Investigation of the function of methylated DNA binding protein in reprogramming

甲基化DNA结合蛋白在重编程中的功能研究

• 批准号: 8752215
• 负责人: In-Hyun Park
• 金额: $ 32.05万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8752215
• 关键词: Address; Adverse effects; Autologous; Behavior; Binding; Binding Sites; Cell Therapy; Cells; ChIP-seq; Clinical; Complex; DNA; DNA Methylation; DNA-Binding Proteins; Data; Developmental Gene; Disease model; Ectopic Expression; Environment; Epigenetic Process; Euchromatin; Event; Gene Activation; Genes; Genetic; Genomics; Germ Layers; Goals; Heterochromatin; Histone H3; Histones; In Vitro; Investigation; Knowledge; Maintenance; Mediating; Memory; Methylation; Molecular; Pluripotent Stem Cells; Process; Property; Proteins; Reading; Recruitment Activity; Regenerative Medicine; Reporting; Research; Role; Somatic Cell; Staging; Testing; Time; Transcriptional Activation; demethylation; embryonic stem cell; epigenomics; histone modification; induced pluripotent stem cell; insight; novel; overexpression; pluripotency; protein complex; public health relevance; self-renewal

DESCRIPTION (provided by applicant): We now have the capability to reprogram a somatic cell to a pluripotent state, a so-called "induced pluripotent stem cells" (iPSCs), which have many of the attributes of embryonic stem cells (ESCs). These include self- renewal and the ability to be directed to the three germ layers. Importantly, iPSCs retain the genetic composition of parental cells, and as a consequence their potential utility as autologous donors for cell therapy and in vitro disease modeling has been recognized. The epigenetic states in iPSCs are similar to ESCs but they differ, especially with respect to methylation. These include differentially methylated regions and incomplete erasure of parental DNA methylation (epigenetic memory), these in turn have repercussions in differentiation potential resulting in unpredictable behavior o iPSC-derivatives. To safeguard against these undesirable side-effects it is crucial to investigate the reprogramming process at the epigenetic level, with the ultimate goal of generating desirable iPSCs. Among the proteins involved in DNA methylation and demethylation, is the hemi-methylated DNA binding protein NP95, which increases reprogramming efficiency and can substitute for c-Myc. Accompanied with these observations, NP95 increases H3K4me3 as well as hydroxymethylcytosine (hmC). These implicate a novel function for NP95 in transcriptional activation during reprogramming, contrary to reported involvement in maintenance of methylated DNA and heterochromatic regions. This proposal will address a number of gaps in our knowledge of reprogramming, by providing crucial insight into the function of NP95. This will be achieved by implementing these specific aims: (1) Determine whether NP95 increases SET1 activity. We will establish whether NP95 stabilizes the SET1 complex or directly activates the catalytic activity of Set1/COMPASS complex and identify the domains critical for SET1 activity. (2) Determine whether NP95 recruits Set1a for H3K4me3 marks. We will assess the ability of NP95 to recruit Set1 complex to targets and mediates euchromatin gene activation. This will be achieved with ChIP-seq against NP95, Set1 and H3K4me3. The TTD domain of NP95 is known to interact with modified histone H3. It will be interrogated to assess its role in H3K4me3 formation during reprogramming. (3) Determine whether NP95 reads hmC for H3K4me3 formation. In ESCs, hmC marks the loci of active genes. The hmC marks are produced by TET proteins induced during the reprogramming process. Recent studies have shown that NP95 binds to hmC as well as mC. We will confirm these observations and further develop this by examining the formation of mC and hmC by NP95 during reprogramming. This proposal will significantly impact on the reprogramming field by providing for the first time a detailed study of molecular events during reprogramming. Additionally we will dissect the novel function of NP95 in transcriptional activation during reprogramming and pluripotent stem cells. Ultimately our data will be critical in generating clinically safe, appropriately reprogrammed iPSCs for cell therapy and disease modeling.

描述（由申请人提供）：我们现在有能力将体细胞重新编程为多能状态，即所谓的“诱导多能干细胞”（IPSC），它具有许多胚胎干细胞的属性（ESC）。这些包括自我更新和指向三个细菌层的能力。重要的是，IPSC保留了亲本细胞的遗传组成，因此已经认识到它们作为细胞疗法和体外疾病模型的自体供体的潜在效用。 IPSC中的表观遗传状态与ESC相似，但它们有所不同，尤其是在甲基化方面。这些包括差异化甲基化区域和父母DNA甲基化（表观遗传记忆）的不完全擦除，这些区域又具有分化潜力的影响，从而导致不可预测的行为O IPSC衍生物。为了保护这些不良副作用，在表观遗传水平上研究重编程过程至关重要，最终是产生理想的IPSC。在参与DNA甲基化和脱甲基化的蛋白质中，是半甲基化的DNA结合蛋白NP95，它提高了重编程效率并可以代替C-MYC。伴随着这些观察结果，NP95增加了H3K4me3以及羟基胞嘧啶（HMC）。这些暗示了在重编程过程中NP95在转录激活中的新功能，与报道参与甲基化DNA和异颜色区域的维持相反。该建议将通过提供对NP95功能的关键见解来解决我们重编程知识中的许多差距。这将通过实施这些特定目标来实现：（1）确定NP95是否增加了SET1活动。我们将确定NP95是稳定SET1复合物还是直接激活SET1/Compass复合物的催化活性，并确定对SET1活动至关重要的域。 （2）确定H3K4ME3标记的NP95招募SET1A。我们将评估NP95募集SET1复合物为靶标的能力并介导整体素基因激活。这将通过CHIP-SEQ对NP95，SET1和H3K4ME3实现。已知NP95的TTD结构域与修饰的组蛋白H3相互作用。它将在重新编程过程中评估其在H3K4ME3形成中的作用。 （3）确定NP95是否读取H3K4me3形成的HMC。在ESC中，HMC标志着活性基因的基因座。 HMC标记是由在重编程过程中诱导的TET蛋白产生的。最近的研究表明，NP95与HMC以及MC结合。我们将通过在重编程过程中通过NP95检查MC和HMC的形成来确认这些观察结果，并进一步开发这种观察结果。该建议将首次提供对重编程领域的重大影响 重编程过程中的分子事件。此外，我们将在重编程和多能干细胞过程中剖析NP95在转录激活中的新功能。最终，我们的数据对于生成用于细胞疗法和疾病建模的临床安全，适当重编程的IPSC至关重要。