Combinatorial epigenetic readout of methylated DNA and histone methylation marks by ICBP90/UHRF1

我们身体所有细胞的许多功能都需要它们的遗传物质以一种高度有组织的方式被维持、读取和翻译。基因组调控的分子目标是染色质，其中DNA包裹在组蛋白周围。DNA和组蛋白的大量化学修饰是所谓表观遗传过程的核心。通过局部募集特异性结合蛋白来翻译离散染色质修饰的生化原理目前正在研究中。虽然这些因素构成了染色质修饰模式和染色质功能之间的重要联系，但人们对它们的调控知之甚少。UHRF1（在小鼠中也被称为ICBP90或NP95）是一种多结构域蛋白，它将DNA甲基化与组蛋白甲基化，特别是抑制标记组蛋白H3赖氨酸9三甲基化（H3K9me3）联系在一起。该因子参与复制过程中DNA甲基化模式的维持，在小鼠中其缺失是胚胎致死的。UHRF1/NP95包含不同的结构域，分别与甲基化DNA、未修饰的H3和H3K9me3结合有关。我们已经获得的证据表明，UHRF1/NP95在其异染色质关联中受到一个小细胞分子，即磷脂酰肌醇5-磷酸（PI5P）的控制。虽然磷脂酰肌醇磷酸（PIP）通常以其在细胞膜和细胞质中的信号功能而闻名，但在细胞核中存在着一个独立的脂质池，其生物化学和功能在很大程度上未知。基于我们的初步工作，我们假设PI5P水平响应于特定的细胞过程，如细胞周期和毒性应激，通过变构诱导不同的蛋白质构象来调节UHRF1/NP95的染色质关联。这些不同的构象在UHRF1/NP95的组蛋白修饰和DNA甲基化结合域的可及性方面存在差异。为了研究这一想法的有效性，我们希望(i)利用生物化学、生物物理和结构方法的结合，建立PI5P调节UHRF1染色质关联的分子细节，以及（ii）通过分析单个细胞和细胞池，以及通过观察单个基因位点和整个基因组，确定PI5P在调节UHRF1/NP95染色质关联中的细胞作用。如果成功，这项工作将为重要的表观遗传因子的调控以及PI5P在细胞核中的功能提供新的见解。除此之外，这项研究可能会对我们对染色质调控和核PIP生物学的一般理解产生相当大的影响。此外，我们可能会定义小分子/药物对染色质修饰结合蛋白进行功能操作的新途径，从而为治疗疾病（如癌症、糖尿病）提供表观遗传治疗的新方法。

The many functions that all cells of our body carry out require that their genetic material is maintained, read and translated in a highly organized manner. The molecular target of genome regulation is chromatin, in which DNA is wrapped around histone proteins. A large variety of chemical modifications of DNA and histones are at the core of essential so called epigenetic processes. The biochemical principles of translation of discrete chromatin modifications by local recruitment of specific binding proteins are currently being worked out. While these factors constitute essential links between chromatin modification patterns and chromatin function, little is known about their regulation. UHRF1 (also referred to as ICBP90 or NP95 in mouse) is a multidomain protein that crucially links DNA methylation to histone methylation and in particular the repressive mark histone H3 lysine 9 trimethylation (H3K9me3). The factor is involved in maintenance of DNA methylation patterns during replication and its deletion in mice is embryonic lethal. UHRF1/NP95 contains different domains, which have been implicated in binding methylated DNA, unmodified H3 and H3K9me3, respectively. We have obtained evidence that UHRF1/NP95 is controlled in its heterochromatin association by a small cellular molecule, namely phosphatidylinositol 5-phosphate (PI5P). While phosphatidylinositol phosphates (PIP) are generally well known for their signaling function at the cell membrane and in the cytoplasm, a separate pool of these lipids exists in the cell nucleus with largely unknown biochemistry and function. Based on our preliminary work, we hypothesize that PI5P levels modulated in response to defined cellular processes such as the cell cycle and toxic stresses regulate the chromatin association of UHRF1/NP95 by allosterically inducing distinct conformations of the protein. These distinct conformations differ in the accessibility of the histone modification and DNA methylation binding domains of UHRF1/NP95. To investigate the validity of this idea we want to (i) establish the molecular details of PI5P regulating UHRF1 chromatin association using a combination of biochemical, biophysical and structural approaches, and (ii) define the cellular role of PI5P in regulating UHRF1/NP95 chromatin association by analyzing individual cells as well as cell pools and by looking at single gene loci as well as at the whole genome.If successful, this work will provide novel insights into regulation of an important epigenetic factor as well as the function of PI5P in the cell nucleus. Beyond that the study might have considerable impact onto our general understanding of chromatin regulation and the biology of nuclear PIP. Also, we might define new avenues of functional manipulation of chromatin modification binding proteins by small molecules/drugs thereby providing novel approaches of epigenetic therapy in curing diseases (e.g. cancer, diabetes).