Dissecting the functions of the novel factors Pdp3 and Lem2 in heterochromatin regulation

真核细胞的基因组被组织成不同的染色质结构域。转录活性域（常染色质）和沉默域（异染色质）的功能划分对于不同的细胞功能和基因组稳定性至关重要。它还允许在发育过程中及时表达基因。此外，沉默染色质在拓扑结构上不同于活性染色质，并且在许多模型系统中位于核外围。这种真染色质和异染色质的分化是可逆的，并且在很大程度上独立于潜在的DNA序列，然而获得的染色质状态是相当稳定的，并且可以遗传（表观遗传学）。在鉴定参与在染色质上建立表观遗传标记的酶活性和结构组分方面已经取得了重大进展。然而，协调这些因素的调节机制仍然知之甚少。最近，我们已经通过无偏见的遗传筛选分离出几个新的候选人，在模式生物裂殖酵母（裂殖酵母），影响异染色质沉默，并可能控制异染色质内的染色质相关因子的空间分布及其定位到核周边。在这项提案中，我们将专注于破译异染色质调控中两个新因子的特定作用：Pdp3和Lem2。Pdp3含有一个假定的甲基结合PWWP结构域，是组蛋白乙酰转移酶（HAT）复合物的一部分。我们未发表的数据表明，Pdp3通过控制HAT的活性而有助于沉默。我们将测试Pdp3是否通过其PWWP结构域区分不同的染色质区域，从而调节HAT复合物在染色质上的空间分布。核膜蛋白Lem2是后生动物核纤层蛋白相关蛋白的同源物。我们发现Lem2有助于沉默各种异染色质结构域，并与其他外周因子冗余地起作用。因此，我们将测试的假设是否Lem2促进沉默通过锚定异染色质的核外围，并检查Lem2如何介导的沉默染色质的核被膜的桥接。我们将在强大的裂变酵母模型中进行这些研究，该模型具有后生动物异染色质的基本特征，非常适合应用功能遗传学，高通量研究和全基因组分析。总之，这些努力将为异染色质在裂殖酵母中的控制和协调提供深刻的机制见解。虽然这种分子机制很难在多细胞系统中研究，但它们可能反映了由于异染色质的强烈保守性而可能也转移到后生动物和人类的基本原理。

The genome of eukaryotic cells is organized into distinct chromatin domains. The functional partitioning into transcriptionally active (euchromatin) and silent domains (heterochromatin) is crucial for diverse cellular functions and genome stability. It allows also the timely gene expression during development. Furthermore, silent chromatin differs topologically from active chromatin and is localized in many model systems at the nuclear periphery. This differentiation of eu- and heterochromatin is reversible and largely independent of the underlying DNA sequence, yet the acquired chromatin states are fairly stable and can be inherited (epigenetics). Significant advances have been made in identifying enzymatic activities and structural components that are involved in establishing epigenetic marks on chromatin. However, the regulatory mechanisms that coordinate these factors are still poorly understood. Recently, we have isolated through unbiased genetic screens in the model organism fission yeast (Schizosaccharomyces pombe) several new candidates that affect heterochromatic silencing and potentially control the spatial distribution of chromatin-associated factors within heterochromatin and its localization to the nuclear periphery. In this proposal, we will focus on deciphering the specific roles of two novel factors in heterochromatin regulation: Pdp3 and Lem2. Pdp3 contains a putative methyl-binding PWWP domain and is part of a histone acetyltransferase (HAT) complex. Our unpublished data suggest that Pdp3 contributes to silencing by controlling the activity of the HAT. We will test whether Pdp3 discriminates between different chromatin regions through its PWWP domain and thereby regulates the spatial distribution of the HAT complex on chromatin. The nuclear envelope protein Lem2 is a homolog of metazoan lamin-associated proteins. We found that Lem2 contributes to silencing of various heterochromatic domains and acts redundantly with other peripheral factors. We will therefore test the hypothesis whether Lem2 promotes silencing through anchoring heterochromatin to the nuclear periphery and examine how Lem2 mediates the bridging of silent chromatin to the nuclear envelope. We will perform these studies in the powerful fission yeast model that harbors essential hallmarks of metazoan heterochromatin and is ideally suited for applying functional genetics, high-throughput studies, and genome-wide analysis. Together, these efforts will provide profound mechanistic insights into how heterochromatin is controlled and coordinated in fission yeast. While such molecular mechanisms are difficult to study in multicellular systems, they may reflect basic principles that due to the strong conservation of heterochromatin can likely be transferred also to metazoans and humans.