Survival of Drosophila germline stem cells requires the chromatin-binding protein Barrier-to-autointegration factor.
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ABSTRACT:
SUBMITTER: Duan T
PROVIDER: S-EPMC7240298 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell nuclei rupture following exposure to mechanical force and/or upon weakening of nuclear integrity, but nuclear ruptures are repairable. Barrier-to-autointegration factor (BAF), a small DNA-binding protein, rapidly localizes to nuclear ruptures; however, its role at these rupture sites is unknown. Here, we show that it is predominantly a nonphosphorylated cytoplasmic population of BAF that binds nuclear DNA to rapidly and transiently localize to the sites of nuclear rupture, resulting in BAF accumulation in the nucleus. BAF subsequently recruits transmembrane LEM-domain proteins, causing their accumulation at rupture sites. Loss of BAF impairs recruitment of LEM-domain proteins and nuclear envelope membranes to nuclear rupture sites and prevents nuclear envelope barrier function restoration. Simultaneous depletion of multiple LEM-domain proteins similarly inhibits rupture repair. LEMD2 is required for recruitment of the ESCRT-III membrane repair machinery to ruptures; however, neither LEMD2 nor ESCRT-III is required to repair ruptures. These results reveal a new role for BAF in the response to and repair of nuclear ruptures.
| S-EPMC6605789 | biostudies-literature
Project description:Nuclear lamin filaments and associated proteins form a nucleoskeletal ("lamina") network required for transcription, replication, chromatin organization and epigenetic regulation in metazoans. Lamina defects cause human disease ("laminopathies") and are linked to aging. Barrier-to-autointegration factor (BAF) is a mobile and essential component of the nuclear lamina that binds directly to histones, lamins and LEM-domain proteins, including the inner nuclear membrane protein emerin, and has roles in chromatin structure, mitosis and gene regulation. To understand BAF's mechanisms of action, BAF associated proteins were affinity-purified from HeLa cell nuclear lysates using BAF-conjugated beads, and identified by tandem mass spectrometry or independently identified and quantified using the iTRAQ method. We recovered A- and B-type lamins and core histones, all known to bind BAF directly, plus four human transcription factors (Requiem, NonO, p15, LEDGF), disease-linked proteins (e.g., Huntingtin, Treacle) and several proteins and enzymes that regulate chromatin. Association with endogenous BAF was independently validated by co-immunoprecipitation from HeLa cells for seven candidates including Requiem, poly(ADP-ribose) polymerase 1 (PARP1), retinoblastoma binding protein 4 (RBBP4), damage-specific DNA binding protein 1 (DDB1) and DDB2. Interestingly, endogenous BAF and emerin each associated with DDB2 and CUL4A in a UV- and time-dependent manner, suggesting BAF and emerin have dynamic roles in genome integrity and might help couple DNA damage responses to the nuclear lamina network. We conclude this proteome is a rich source of candidate partners for BAF and potentially also A- and B-type lamins, which may reveal how chromatin regulation and genome integrity are linked to nuclear structure.
| S-EPMC2739719 | biostudies-literature
Project description:Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.
| S-EPMC2173821 | biostudies-literature
Project description:Chromatin disorganization is one of the major alterations linked to prelamin A processing impairment. In this study we demonstrate that BAF is necessary to modulate prelamin A effects on chromatin structure. We show that when prelamin A and BAF cannot properly interact no prelamin A-dependent effects on chromatin occur; similar to what is observed in human Nestor Guillermo Progeria Syndrome cells harboring a BAF mutation, in HEK293 cells expressing a BAF mutant unable to bind prelamin A, or in siRNA mediated BAF-depleted HEK293 cells expressing prelamin A. BAF is necessary to induce histone trimethyl-H3K9 as well as HP1-alpha and LAP2-alpha nuclear relocalization in response to prelamin A accumulation. These findings are enforced by electron microscopy evaluations showing how the prelamin A-BAF interaction governs overall chromatin organization. Finally, we demonstrate that the LAP2-alpha nuclear localization defect observed in HGPS cells involves the progerin-BAF interaction, thus establishing a functional link between BAF and prelamin A pathological forms.
| S-EPMC4941268 | biostudies-literature
Project description:Defects in the nuclear envelope or nuclear 'lamina' networks cause disease and can perturb histone posttranslational (epigenetic) regulation. Barrier-to-Autointegration Factor (BAF) is an essential but enigmatic lamina component that binds lamins, LEM-domain proteins, DNA and histone H3 directly. We report that BAF copurified with nuclease-digested mononucleosomes and associated with modified histones in vivo. BAF overexpression significantly reduced global histone H3 acetylation by 18%. In cells that stably overexpressed BAF 3-fold, silencing mark H3-K27-Me1/3 and active marks H4-K16-Ac and H4-Ac5 decreased significantly. Significant increases were also seen for silencing mark H3-K9-Me3, active marks H3-K4-Me2, H3-K9/K14-Ac and H4-K5-Ac and a mark (H3-K79-Me2) associated with both active and silent chromatin. Other increases (H3-S10-P, H3-S28-P and silencing mark H3-K9-Me2) did not reach statistical significance. BAF overexpression also significantly influenced cell cycle distribution. Moreover, BAF associated in vivo with SET/I2PP2A (protein phosphatase 2A inhibitor; blocks H3 dephosphorylation) and G9a (H3-K9 methyltransferase), but showed no detectable association with HDAC1 or HATs. These findings reveal BAF as a novel epigenetic regulator and are discussed in relation to BAF deficiency phenotypes, which include a hereditary progeria syndrome and loss of pluripotency in embryonic stem cells.
| S-EPMC3324346 | biostudies-literature
Project description:Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy. We found that BAF compacts DNA by a looping mechanism. Dissociation of BAF from DNA occurs with multiphasic kinetics; an initial fast phase is followed by a much slower dissociation phase. The mechanistic basis of the broad timescale of dissociation is discussed. This behavior mimics the dissociation of BAF from retroviral DNA within preintegration complexes as monitored by functional assays. Thus the DNA binding properties of BAF may alone be sufficient to account for its association with the preintegration complex.
| S-EPMC2748091 | biostudies-literature
Project description:Barrier-to-autointegration factor (BAF/BANF) is a nuclear lamina protein essential for nuclear integrity, chromatin structure, and genome stability. Whereas complete loss of BAF causes lethality in multiple organisms, the A12T missense mutation of the BANF1 gene in humans causes a premature aging syndrome, called Néstor-Guillermo Progeria Syndrome (NGPS). Here, we report the first in vivo animal investigation of progeroid BAF, using CRISPR editing to introduce the NGPS mutation into the endogenous Drosophila baf gene. Progeroid BAF adults are born at expected frequencies, demonstrating that this BAF variant retains some function. However, tissue homeostasis is affected, supported by studies of the ovary, a tissue that depends upon BAF for stem cell survival and continuous oocyte production. We find that progeroid BAF causes defects in germline stem cell mitosis that delay anaphase progression and compromise chromosome segregation. We link these defects to decreased recruitment of centromeric proteins of the kinetochore, indicating dysfunction of cenBAF, a localized pool of dephosphorylated BAF produced by Protein Phosphatase PP4. We show that DNA damage increases in progenitor germ cells, which causes germ cell death due to activation of the DNA damage transducer kinase Chk2. Mitotic defects appear widespread, as aberrant chromosome segregation and increased apoptosis occur in another tissue. Together, these data highlight the importance of BAF in establishing centromeric structures critical for mitosis. Further, these studies link defects in cenBAF function to activation of a checkpoint that depletes progenitor reserves critical for tissue homeostasis, aligning with phenotypes of NGPS patients.
| S-EPMC9968693 | biostudies-literature
Project description:China is a country with a high incidence of gastric cancer (GC), where the GC incidence and the resultant mortality rates account for 50% of those worldwide. Surgical resection remains the primary treatment for GC. However, postoperative patients have a poor prognosis as the majority of patients present with metastases at the time of diagnosis. Therefore, the identification of novel treatment targets is required. The present study aimed to determine the effects of barrier-to-autointegration factor 1 (BANF1) on the clinical features and prognosis of GC, which may aid in discovering a novel tumor diagnostic biomarker and treatment target. The BANF1 gene expression profiles for normal and gastric tumor tissues were downloaded from the Gene Expression Omnibus GSE54129 data set to analyse the expression of BANF1 at the mRNA levels. Then, online survival analysis was performed using the GC database with the Kaplan-Meier Plotter (http://kmplot.com/analysis/) data. To examine the association between BANF1 and clinical features and prognosis, 132 postoperative GC pathological specimens were collected for immunohistochemical analyses. In the GSE54129 data sets, BANF1 expression at the mRNA level was significantly higher in the tumor tissue compared with that in the normal tissue. The same result was obtained in following the immunohistochemical analyses. In addition, BANF1 expression was associated with the patient age, tumor differentiation and infiltration depth. The survival time of BANF1 high-expression patients was shorter compared with that of the low-expression patients, and tumor differentiation status and tumor node metastasis stage were independent prognostic factors of the overall survival of patients with GC. The results of the present study suggest that BANF1 is associated with the clinical features and prognosis of GC. It may be a novel indicator of tumor prognosis and a potential therapeutic target for GC.
| S-EPMC6202538 | biostudies-literature
Project description:Stem cells reside in specialized niches and are regulated by a variety of physiological inputs. Adipocytes influence whole-body physiology and stem cell lineages; however, the molecular mechanisms linking adipocytes to stem cells are poorly understood. Here, we report that collagen IV produced in adipocytes is transported to the ovary to maintain proper germline stem cell (GSC) number in adult Drosophila females. Adipocyte-derived collagen IV acts through ?-integrin signaling to maintain normal levels of E-cadherin at the niche, thereby ensuring proper adhesion to GSCs. These findings demonstrate that extracellular matrix components produced in adipocytes can be transported to and incorporated into an established adult tissue to influence stem cell number.
| S-EPMC6063239 | biostudies-literature
Project description:Although the pathogen recognition receptor pathways that activate cell-intrinsic antiviral responses are well delineated, less is known about how the host regulates this response to prevent sustained signaling and possible immune-mediated damage. Using a genome-wide CRISPR-Cas9 screening approach to identify host factors that modulate interferon-stimulated gene (ISG) expression, we identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1), a previously described inhibitor of retrovirus integration, as a modulator of basal cell-intrinsic immunity. Ablation of Banf1 by gene editing resulted in chromatin activation near host defense genes with associated increased expression of ISGs, including Oas2, Rsad2 (viperin), Ifit1, and ISG15 The phenotype in Banf1-deficient cells occurred through a cGAS-, STING-, and IRF3-dependent signaling axis, was associated with reduced infection of RNA and DNA viruses, and was reversed in Banf1 complemented cells. Confocal microscopy and biochemical studies revealed that a loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline. Our study identifies an undescribed role for Banf1 in regulating the levels of cytoplasmic DNA and cGAS-dependent ISG homeostasis and suggests possible therapeutic directions for promoting or inhibiting cell-intrinsic innate immune responses.IMPORTANCE Although the interferon (IFN) signaling pathway is a key host mechanism to restrict infection of a diverse range of viral pathogens, its unrestrained activity either at baseline or in the context of an immune response can result in host cell damage and injury. Here, we used a genome-wide CRISPR-Cas9 screen and identified the DNA binding protein Barrier-to-autointegration factor 1 (Banf1) as a modulator of basal cell-intrinsic immunity. A loss of Banf1 expression resulted in higher level of cytosolic double-stranded DNA at baseline, which triggered IFN-stimulated gene expression via a cGAS-STING-IRF3 axis that did not require type I IFN or STAT1 signaling. Our experiments define a regulatory network in which Banf1 limits basal inflammation by preventing self DNA accumulation in the cytosol.
| S-EPMC7064753 | biostudies-literature