bims-micesi 2024-12-15 papers

bims-micesi

Biomed News

on Mitotic cell signalling

Issue of 2024–12–15
nine papers selected by
Valentina Piano, Uniklinik Köln

TTF2 promotes replisome eviction from stalled forks in mitosis.
Lamin B1-dependent regulation of human separase in mitosis.
MLL/WDR5 complex recruits centriolar satellite protein Cep72 to regulate microtubule nucleation and spindle formation.
Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint.
TBP bookmarks and preserves neural stem cell fate memory by orchestrating local chromatin architecture.
Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex.
Novel BRCA1-PLK1-CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis.
A missense variant effect map for the human tumor-suppressor protein CHK2.
Fission yeast essential nuclear pore protein Nup211 regulates the expression of genes involved in cytokinesis.

bioRxiv. 2024 Nov 30. pii: 2024.11.30.626186. [Epub ahead of print]

TTF2 promotes replisome eviction from stalled forks in mitosis.

Geylani Can, Maksym Shyian, Archana Krishnamoorthy, Yang Lim, R Alex Wu, Manal S Zaher, Markus Raschle, Johannes C Walter, David S Pellman.

When cells enter mitosis with under-replicated DNA, sister chromosome segregation is compromised, which can lead to massive genome instability. The replisome-associated E3 ubiquitin ligase TRAIP mitigates this threat by ubiquitylating the CMG helicase in mitosis, leading to disassembly of stalled replisomes, fork cleavage, and restoration of chromosome structure by alternative end-joining. Here, we show that replisome disassembly requires TRAIP phosphorylation by the mitotic Cyclin B-CDK1 kinase, as well as TTF2, a SWI/SNF ATPase previously implicated in the eviction of RNA polymerase from mitotic chromosomes. We find that TTF2 tethers TRAIP to replisomes using an N-terminal Zinc finger that binds to phosphorylated TRAIP and an adjacent TTF2 peptide that contacts the CMG-associated leading strand DNA polymerase ε. This TRAIP-TTF2-pol ε bridge, which forms independently of the TTF2 ATPase domain, is essential to promote CMG unloading and stalled fork breakage. Conversely, RNAPII eviction from mitotic chromosomes requires the ATPase activity of TTF2. We conclude that in mitosis, replisomes undergo a CDK- and TTF2-dependent structural reorganization that underlies the cellular response to incompletely replicated DNA.

DOI: https://doi.org/10.1101/2024.11.30.626186
bioRxiv. 2024 Nov 28. pii: 2024.11.28.625860. [Epub ahead of print]

Lamin B1-dependent regulation of human separase in mitosis.

Julien Picotto, Francesca Cipressa, Didier Busso, Giovanni Cenci, Pascale Bertrand, Gaëlle Pennarun.

Separase plays a central role in chromosome separation during mitosis and in centrosome cycle. Tight control of separase activity is required to prevent unscheduled resolution of sister chromatid cohesion and centrosome aberrations, thereby preserving genome stability. In mammals, despite their disassembly in early mitosis, some nuclear envelope components possess mitotic roles, but links with separase activity remain unexplored. Here, we uncover a new mechanism of separase regulation involving lamin B1, a key nuclear envelope factor. We show that separase and lamin B1 associate preferentially during early stages of mitosis. Importantly, lamin B1 depletion leads to an increase in separase recruitment on chromosomes together with premature chromatid separation, a phenotype reminiscent of separase overexpression. Conversely, similar to separase depletion, lamin B1 overexpression induces formation of diplochromosomes- resulting from chromatid separation failure-, in association with centrosome amplification. Importantly, increasing separase level prevents lamin B1-induced centrosome aberrations, suggesting a separase defect at their origin. Indeed, we show that overexpression of lamin B1 leads to a decrease in the recruitment of separase to the chromosome and a delay in its activity. Taken together, this study unveils a novel mechanism of separase regulation involving the nuclear envelope factor lamin B1, that is crucial for genome integrity maintenance.

DOI: https://doi.org/10.1101/2024.11.28.625860
Sci Adv. 2024 Dec 13. 10(50): eadn0086

MLL/WDR5 complex recruits centriolar satellite protein Cep72 to regulate microtubule nucleation and spindle formation.

Swathi Chodisetty, Aditi Arora, Kausika Kumar Malik, Himanshu Goel, Shweta Tyagi.

Dysfunction of the centrosome, the major microtubule-organizing center of the cell, is implicated in microcephaly. Haploinsufficiency of mixed-lineage leukemia (MLL/KMT2A) protein causes Wiedemann-Steiner syndrome (WSS), a neurodevelopmental disorder associated with microcephaly. However, whether MLL has a function at the centrosome is not clear. Here, we show that loss of the MLL/WDR5 complex affects microtubule nucleation and regrowth. MLL/WDR5 localize to the pericentriolar material and interact with centriolar satellite protein Cep72 and γ-tubulin ring complex proteins (γ-TuRCs). MLL/WDR5 promote the localization of γ-TuRCs and structural proteins like AKAP9 to the centrosome during interphase and mitosis, a phenotype also observed in cells derived from patients with WSS. During mitosis, loss of MLL, WDR5, and Cep72 affects spindle formation and leads to misaligned chromosomes. Last, we show that MLL and WDR5 recruit Cep72 to the centrosome. Our studies provide insight into an undiscovered role of MLL at the centrosome and elucidate how centriolar satellite proteins like Cep72 can be recruited to the centrosome.

DOI: https://doi.org/10.1126/sciadv.adn0086
Elife. 2024 Dec 10. pii: RP94334. [Epub ahead of print]13

Prolonged cell cycle arrest in response to DNA damage in yeast requires the maintenance of DNA damage signaling and the spindle assembly checkpoint.

Felix Y Zhou, David P Waterman, Marissa Ashton, Suhaily Caban-Penix, Gonen Memisoglu, Vinay V Eapen, James E Haber.

  Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12-15 hr, after which cells 'adapt' to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and maintain the arrest. Dun1 is required for the establishment, but not the maintenance, of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with two persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2, Rad9, and Rad53; however, after 15 hr these proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest require overlapping but different sets of factors.

Keywords:  DDC; DNA damage checkpoint; DSB; HO endonuclease; S. cerevisiae; S. cerevisiae budding yeast; SAC; auxin-inducible degron; chromosomes; double-strand break; gene expression; spindle assembly 3 checkpoint

DOI:  https://doi.org/10.7554/eLife.94334
Mol Cell. 2024 Dec 05. pii: S1097-2765(24)00944-4. [Epub ahead of print]

TBP bookmarks and preserves neural stem cell fate memory by orchestrating local chromatin architecture.

Yuying Shen, Kun Liu, Jie Liu, Jingwen Shen, Tongtong Ye, Runxiang Zhao, Rulan Zhang, Yan Song.

  Mitotic bookmarking has been posited as an important strategy for cells to faithfully propagate their fate memory through cell generations. However, the physiological significance and regulatory mechanisms of mitotic bookmarking in neural development remain unexplored. Here, we identified TATA-binding protein (TBP) as a crucial mitotic bookmarker for preserving the fate memory of Drosophila neural stem cells (NSCs). Phosphorylation by the super elongation complex (SEC) is important for TBP to retain as discrete foci at mitotic chromosomes of NSCs to effectively transmit their fate memory. TBP depletion leads to drastic NSC loss, whereas TBP overexpression enhances the ability of SEC to induce neural progenitor dedifferentiation and tumorigenesis. Importantly, TBP achieves its mitotic retention through recruiting the chromatin remodeler EP400, which in turn increases local chromatin accessibility via depositing H2A.Z. Thus, local chromatin remodeling ensures mitotic bookmarking, which may represent a general principle underlying the preservation of cell fate memory.

Keywords:  Drosophila melanogaster; EP400; H2A.Z; TBP; cell fate memory; chromatin remodeler; mitotic bookmarking; mitotic retention; neural development; neural stem cell

DOI:  https://doi.org/10.1016/j.molcel.2024.11.019
bioRxiv. 2024 Dec 03. pii: 2024.11.26.625568. [Epub ahead of print]

Structural and functional insights into activation and regulation of the dynein-dynactin-NuMA complex.

Merve Aslan, Ennio A d'Amico, Nathan H Cho, Aryan Taheri, Yuanchang Zhao, Xinyue Zhong, Madeline Blaauw, Andrew P Carter, Sophie Dumont, Ahmet Yildiz.

During cell division, NuMA orchestrates the focusing of microtubule minus-ends in spindle poles and cortical force generation on astral microtubules by interacting with dynein motors, microtubules, and other cellular factors. Here we used in vitro reconstitution, cryo-electron microscopy, and live cell imaging to understand the mechanism and regulation of NuMA. We determined the structure of the processive dynein/dynactin/NuMA complex (DDN) and showed that the NuMA N-terminus drives dynein motility in vitro and facilitates dynein-mediated transport in live cells. The C-terminus of NuMA directly binds to and suppresses the dynamics of the microtubule minus-end. Full-length NuMA is autoinhibited, but mitotically phosphorylated NuMA activates dynein in vitro and interphase cells. Together with dynein, activated full-length NuMA focuses microtubule minus-ends into aster-like structures. The binding of the cortical protein LGN to the NuMA C-terminus results in preferential binding of NuMA to the microtubule plus-end. These results provide critical insights into the activation of NuMA and dynein for their functions in the spindle body and the cell cortex.

DOI: https://doi.org/10.1101/2024.11.26.625568
Nucleic Acids Res. 2024 Dec 09. pii: gkae1207. [Epub ahead of print]

Novel BRCA1-PLK1-CIP2A axis orchestrates homologous recombination-mediated DNA repair to maintain chromosome integrity during oocyte meiosis.

Crystal Lee, Jeong Su Oh.

Double-strand breaks (DSBs) are a formidable threat to genome integrity, potentially leading to cancer and various genetic diseases. The prolonged lifespan of mammalian oocytes increases their susceptibility to DNA damage over time. While somatic cells suppress DSB repair during mitosis, oocytes exhibit a remarkable capacity to repair DSBs during meiotic maturation. However, the precise mechanisms underlying DSB repair in oocytes remain poorly understood. Here, we describe the pivotal role of the BRCA1-PLK1-CIP2A axis in safeguarding genomic integrity during meiotic maturation in oocytes. We found that inhibition of homologous recombination (HR) severely impaired chromosome integrity by generating chromosome fragments during meiotic maturation. Notably, HR inhibition impaired the recruitment of CIP2A to damaged chromosomes, and the depletion of CIP2A led to chromosome fragmentation following DSB induction. Moreover, BRCA1 depletion impaired chromosomal recruitment of CIP2A, but not vice versa. Importantly, the impaired chromosomal recruitment of CIP2A could be rescued by PLK1 inhibition. Consequently, our findings not only underscore the importance of the chromosomal recruitment of CIP2A in preventing chromosome fragmentation, but also demonstrate the regulatory role of the BRCA1-PLK1-CIP2A axis in this process during oocyte meiotic maturation.

DOI: https://doi.org/10.1093/nar/gkae1207
Am J Hum Genet. 2024 Dec 05. pii: S0002-9297(24)00382-3. [Epub ahead of print]111(12): 2675-2692

A missense variant effect map for the human tumor-suppressor protein CHK2.

Marinella Gebbia, Daniel Zimmerman, Rosanna Jiang, Maria Nguyen, Jochen Weile, Roujia Li, Michelle Gavac, Nishka Kishore, Song Sun, Rick A Boonen, Rayna Hamilton, Jennifer N Dines, Alexander Wahl, Jason Reuter, Britt Johnson, Douglas M Fowler, Fergus J Couch, Haico van Attikum, Frederick P Roth.

  The tumor suppressor CHEK2 encodes the serine/threonine protein kinase CHK2 which, upon DNA damage, is important for pausing the cell cycle, initiating DNA repair, and inducing apoptosis. CHK2 phosphorylation of the tumor suppressor BRCA1 is also important for mitotic spindle assembly and chromosomal stability. Consistent with its cell-cycle checkpoint role, both germline and somatic variants in CHEK2 have been linked to breast and other cancers. Over 90% of clinical germline CHEK2 missense variants are classified as variants of uncertain significance, complicating diagnosis of CHK2-dependent cancer. We therefore sought to test the functional impact of all possible missense variants in CHK2. Using a scalable multiplexed assay based on the ability of human CHK2 to complement DNA sensitivity of Saccharomyces cerevisiae cells lacking the CHEK2 ortholog, RAD53, we generated a systematic "missense variant effect map" for CHEK2 missense variation. The map reflects known biochemical features of CHK2 while offering new biological insights. It also provides strong evidence toward pathogenicity for some clinical missense variants and supporting evidence toward benignity for others. Overall, this comprehensive missense variant effect map contributes to understanding of both known and yet-to-be-observed CHK2 variants.

Keywords:  CHEK2; CHK2; DNA damage repair; Saccharomyces cerevisiae; breast cancer; deep mutational scanning; tumor suppressor; variant effect mapping; yeast

DOI:  https://doi.org/10.1016/j.ajhg.2024.10.013
PLoS One. 2024 ;19(12): e0312095

Fission yeast essential nuclear pore protein Nup211 regulates the expression of genes involved in cytokinesis.

Domenick Kamel, Ayisha Sookdeo, Ayana Ikenouchi, Hualin Zhong.

Nuclear pore proteins control nucleocytoplasmic transport; however, certain nucleoporins play regulatory roles in activities such as transcription and chromatin organization. The fission yeast basket nucleoporin Nup211 is implicated in mRNA export and is essential for cell viability. Nup211 preferentially associates with heterochromatin, however, it is unclear whether it plays a role in regulating transcription. To better understand its functions, we constructed a nup211 "shut-off" strain and observed that Nup211 depletion led to severe defects in cell cycle progression, including septation and cytokinesis. Using RNA-Seq and RT-qPCR, we revealed that loss of Nup211 significantly altered the mRNA levels of a set of genes crucial for cell division. Using domain analysis and CRISPR/cas9 technology, we determined that the first 655 residues of Nup211 are sufficient for viability. This truncated protein was detected at the nuclear periphery. Furthermore, exogenous expression of this domain in nup211 shut-off cells effectively restored both cell morphology and transcript abundance for some selected genes. Our findings unveil a novel role for Nup211 in regulating gene expression.

DOI: https://doi.org/10.1371/journal.pone.0312095