Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase [ChIP-Seq]

Pch2 is an AAA+ protein that controls DNA break formation, recombination and checkpoint signaling during meiotic G2/prophase. Chromosomal association of Pch2 is linked to these processes, and several factors influence the association of Pch2 to euchromatin and the specialized chromatin of the ribosomal (r)DNA array of budding yeast. Here, we describe a comprehensive mapping of Pch2 localization across the budding yeast genome during meiotic G2/prophase. Within non-rDNA chromatin, Pch2 associates with a subset of actively RNA Polymerase II (RNAPII)-dependent transcribed genes. Chromatin immunoprecipitation (ChIP)- and microscopy-based analysis reveals that active transcription is required for chromosomal recruitment of Pch2. Similar to what was previously established for association of Pch2 with rDNA chromatin, we find that Orc1, a component of the Origin Recognition Complex (ORC), is required for the association of Pch2 to these euchromatic, transcribed regions, revealing a broad connection between chromosomal association of Pch2 and Orc1/ORC function. Ectopic mitotic expression is insufficient to drive recruitment of Pch2, despite the presence of active transcription and Orc1/ORC in mitotic cells. This suggests meiosis-specific ‘licensing’ of Pch2 recruitment to sites of transcription, and accordingly, we find that the synaptonemal complex (SC) component Zip1 is required for the recruitment of Pch2 to transcription-associated binding regions. Interestingly, Pch2 binding patterns are distinct from meiotic axis enrichment sites (as defined by Red1, Hop1 and Rec8). This suggests that although Pch2 is linked to axis/SC-directed recruitment and function, the chromosomal population of Pch2 described here is not directly associated with chromosomal axis sites. In line with this observation, interfering with the pool of Pch2 that associates with active RNAPII transcription does not lead to effects on the chromosomal abundance of Hop1, a known axial client of Pch2. We thus report characteristics and dependencies for Pch2 recruitment to meiotic chromosomes, and reveal an unexpected link between Pch2, SC formation, chromatin and active transcription. 3X-FLAG Pch2 wild type and 3X-FLAGPch2-E399Q (samples collected at 4h in SPO medium) binding sites were identified by ChIP-seq in triplicate using a Illumina HiSeq 3000 platform

Pch2是一类AAA+蛋白（AAA+ protein），可在减数分裂G2/早前期（meiotic G2/prophase）中调控DNA断裂形成、重组过程与检查点信号传导。Pch2的染色体结合与上述生理过程密切相关，且多种因素可影响其与出芽酵母常染色质及核糖体（r）DNA阵列特化染色质的缔合。本研究对减数分裂G2/早前期出芽酵母全基因组范围内的Pch2定位进行了全面图谱绘制。在非rDNA染色质区域中，Pch2可结合于一类由RNA聚合酶II（RNA Polymerase II, RNAPII）介导的活跃转录基因子集。基于染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）与显微镜成像的分析结果显示，Pch2的染色体招募依赖于活跃的转录过程。与此前已阐明的Pch2与rDNA染色质缔合的调控机制类似，本研究发现起源识别复合物（Origin Recognition Complex, ORC）的组分Orc1，是Pch2结合上述常染色质转录区域的必需因子，这一发现揭示了Pch2的染色体缔合与Orc1/ORC功能之间存在广泛的调控关联。尽管有丝分裂细胞中同样存在活跃转录与Orc1/ORC蛋白，但异位表达Pch2不足以驱动其染色体招募。这提示Pch2向转录位点的招募存在减数分裂特异性的“许可”调控机制；据此我们发现，联会复合体（synaptonemal complex, SC）组分Zip1是Pch2招募至转录相关结合区域的必需因子。有趣的是，Pch2的全基因组结合模式与减数分裂轴富集位点（以Red1、Hop1及Rec8为标记蛋白）存在显著差异。这表明尽管Pch2与染色体轴/联会复合体介导的招募及功能相关，但本研究中所鉴定的Pch2染色体结合群体并不直接结合染色体轴区域。与这一观察结果相符，干扰与活跃RNAPII转录相关的Pch2蛋白池，并不会影响已知的轴下游靶标Hop1的染色体丰度。综上，本研究阐明了Pch2向减数分裂染色体招募的特征与调控依赖机制，并揭示了Pch2、联会复合体形成、染色质与活跃转录之间此前未被发现的关联。本研究利用Illumina HiSeq 3000测序平台，通过三次生物学重复的染色质免疫沉淀测序（ChIP-seq），鉴定了3X-FLAG标记的野生型Pch2与3X-FLAG标记的Pch2-E399Q突变体（在SPO培养基中培养4小时后收集的样本）的全基因组结合位点。