Biology subjects

Michael, W. M.

Publications and source records attributed to Michael, W. M..

5 recordsLinked to original sources

bioRxiv2025

A role for the nuclear RNAi pathway in global transcriptional silencing in C. elegans primordial germ cells

In C. elegans, if embryos hatch into an environment lacking nutrients the primordial germ cells (PGCs) will arrest mRNA transcription and enter quiescence until food becomes available. Our previous work had shown that the global transcriptional silencing (GTS) that occurs in PGCs during L1 starvation requires a "hyperdeposition" of the repressive histone mark H3K9me3 in germline chromatin. Hyperdeposition is defined as an increase in the amount of H3K9me3 embedded in germline chromatin, relative to neighboring somatic nuclei. Hyperdeposition initiates in embryonic PGCs and is maintained in starved L1s. Here, we show that the nuclear RNAi pathway contributes to both the initiation and maintenance of H3K9me3 hyperdeposition. Interestingly, both known nuclear Argonautes, HRDE-1 and NRDE-3, play a role in hyperdeposition and they do so in a sequential manner. We show that HRDE-1 acts in embryonic PGCs to initiate hyperdeposition and then, at the embryo-to-L1 transition, HRDE-1 becomes dispensable and NRDE-3 is required to maintain H3K9me3 levels, and for GTS. We also examine the timing of H3K9me3 hyperdeposition and find that it initiates soon after germline zygotic genome activation (ZGA) occurs. Our data suggest a model where ZGA promotes both gene expression and H3K9me3 deposition at active loci, and under starvation conditions these H3K9me3 marks are then employed to silence the germline genome.

developmental biology↗

bioRxiv2024

Transcriptome analysis in C. elegans early embryos upon depletion of the Topoisomerase 2/condensin II axis.

In C. elegans, the chromosome compaction factors topoisomerase 2 (TOP-2) and condensin II have been shown to globally repress transcription in multiple contexts. Our group has previously reported that TOP-2 and condensin II repress transcription in the C. elegans germline during larval starvation, oocyte maturation, and in germline progenitor cells of the early embryo. Here, we assess the transcriptome of early embryos treated with RNAi against TOP-2 and the condensin II subunit CAPG-2. We found 144 upregulated and 172 downregulated genes. Further analysis showed that the upregulated genes are mostly somatic, with a host of neuronal cells present in our tissue enrichment analysis.

developmental biology↗

bioRxiv2022

NBS1 binds directly to TOPBP1 via disparate interactions between the NBS1 BRCT1 domain and the TOPBP1 BRCT1 and BRCT2 domains

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. In this work we examine the interaction between TOPBP1 and NBS1 in detail. We report that NBS1 uses its BRCT1 domain to interact with TOPBP1s BRCT1 domain and, separately, with TOPBP1s BRCT2 domain. Thus, NBS1 can make two distinct contacts with TOPBP1. We report that recombinant TOPBP1 and NBS1 proteins bind one another in a purified system, showing that the interaction is direct and does not require post-translational modifications. Surprisingly, we also report that intact BRCT domains are not required for these interactions, as truncated versions of the domains are sufficient to confer binding. For TOPBP1, we find that small 24-29 amino acid sequences within BRCT1 or BRCT2 allow binding to NBS1, in a transferrable manner. These data expand our knowledge of how the crucial DNA damage response proteins TOPBP1 and NBS1 interact with one another and set the stage for functional analysis of the two disparate binding sites for NBS1 on TOPBP1.

cell biology↗

bioRxiv2022

Multi-site phosphorylation of the TOPBP1 ATR Activation Domain propels ATR signaling during the response to DNA breaks

TOPBP1 is a BRCT domain-containing scaffold protein that plays important roles in a diverse array of cellular processes, including DNA damage signaling, DNA repair, DNA replication, and mRNA transcription. For DNA damage signaling, TOPBP1 activates the crucial damage response kinase ATR and this occurs during replication stress as well as during a DNA double-strand break response. ATR signaling allows cells to survive genotoxic issues and represents a formidable barrier to transformation to a tumorigenic state. Despite its importance to genome stability, the biochemical mechanism for how TOPBP1 activates ATR is not fully understood. TOPBP1 uses a discrete domain, termed the ATR activation domain, to stimulate ATR kinase. Recent work has shown that the AAD must be in a multimeric state to activate ATR. Other work has shown that phosphorylation of the AAD on serine 1131 (in Xenopus) is important for its function, and some have suggested that this is linked to the formation of TOPBP1 condensates during a DNA damage response. In this study we examine AAD phosphorylation in detail and we report three important new findings. One, S1131 phosphorylation promotes ATR activation in a manner independent of condensate formation and is instead linked to promoting multimerization of the AAD. Two, we identify a novel sight of AAD phosphorylation, on T1098, and show that is required for ATR activation. Three, we identify additional, candidate phosphorylation sites, some of which fit the consensus for casein kinase 2, and we show that casein kinase 2 activity is required for the AAD to perform its function. These studies show that multi-site phosphorylation of the AAD is an important component of the mechanism by which TOPBP1 activates ATR.

cell biology↗

bioRxiv2022

The TOP-2-condensin II axis silences transcription during germline specification in C. elegans

In C. elegans, the germline is specified via a preformation mechanism that relies on the PIE-1 proteins ability to globally silence mRNA transcription in germline precursor cells, also known as the P-lineage. Recent work from our group has identified additional genome silencing events in C. elegans during oogenesis and in starved L1 larvae, and these require the condensin II complex, topoisomerase II (TOP-2), and components of the H3K9me/heterochromatin pathway. Interestingly, silencing in oocytes also requires PIE-1, but this is not the case in starved L1s. Here, we ask if additional genome silencing components besides PIE-1 are required to repress gene expression in the P-lineage of early embryos, and we find that condensin II and TOP-2 are required and the H3K9me/heterochromatin pathway is not. We show that depletion of condensin II/TOP-2 activates the normally suppressed RNA polymerase II to inappropriately transcribe somatic genes in the P-lineage. We also present evidence that while both PIE-1 and condensin II/TOP-2 are required for genome silencing in the P-lineage, PIE-1 can silence transcription independently of condensin II/TOP-2 when misexpressed in somatic cells. Thus, in oocytes, all three genome silencing systems (TOP-2/condensin II, H3K9me, and PIE-1) are operational while in both early embryos and starved L1s two of the three are active (TOP- 2/condensin II and PIE-1 for early embryos, TOP-2/condensin II and H3K9me for starved L1s). Our data show that multiple, redundantly acting genome silencing mechanisms act in a mix and match manner to repress transcription at different developmental stages in the C. elegans germline.

developmental biology↗