Characterization of Pch2 localization determinants reveals a nucleolar-independent role in the meiotic recombination checkpoint

Herruzo, Esther; Santos, Beatriz Tamargo; Freire, Raimundo; Carballo, Jesús A.; San-Segundo, Pedro A.

doi:10.1101/541367

Cited by 4 publications

(16 citation statements)

References 74 publications

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“…In line with previous observations [43], Pch2 was required for phosphorylation of Hop1 in checkpoint activating conditions: we observed that the abundance of Hop1 and presence of Pch2 affected the phosphorylated pool of Hop1 (for example, compare wild type, pch2 dmc1, HOP1/hop1 dmc1 and pch2 HOP1/hop1 dmc1 in Figure 2F). Combined with our findings (see also above; Figure 1) and earlier work [41], these observations suggest a dynamic exchange between non-chromosomal and chromosomal ( i.e. active) Hop1, driven by (non-chromosomal) Pch2 function influence checkpoint function.…”

Section: Resultssupporting

confidence: 91%

“…In zip1Δ cells, Pch2 is not associated with chromosomes, except with nucleolar chromatin [5]. The nucleolar pool of Pch2 is unlikely to be involved in checkpoint function [41], and work in Drosophila suggests that Pch2 is associated with nuclear lamina [41, 42]. In line with previous observations [43], Pch2 was required for phosphorylation of Hop1 in checkpoint activating conditions: we observed that the abundance of Hop1 and presence of Pch2 affected the phosphorylated pool of Hop1 (for example, compare wild type, pch2 dmc1, HOP1/hop1 dmc1 and pch2 HOP1/hop1 dmc1 in Figure 2F).…”

Section: Resultsmentioning

confidence: 99%

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Homeostatic control of meiotic G2/prophase checkpoint function by Pch2 and Hop1

Raina

Vader

2020

Preprint

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17 Checkpoints cascades coordinate cell cycle progression with essential chromosomal 18 processes. During meiotic G2/prophase, recombination and chromosome synapsis are 19 monitored by what are considered distinct checkpoints [1-3]. In budding yeast, the AAA+ 20 ATPase Pch2 is thought to specifically promote cell cycle delay in response to synapsis defects 21 [4-6]. However, unperturbed pch2D cells are delayed in meiotic G2/prophase [6], suggesting 22 paradoxical roles for Pch2 in cell cycle progression. Here, we provide insight into the checkpoint 23 roles of Pch2 and its connection to Hop1, a HORMA domain-containing client protein. 24 Contrary to current understanding, we find that the Pch2-Hop1 module is crucial for 25 checkpoint function in response to both recombination and synapsis defects, thus revealing a 26 shared meiotic checkpoint cascade. Meiotic checkpoint responses are transduced by DNA 27 break-dependent phosphorylation of Hop1 [7, 8]. Based on our data and on the effect of Pch2 28 on HORMA topology [9-11], we propose that Pch2 promotes checkpoint proficiency by 29 catalyzing the availability of signaling-competent Hop1. Conversely, we demonstrate that Pch2 30 can act as a checkpoint silencer, also in the face of persistent DNA repair defects. We establish 31 a framework in which Pch2 and Hop1 form a homeostatic module that governs general meiotic 32 checkpoint function. We show that this module can -depending on the cellular context -fuel 33 or extinguish meiotic checkpoint function, which explains the contradictory roles of Pch2 in cell 34 cycle control. Within the meiotic checkpoint, the Pch2-Hop1 module thus operates analogous 35 to the Pch2/TRIP13-Mad2 module in the spindle assembly checkpoint that monitors 36 chromosome segregation [12-16]. 37 38 repair 40 41 Results and discussion 42 43 Feedback regulation between Hop1, Zip1 and Pch2 influences SC assembly 44 In G2/prophase of the meiotic program, double strand break (DSB) formation 45 recombinational repair, synapsis of homologous chromosomes is integrated with cell cycle 46 progression [17]. In budding yeast, Zip1-mediated Synaptonemal Complex (SC) assembly promotes 47 cell cycle progression by regulating chromosome-based checkpoint activity [18-20]. The 48 chromosome axis, of which Hop1 is a key component, forms the structural foundation for recruitment 49 of the SC [21]. The functionality of meiotic checkpoints that monitor recombination and synapsis is 50 linked to successful recombination and SC establishment, and the function of Pch2 is associated with 51 both Hop1 and Zip1: Hop1 is a client of Pch2 [22][23][24][25], and chromosomal association of Pch2 is tightly 52 linked to Zip1/SC establishment [5, 20, 23,[26][27][28][29]. To understand the wiring between these numerous 53 crucial events during meiotic G2/prophase and the role of Pch2 herein, we initially investigated SC 54 formation and its relation to Pch2, Hop1 and Zip1. 55 SC components, like Zip1, can form extrachromosomal aggregates known as polycomplexes 56 (PCs) [4]. Cells ...

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Homeostatic control of meiotic G2/prophase checkpoint function by Pch2 and Hop1

Raina

Vader

2020

Preprint

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“…In addition, it has been reported that the NTD of Pch2 harbors a nuclear localization signal [39]. As such, Pch2 243-564 is likely inefficiently localized to the nucleus [39]. In conclusion, we find that during meiotic G2/prophase, Pch2 associates within the body of a selected group of RNAPII-associated genes, and that recruitment depends on characteristics of AAA+ proteins.…”

Section: Gse144835supporting

confidence: 56%

“…In this regard, it is important to note that the expression of Pch2 lacking its NTD (Pch2 243-564) was significantly lower as compared to wild type Pch2 (see also S4 Table for a spreadsheet containing, in separate sheets, selected western blot signals for representative western blot images presented in the manuscript). In addition, it has been reported that the NTD of Pch2 harbors a nuclear localization signal [39]. As such, Pch2 243-564 is likely inefficiently localized to the nucleus [39].…”

Section: Gse144835mentioning

confidence: 99%

Active transcription and Orc1 drive chromatin association of the AAA+ ATPase Pch2 during meiotic G2/prophase

2020

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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 meiosisspecific '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 RNA-PII-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.

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“…However, using either an inactivating mutant of ORC1, which is required for Pch2 nucleolar localization or a nucleolar localization-deficient mutant of PCH2, San-Segundo et colleagues observed that, surprisingly, the pachytene checkpoint was still active. This work reveals that the checkpoint functions of Pch2 are independent of its localization to the nucleolus, which reveals a puzzling aspect of the pachytene checkpoint (Herruzo et al, 2019).…”

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confidence: 94%