (325) GATA-1 target genes and their GATA-1 bound cis-regulatory elements. Bioinformatic analysis revealed candidate features that distinguish GATA-1 activated versus repressed genes. Consistent with the recent work of others, we found that combinatorial occupancy by SCL complexes strongly correlates with gene activation. However, the simple presence of composite GATA:E-box (SCL binding) DNA binding motifs by themselves does not fully distinguish between activated and repressed genes. Therefore, additional information must be required to specify GATA-1 activated genes. The specific aims of this proposal are to: (1) identify additional factors that cooperate with SCL to distinguish between activated and repressed GATA-1 target genes; and (2) further understand the mechanisms by which SCL co-occupancy results in GATA-1 positive transcriptional activity. Based on our preliminary studies, we hypothesize that certain GC-rich and CAAT binding transcription factors contribute to distinguishing GATA-1 activated genes. We also hypothesize that SCL complexes block interactions between GATA-1 and Polycomb Repressive Complex 2, and recruit positive transcriptional elongation regulators. These hypotheses will be tested using ChlP-seq, gene expression analysis, transgenic reporter assays, and biochemical techniques. The expected outcome from these studies is the identification of novel mechanisms involved in specifying GATA-1 gene activation versus repression.