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编程为多能状态，即所谓的“诱导多能干细胞”（iPSC），它具有胚胎干细胞（ESC）的许多属性。这些包括自我更新和定向到三个胚层的能力。重要的是，iPSC 保留了亲本细胞的遗传组成，因此它们作为细胞治疗和体外疾病模型的自体供体的潜在用途已得到认可。 iPSC 的表观遗传状态与 ESC 相似，但也有所不同，特别是在甲基化方面。这些包括差异甲基化区域和亲本 DNA 甲基化（表观遗传记忆）的不完全擦除，这些反过来又对分化潜力产生影响，导致 iPSC 衍生物出现不可预测的行为。为了防止这些不良副作用，在表观遗传水平上研究重编程过程至关重要，最终目标是产生理想的 iPSC。在参与 DNA 甲基化和去甲基化的蛋白质中，半甲基化 DNA 结合蛋白 NP95 可以提高重编程效率，并且可以替代 c-Myc。伴随这些观察结果，NP95 增加了 H3K4me3 以及羟甲基胞嘧啶 (hmC)。这些表明 NP95 在重编程过程中的转录激活中具有新功能，这与报道的参与甲基化 DNA 和异染色质区域的维持相反。该提案将通过提供对 NP95 功能的重要见解来弥补我们在重编程知识方面的一些空白。这将通过实现以下具体目标来实现： (1) 确定 NP95 是否增加 SET1 活性。我们将确定 NP95 是否稳定 SET1 复合物或直接激活 Set1/COMPASS 复合物的催化活性，并确定对 SET1 活性至关重要的结构域。 (2)确定NP95是否为H3K4me3标记招募Set1a。我们将评估 NP95 招募 Set1 复合物靶向并介导常染色质基因激活的能力。这将通过针对 NP95、Set1 和 H3K4me3 的 ChIP-seq 来实现。已知 NP95 的 TTD 结构域与修饰的组蛋白 H3 相互作用。将对其进行询问以评估其在重编程过程中 H3K4me3 形成中的作用。 (3)确定NP95是否读取hmC以形成H3K4me3。在 ESC 中，hmC 标记活性基因的位点。 hmC 标记是由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 和 mC 结合。我们将通过检查重编程期间 NP95 形成的 mC 和 hmC 来确认这些观察结果并进一步发展这一点。该提案首次提供了详细的研究，将对重编程领域产生重大影响。 重编程期间的分子事件。此外，我们将剖析 NP95 在重编程和多能干细胞过程中转录激活中的新功能。最终，我们的数据对于生成用于细胞治疗和疾病建模的临床安全、适当重编程的 iPSC 至关重要。