Abstract
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). Inactivating RNAPII-dependent transcription/Orc1 does not lead to effects on the chromosomal abundance of Hop1, a known chromosomal client of Pch2, suggesting a complex relationship between SC formation, Pch2 recruitment and Hop1 chromosomal association. 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.
Highlights
Meiosis is a specialized developmental program dedicated to the production of genetically unique haploid gametes [1]
Our results show that Pch2 is recruited to a subset of actively transcribed genes, and we find that active RNA Polymerase II (RNAPII) transcription contributes to Pch2 chromosomal association
These findings reveal a connection between Pch2, Orc1 and RNAPII activity during meiosis
Summary
Meiosis is a specialized developmental program dedicated to the production of genetically unique haploid gametes [1]. DSB formation happens in the context of a specialized, meiosis-specific chromosome architecture [3] [4]. Several protein factors, such as Hop and Red in budding yeast, (whose functional and structural homologs are HORMAD1/ 2 and SYCP2/3 in mammalians, respectively) [5] [6] [7] drive the assembly of chromosomes into linear arrays of chromatin loops that emanate from a proteinaceous structure termed the meiotic chromosome axis. In zip1Δ cells, Pch cannot be recruited to meiotic chromosomes (except to the nucleolus/rDNA; see below) [18] This is unlikely via a direct molecular interaction. A recent report has linked topoisomerase II (Top2) function to Pch association with synapsed chromosomes [24], hinting at a connection between chromosome topology and Pch recruitment
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