Using AKT1 and 2 and Rb1 to approach MyoD binding mechanisms genome-wide

使用 AKT1 和 2 以及 Rb1 探讨全基因组范围内的 MyoD 结合机制

• 批准号: 8062116
• 负责人: SANDRA B SHARP
• 金额: $ 10.73万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8062116
• 关键词: AKT1 gene; AKT2 gene; Acetylation; Address; Affect; Binding; Binding Sites; Bioinformatics; Biological; Biological Assay; California; Cells; Chromatin; Collaborations; Computers; DNA; DNA Binding; DNA Sequencing Facility; Data; Dependence; Development; E1A-associated p300 protein; EP300 gene; Engineering; Excision; Exhibits; Failure; Funding; Gene Expression; Gene Expression Regulation; Genes; Genome; Goals; Histone Acetylation; Histones; Informatics; Knowledge; Learning; Life; Literature; Location; Malignant Neoplasms; Manuscripts; Molecular; Muscle; Muscle Cells; Muscle Development; Nature; Phosphorylation; Play; Preparation; Process; Protein Kinase; Proteins; Proto-Oncogene Proteins c-akt; Quantitative Reverse Transcriptase PCR; Research; Research Support; Retinoblastoma; Role; Site; Students; System; Systems Biology; Technical Expertise; Testing; Tissues; Transferase; Universities; Work; Writing; base; genome-wide; genome-wide analysis; human HDAC1 protein; in vivo; inhibitor/antagonist; insight; interest; muscle regeneration; myogenesis; public health relevance; research study; tissue processing; transcription factor

DESCRIPTION (provided by applicant): To achieve the larger goal of understanding the regulatory network that controls myogenesis, it is important to understand the mechanisms governing DNA binding by transcription factor MyoD, which plays a defining role in muscle development. MyoD binding at numerous muscle-specific genes is facilitated by phosphorylation of the histone acetyl transferase p300 by the protein kinases AKTs 1 and 2, the consequent association of p300 with MyoD, and the removal of HDAC1 from MyoD by Rb1. Our recent determination of precise locations of in vivo MyoD genome-wide binding in differentiated muscle cells in culture has identified binding sites in apparent association with genes that are not expressed or that are down-regulated. These results raise the possibility that histone acetylation, commonly associated with active genes, is not a contributor to MyoD binding at these sites. Thus, rather than being dependent upon Rb1 and/or AKT 1 and 2, MyoD binding at these sites may be facilitated by different coregulatory mechanisms. We propose to begin a search for alternate binding mechanisms using a combination of molecular biological and bioinformatic approaches. This proposal has 3 specific aims. 1) To informatically analyze our current genome-wide MyoD binding data to identify candidate sites for those at which binding of MyoD may be independent of Rb1 and/or AKT 1 and 2. 2) Using leads from the literature, from our preliminary results, and the proposed informatic analysis, to a) identify a small set of sites for which MyoD binding is differentially affected in vivo Rb1 at only some of the sites, and b) identify a small set of sites for which MyoD binding is differentially affected in vivo AKT1/2 at only some of the sites. MyoD binding will be assayed by ChIP followed by quantitative PCR and gene expression will be assayed by quantitative reverse transcriptase PCR. Once a set of binding sites has been identified, they will be used to vet preparations of ChIP fragments for genome-wide ChIP-Seq. 3) To use ChIP-Seq to determine on a genome- wide scale the sites at which MyoD is bound a) Rb1 and b) AKT1 and 2 activities. The results will be examined informatically for selective enrichment of one or more sequence motifs in those regions containing sites that remain bound in the absence of AKT 1and 2 activity or of Rb1. Such enrichment would suggest differential use of coregulators to facilitate DNA binding. The proposed work incorporates tests for 3 hypotheses. 1) Mechanisms of MyoD binding at sites throughout the genome differ with respect to their dependence upon the presence of Rb1 or AKT 1 and 2 activities; the difference will be apparent in sequence features near MyoD binding sites. 2) At genes for which expression has been shown to be reduced in the absence of Rb1, MyoD binding will also be reduced. If true, binding will be restored in the presence of histone deacetylase 1 inhibitors. 3) Given their mutual influence on chromatin and MyoD acetylation, the increased expression of the Rb1 and Akt2 genes during myogenesis may be regulated by crosstalk. The results of our work can be expected to generate testable hypotheses regarding alternate mechanisms of MyoD binding. PUBLIC HEALTH RELEVANCE: The proposed work will contribute to our understanding of muscle development at the molecular level. What we learn will also provide insights into the nature of molecular mechanisms that operate in other process of tissue formation, including muscle regeneration. The more we understand about normal processes, the more quickly we will be able to understand and address the molecular failures that occur in abnormal development and in cancers, which frequently exhibit loss of the developed nature of the tissues in which they arise.

描述（由申请人提供）：为了实现了解控制肌发生的调节网络的更大目标，重要的是要了解通过转录因子Myod控制DNA结合的机制，该机制在肌肉发育中起着决定性作用。蛋白激酶Akts 1和2磷酸化组蛋白乙酰转移酶p300促进了许多肌肉特异性基因的Myod结合，随之而来的p300与Myod的关联以及RB1从MYOD中删除了HDAC1。我们最近确定培养物中分化肌肉细胞中整个体内基因组结合的精确位置已鉴定出与未表达或下调的基因的明显相关性的结合位点。这些结果增加了通常与活性基因相关的组蛋白乙酰化并不是在这些位点结合MYOD结合的原因。因此，在这些位点上的Myod结合而不是依赖RB1和/或AKT 1和2，可以通过不同的核心调节机制来促进。我们建议使用分子生物学和生物信息学方法的组合开始搜索替代结合机制。该提案具有3个具体目标。 1）通过信息分析我们当前的全基因组MYOD结合数据，以识别MYOD结合的候选站点可能独立于RB1和/或AKT 1和2。2。2）使用来自初步结果的文献中的潜在客户，我们的初步结果，以及建议的信息分析，与一定的网站相关的位置，并确定了一定的位点，并且在某些位置识别了一定的位置，并且BB差异是差异的1个差异。仅在某些位点，在体内Akt1/2中，Myod结合的小位点会受到差异影响。 MYOD结合将通过芯片进行测定，然后进行定量PCR，基因表达将通过定量逆转录酶PCR分析。一旦确定了一组结合位点，它们将用于审查全基因组芯片seq的芯片片段的制备。 3）使用chip-seq在基因组大规模上确定myod绑定的位点a）rb1和b）akt1和2个活动。将对结果进行信息检查，以在没有AKT 1和2活性或RB1的情况下选择性地富集一个或多个序列基序。这种富集将表明将核心节剂的不同使用以促进DNA结合。拟议的工作包含了3个假设的测试。 1）在整个基因组中，MYOD结合的机制在依赖RB1或AKT 1和2活动的依赖方面有所不同；在Myod结合位点附近的序列特征中，差异将显而易见。 2）在没有RB1的情况下，在表达降低的基因上，Myod结合也将减少。如果是真的，则在组蛋白脱乙酰基酶1抑制剂的情况下将恢复结合。 3）鉴于它们对染色质和Myod乙酰化的相互影响，在肌发生过程中，RB1和AKT2基因的表达增加可能受串扰调节。可以预期，我们的工作结果将产生有关MYOD结合的替代机制的可检验假设。 公共卫生相关性：拟议的工作将有助于我们对分子水平上的肌肉发育的理解。我们所学的还将提供有关在组织形成的其他过程（包括肌肉再生过程）中发挥作用的分子机制本质的见解。我们对正常过程的了解越多，我们就越能够越快理解和解决异常发育和癌症中发生的分子失败，这些分子失败经常表现出发达的组织性质的损失。