Investigation of the function of methylated DNA binding protein in reprogramming

描述(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”(IPSCs)，它具有胚胎干细胞(ESCs)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，IPSCs保留了亲代细胞的遗传成分，因此，它们作为细胞治疗和体外疾病建模的自体供体的潜在用途已经被认识到。IPSCs的表观遗传状态类似于ESCs，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父母DNA甲基化的不完全擦除(表观遗传记忆)，这些反过来又对分化潜力产生影响，导致iPSC-衍生物的不可预测行为。为了防止这些不良的副作用，至关重要的是在表观遗传学水平上研究重新编程过程，最终目标是产生理想的ipscs。在参与DNA甲基化和去甲基化的蛋白质中，有一种是半甲基化的DNA结合蛋白NP95，它可以提高重编程效率，并可以取代c-Myc。伴随着这些观察，NP95增加了H3K4me3和羟甲基胞嘧啶(HMC)。这意味着NP95在重编程过程中的转录激活中有一种新的功能，与已报道的参与维持DNA甲基化和异染色质区域相反。这项提案将通过提供对NP95功能的关键洞察，解决我们在重新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是否会增加SET1的活性。我们将确定NP95是稳定SET1复合体还是直接激活Set1/COMPASS复合体的催化活性，并确定对SET1活性至关重要的结构域。(2)确定NP95是否为H3K4me3分数招募Set1a。我们将评估NP95将Set1复合体招募到靶点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实现。已知NP95的TTD结构域与修饰组蛋白H3相互作用。在重新编程期间，将对其进行讯问，以评估其在H3K4me3形成中的作用。(3)确定NP95是否读取H3K4me3地层的HMC。在胚胎干细胞中，HMC标记活性基因的基因座。HMC标记是由重新编程过程中诱导的Tet蛋白产生的。最近的研究表明，NP95与HMC和MC结合。我们将证实这些观察结果，并通过研究NP95在重新编程期间形成MC和HMC来进一步发展这一点。这项提案将首次对以下方面进行详细研究，从而对重新编制方案领域产生重大影响 重新编程过程中的分子事件。此外，我们还将剖析NP95在重编程和多能干细胞转录激活中的新功能。最终，我们的数据将对产生临床安全的、适当重新编程的ipscs用于细胞治疗和疾病建模至关重要。

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.