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1.
Nucleic Acids Res ; 52(12): 7063-7080, 2024 Jul 08.
Article in English | MEDLINE | ID: mdl-38808662

ABSTRACT

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs), which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here, we discover that the histone demethylase PHF2 associates with RAD21, a core subunit of cohesin, to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably, the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient, active replication origins, and can act as boundaries to separate adjacent domains. Accordingly, PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore, we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation, and that PHF2's histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops, where cohesin represses dormant replication origins directly or indirectly, to sustain DNA replication in NSC.


Subject(s)
Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , Cohesins , DNA Replication , DNA-Binding Proteins , Neural Stem Cells , Animals , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Mice , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Chromatin/metabolism , Replication Origin , Histone Demethylases/metabolism , Histone Demethylases/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Genome/genetics , CCCTC-Binding Factor/metabolism , CCCTC-Binding Factor/genetics , Mice, Knockout
2.
Cell Death Differ ; 30(8): 1973-1987, 2023 08.
Article in English | MEDLINE | ID: mdl-37468549

ABSTRACT

MAD2 is a spindle assembly checkpoint protein that participates in the formation of mitotic checkpoint complex, which blocks mitotic progression. RNF8, an established DNA damage response protein, has been implicated in mitotic checkpoint regulation but its exact role remains poorly understood. Here, RNF8 proximity proteomics uncovered a role of RNF8-MAD2 in generating the mitotic checkpoint signal. Specifically, RNF8 competes with a small pool of p31comet for binding to the closed conformer of MAD2 via its RING domain, while CAMK2D serves as a molecular scaffold to concentrate the RNF8-MAD2 complex via transient/weak interactions between its p-Thr287 and RNF8's FHA domain. Accordingly, RNF8 overexpression impairs glioma stem cell (GSC) mitotic progression in a FHA- and RING-dependent manner. Importantly, low RNF8 expression correlates with inferior glioma outcome and RNF8 overexpression impedes GSC tumorigenicity. Last, we identify PLK1 inhibitor that mimics RNF8 overexpression using a chemical biology approach, and demonstrate a PLK1/HSP90 inhibitor combination that synergistically reduces GSC proliferation and stemness. Thus, our study has unveiled a previously unrecognized CAMK2D-RNF8-MAD2 complex in regulating mitotic checkpoint with relevance to gliomas, which is therapeutically targetable.


Subject(s)
Cell Cycle Proteins , Glioma , Mad2 Proteins , Humans , Adaptor Proteins, Signal Transducing/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Glioma/genetics , Glioma/metabolism , M Phase Cell Cycle Checkpoints , Mad2 Proteins/genetics , Mad2 Proteins/metabolism , Mitosis , Nuclear Proteins/metabolism , Spindle Apparatus/metabolism , Ubiquitin-Protein Ligases/metabolism
3.
Cell Death Differ ; 29(7): 1379-1394, 2022 07.
Article in English | MEDLINE | ID: mdl-35058574

ABSTRACT

The histone variant H2AZ is overexpressed in diverse cancer types where it facilitates the accessibility of transcriptional regulators to the promoters of cell cycle genes. However, the molecular basis for its dysregulation in cancer remains unknown. Here, we report that glioblastomas (GBM) and glioma stem cells (GSCs) preferentially overexpress H2AZ for their proliferation, stemness and tumorigenicity. Chromatin accessibility analysis of H2AZ2 depleted GSC revealed that E2F1 occupies the enhancer region within H2AZ2 gene promoter, thereby activating H2AZ2 transcription. Exploration of other H2AZ2 transcriptional activators using a customized "anti-H2AZ2" query signature for connectivity map analysis identified STAT3. Co-targeting E2F and STAT3 synergistically reduced the levels of H2AZ, histone 3 lysine 27 acetylation (H3K27ac) and cell cycle gene transcription, indicating that E2F1 and STAT3 synergize to activate H2AZ gene transcription in GSCs. Remarkably, an E2F/STAT3 inhibitor combination durably suppresses GSC tumorigenicity in an orthotopic GBM xenograft model. In glioma patients, high STAT3 signaling is associated with high E2F1 and H2AZ2 expression. Thus, GBM has uniquely opted the use of E2F1- and STAT3-containing "enhanceosomes" that integrate multiple signaling pathways to achieve H2AZ gene activation, supporting a translational path for the E2F/STAT3 inhibitor combination to be applied in GBM treatment.


Subject(s)
Brain Neoplasms , E2F1 Transcription Factor , Glioblastoma , Glioma , Histones , STAT3 Transcription Factor , Acetylation , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Chromatin/genetics , Chromatin/metabolism , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/metabolism , Glioblastoma/genetics , Glioblastoma/metabolism , Glioma/genetics , Glioma/metabolism , Histones/metabolism , Humans , Neoplastic Stem Cells/metabolism , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism
4.
Proc Natl Acad Sci U S A ; 117(27): 15923-15934, 2020 07 07.
Article in English | MEDLINE | ID: mdl-32571920

ABSTRACT

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease in the tropics and subtropics with high morbidity and mortality. The facultative intracellular bacterium induces host cell fusion through its type VI secretion system 5 (T6SS5) as an important part of its pathogenesis in mammalian hosts. This allows it to spread intercellularly without encountering extracellular host defenses. We report that bacterial T6SS5-dependent cell fusion triggers type I IFN gene expression in the host and leads to activation of the cGAMP synthase-stimulator of IFN genes (cGAS-STING) pathway, independent of bacterial ligands. Aberrant and abortive mitotic events result in the formation of micronuclei colocalizing with cGAS, which is activated by double-stranded DNA. Surprisingly, cGAS-STING activation leads to type I IFN transcription but not its production. Instead, the activation of cGAS and STING results in autophagic cell death. We also observed type I IFN gene expression, micronuclei formation, and death of chemically induced cell fusions. Therefore, we propose that the cGAS-STING pathway senses unnatural cell fusion through micronuclei formation as a danger signal, and consequently limits aberrant cell division and potential cellular transformation through autophagic death induction.


Subject(s)
Membrane Proteins/metabolism , Nucleotidyltransferases/genetics , Burkholderia pseudomallei/metabolism , Cell Fusion , DNA Damage , Gene Expression Regulation , Genomic Instability , Hep G2 Cells , Humans , Immunity, Innate , Interferon Type I/genetics , Interferon Type I/metabolism , Membrane Proteins/genetics , Microscopy, Confocal , Nucleotidyltransferases/metabolism , Signal Transduction
5.
Mutat Res Rev Mutat Res ; 777: 29-51, 2018.
Article in English | MEDLINE | ID: mdl-30115429

ABSTRACT

In recent years, the paradigm that genomic abnormalities in cancer cells arise through progressive accumulation of mutational events has been challenged by the discovery of single catastrophic events. One such phenomenon termed chromothripsis, involving massive chromosomal rearrangements arising all at once, has emerged as a major mutational game changer. The strong interest in this process stems from its widespread association with a range of cancer types and its potential as a mutational driver. In this review, we first describe chromothripsis detection and incidence in cancers. We then explore recently proposed underlying mechanistic origins, which explain the curious observations of the highly localised nature of the rearrangements on chromothriptic chromosomes. Detection of chromothriptic patterns following incorporation of single chromosomes into micronuclei or following telomere attrition have greatly contributed to our understanding of the reasons behind this chromosomal restriction. These underlying cellular events have been found to be participants in the tumourigenic process, strongly suggesting a potential role for chromothripsis in cancer development. Thus, we discuss potential implications of chromothripsis for cancer progression and therapy.


Subject(s)
Chromothripsis , Mutation , Neoplasms/genetics , Animals , DNA Damage , Humans , Neoplasms/etiology , Polymorphism, Single Nucleotide , Telomere , Translocation, Genetic
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