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），其具有胚胎干细胞（ESC）的许多属性。这些包括自我更新和被引导到三个胚层的能力。重要的是，iPSC保留了亲本细胞的遗传组成，因此它们作为自体供体用于细胞治疗和体外疾病建模的潜在效用已经得到认可。iPSC中的表观遗传状态与ESC相似，但它们不同，特别是在甲基化方面。这些包括差异甲基化区域和亲本DNA甲基化的不完全消除（表观遗传记忆），这些又对分化潜力产生影响，导致iPSC衍生物的不可预测的行为。为了防止这些不良副作用，至关重要的是在表观遗传水平上研究重编程过程，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，半甲基化的DNA结合蛋白NP 95增加了重编程效率并可以替代c-Myc。伴随着这些观察结果，NP 95增加H3 K4 me 3以及羟甲基胞嘧啶（hmC）。这些暗示NP 95在重编程期间的转录激活中具有新的功能，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NP 95功能的重要见解，解决我们对重编程知识的一些空白。这将通过实现以下具体目标来实现：（1）确定NP 95是否增加SET 1活性。我们将确定NP 95是否稳定了SET 1复合物或直接激活了SET 1/COMPASS复合物的催化活性，并确定了SET 1活性的关键结构域。(2)确定NP 95是否为H3 K4 me 3标记招募Set 1a。我们将评估NP 95募集Set 1复合物的能力，并介导常染色质基因激活。这将通过针对NP 95、Set 1和H3 K4 me 3的ChIP-seq实现。已知NP 95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期间H3 K4 me 3形成中的作用。 (3)确定NP 95是否读取H3 K4 me 3形成的hmC。在ESC中，hmC标记活性基因的位点。hmC标记由重编程过程中诱导的泰特蛋白产生。最近的研究表明，NP 95结合hmC以及mC。我们将证实这些观察结果，并通过检查在重编程过程中NP 95对mC和hmC的形成来进一步发展这一点。这项建议将对重新编程领域产生重大影响，因为它首次提供了对以下方面的详细研究： 重编程过程中的分子事件此外，我们将剖析新的功能NP 95在转录激活过程中重编程和多能干细胞。最终，我们的数据将在产生临床安全，适当重编程的iPSC用于细胞治疗和疾病建模方面发挥关键作用。