ZBTB2 protein is a new partner of the Nucleosome Remodeling and Deacetylase (NuRD) complex

2021 ◽  
Vol 168 ◽  
pp. 67-76
Author(s):  
Rosita Russo ◽  
Veronica Russo ◽  
Francesco Cecere ◽  
Mariangela Valletta ◽  
Maria Teresa Gentile ◽  
...  
2019 ◽  
Author(s):  
Matteo Vecellio ◽  
Adrian Cortes ◽  
Sarah Bonham ◽  
Carlo Selmi ◽  
Julian C Knight ◽  
...  

ABSTRACTObjectivesTo investigate the functional consequences of the single nucleotide polymorphism rs4648889 in a putative enhancer upstream of the RUNX3 promoter strongly associated with ankylosing spondylitis (AS).MethodsThe effects of rs4648889 on transcription factor (TF) binding were tested by DNA pull-down and quantitative mass spectrometry. The results were validated by electrophoretic mobility gel shift assays (EMSA), Western blot (WB) analysis of the pulled-down eluates, and chromatin immuno-precipitation (ChIP)-qPCR.ResultsSeveral TFs showed differential allelic binding to a 50bp DNA probe spanning rs4648889. Binding was increased to the AS-risk A allele for IKZF3 (aiolos) in nuclear extracts from CD8+ T-cells (3.7-fold, p<0.03) and several components of the NUcleosome Remodeling Deacetylase (NuRD) complex, including Chromodomain-Helicase-DNA-binding protein 4 (3.6-fold, p<0.05) and Retinoblastoma-Binding Protein 4 (4.1-fold, p<0.02). In contrast, binding of interferon regulatory factor (IRF) 5 was increased to the AS-protective G allele. These results were confirmed by EMSA, WB and ChIP-qPCR.ConclusionsThe association of AS with rs4648889 most likely results from its influence on the binding of this enhancer-like region to TFs, including IRF5, IKZF3 and members of the NuRD complex. Further investigation of these factors and RUNX3-related pathways may reveal important new therapeutic possibilities in AS.


2018 ◽  
Vol 115 (26) ◽  
pp. 6727-6732 ◽  
Author(s):  
Caralynn M. Wilczewski ◽  
Austin J. Hepperla ◽  
Takashi Shimbo ◽  
Lauren Wasson ◽  
Zachary L. Robbe ◽  
...  

Cardiac development relies on proper cardiomyocyte differentiation, including expression and assembly of cell-type-specific actomyosin subunits into a functional cardiac sarcomere. Control of this process involves not only promoting expression of cardiac sarcomere subunits but also repressing expression of noncardiac myofibril paralogs. This level of transcriptional control requires broadly expressed multiprotein machines that modify and remodel the chromatin landscape to restrict transcription machinery access. Prominent among these is the nucleosome remodeling and deacetylase (NuRD) complex, which includes the catalytic core subunit CHD4. Here, we demonstrate that direct CHD4-mediated repression of skeletal and smooth muscle myofibril isoforms is required for normal cardiac sarcomere formation, function, and embryonic survival early in gestation. Through transcriptomic and genome-wide analyses of CHD4 localization, we identified unique CHD4 binding sites in smooth muscle myosin heavy chain, fast skeletal α-actin, and the fast skeletal troponin complex genes. We further demonstrate that in the absence of CHD4, cardiomyocytes in the developing heart form a hybrid muscle cell that contains cardiac, skeletal, and smooth muscle myofibril components. These misexpressed paralogs intercalate into the nascent cardiac sarcomere to disrupt sarcomere formation and cause impaired cardiac function in utero. These results demonstrate the genomic and physiological requirements for CHD4 in mammalian cardiac development.


2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Samuel G. Salamun ◽  
Justine Sitz ◽  
Carlos F. De La Cruz-Herrera ◽  
Jaime Yockteng-Melgar ◽  
Edyta Marcon ◽  
...  

ABSTRACT The BMRF1 protein of Epstein-Barr virus (EBV) has multiple roles in viral lytic infection, including serving as the DNA polymerase processivity factor, activating transcription from several EBV promoters and inhibiting the host DNA damage response to double-stranded DNA breaks (DSBs). Using affinity purification coupled to mass spectrometry, we identified the nucleosome remodeling and deacetylation (NuRD) complex as the top interactor of BMRF1. We further found that NuRD components localize with BMRF1 at viral replication compartments and that this interaction occurs through the BMRF1 C-terminal region previously shown to mediate transcriptional activation. We identified an RBBP4 binding motif within this region that can interact with both RBBP4 and MTA2 components of the NuRD complex and showed that point mutation of this motif abrogates NuRD binding as well as the ability of BMRF1 to activate transcription from the BDLF3 and BLLF1 EBV promoters. In addition to its role in transcriptional regulation, NuRD has been shown to contribute to DSB signaling in enabling recruitment of RNF168 ubiquitin ligase and subsequent ubiquitylation at the break. We showed that BMRF1 inhibited RNF168 recruitment and ubiquitylation at DSBs and that this inhibition was at least partly relieved by loss of the NuRD interaction. The results reveal a mechanism by which BMRF1 activates transcription and inhibits DSB signaling and a novel role for NuRD in transcriptional activation in EBV. IMPORTANCE The Epstein-Barr virus (EBV) BMRF1 protein is critical for EBV infection, playing key roles in viral genome replication, activation of EBV genes, and inhibition of host DNA damage responses (DDRs). Here we show that BMRF1 targets the cellular nucleosome remodeling and deacetylation (NuRD) complex, using a motif in the BMRF1 transcriptional activation sequence. Mutation of this motif disrupts the ability of BMRF1 to activate transcription and interfere with DDRs, showing the importance of the NuRD interaction for BMRF1 functions. BMRF1 was shown to act at the same step in the DDR as NuRD, suggesting that it interferes with NuRD function.


Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2253-2253 ◽  
Author(s):  
Jeffrey R Shearstone ◽  
John H van Duzer ◽  
Simon S Jones ◽  
Matthew Jarpe

Abstract Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease (SCD), and could also be effective in treating beta-thalassemia (bT). Fetal beta-like globin gene (HbG) expression is silenced in adults partly through the nucleosome remodeling and histone deacetylase (NuRD) complex, which contains HDAC1/2 (Sankaran VG, Science, 2008). Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of HbG expression (Bradner JE, Proc Natl Acad Sci, 2010). Furthermore, we have previously shown that selective chemical inhibitors of HDAC1 and 2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). However, the mechanism through which HDAC1/2 inhibition leads to activation of HbG remains largely unknown. In this work, we have utilized our proof of concept molecule, ACY-957, to investigate changes in gene expression and chromatin organization that result from inhibition of HDAC1/2. Gene expression profiling was performed on cells treated with ACY-957 (n=3) or vehicle (n=3) using Affymetrix PrimeView GeneChips. Treatment of early erythroblasts (CD71+, GlyA-) resulted in the up and down regulation of 1294 and 681 transcript probe sets, respectively. In comparison, treatment of late erythroblasts (CD71+, GlyA+) resulted in a total of 255 transcript probe set changes. This finding is consistent with follow-up experiments demonstrating that ACY-957 is unable to induce HbG in cells positive for both CD71 and GlyA. Taken together, these results suggest that erythroblasts become less responsive to HDAC inhibition as they mature. Gene set enrichment analysis using public domain data revealed that genes up- or down-regulated by HDAC1/2 shRNA knockdown are significantly overrepresented in the list of genes induced or repressed by ACY-957, respectively; suggesting pharmacologic inhibition of HDAC1/2 recapitulates genetic ablation. We also identified significant enrichment in other gene sets involving targets linked to HbG regulation, including lysine-specific demethylase 1 (LSD1) (Shi L, Nature Medicine, 2012). GeneChip and quantitative real-time PCR time course experiments show ACY-957 treatment leads to a decrease in Bcl11A (2-fold) and Sox6 (10-fold) mRNA, known repressors of fetal globin synthesis, and an increase in Klf2 (2-fold) and Gata2 (8-fold) mRNA, proposed fetal globin activators. This result is consistent with work by others that show Gata2 is suppressed, in part, by the NuRD complex (Hong W, EMBO Journal, 2005) and that Gata2 binding at the HbG promoter leads to increased levels of HbG expression (Zhu X, PLoS One, 2012). Interestingly, Gata2 induction preceded Sox6 suppression in ACY-957 treated cells and the Sox6 promoter contains 8 canonical WGATAR binding sites and one Gata2-specific binding motif, raising the possibility suppression of Sox6 by ACY-957 is mediated by Gata2 induction. To investigate these possibilities, we have performed chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) for HDAC1, HDAC2, Gata2, and the HDAC2-specific histone modification H3K56 in ACY-957 and vehicle treated cells. These experiments will be discussed. ChIP-seq data, both by itself and in combination with gene expression data, will provide further insight into the mechanism through which HDAC1/2 regulates HbF synthesis. Disclosures: Shearstone: Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. van Duzer:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.


2009 ◽  
Vol 30 (4) ◽  
pp. 1067-1076 ◽  
Author(s):  
Mo Li ◽  
Vladimir E. Belozerov ◽  
Haini N. Cai

ABSTRACT Chromatin boundaries facilitate independent gene regulation by insulating genes from the effects of enhancers or organized chromatin. However, the mechanisms of boundary action are not well understood. To investigate whether boundary function depends on a higher order of chromatin organization, we examined the function of several Drosophila melanogaster insulators in cells with reduced chromatin-remodeling activities. We found that knockdown of NURF301 and ISWI, key components of the nucleosome-remodeling factor (NURF), synergistically disrupted the enhancer-blocking function of Fab7 and SF1 and augmented the function of Fab8. Mutations in Nurf301/Ebx and Iswi also affected the function of these boundaries in vivo. We further show that ISWI was localized on the endogenous Fab7 and Fab8 insulators and that NURF knockdown resulted in a marked increase in the nucleosome occupancy at these insulator sites. In contrast to the effect of NURF knockdown, reduction in dMi-2, the ATPase component of the Drosophila nucleosome-remodeling and deacetylation (NuRD) complex, augmented Fab7 and suppressed Fab8. Our results provide the first evidence that higher-order chromatin organization influences the enhancer-blocking activity of chromatin boundaries. In particular, the NURF and NuRD nucleosome-remodeling complexes may regulate Hox expression by modulating the function of boundaries in these complexes. The unique responses by different classes of boundaries to changes in the chromatin environment may be indicative of their distinct mechanisms of action, which may influence their placement in the genome and selection during evolution.


2020 ◽  
Author(s):  
Jonathan Lenz ◽  
Robert Liefke ◽  
Julianne Funk ◽  
Samuel Shoup ◽  
Andrea Nist ◽  
...  

AbstractThe generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.


2019 ◽  
Author(s):  
Rodrigo O. de Castro ◽  
Victor Goitea ◽  
Luciana Previato ◽  
Agustin Carbajal ◽  
Courtney T. Griffin ◽  
...  

AbstractTestis development and sustained germ cell production in adults rely on the establishment of spermatogonia stem cells and their proper differentiation into mature gametes. Control of these processes involves not only promoting the expression of genes required for cell survival and differentiation but also repressing other cell fates. This level of transcriptional control requires chromatin-remodeling complexes that restrict or promote transcription machinery. Here, we investigated the roles of the NUcleosome Remodeling and Deacetylase (NURD) complex during spermatogenesis. Our cellular and biochemical analyses revealed differential expression and composition of NURD subunits in gametocytes at different stages of testis development. Germ cell-specific deletion of the NURD catalytic component CHD4, but not CHD3, resulted in arrested early gamete development due to failed cell survival of the undifferentiated spermatogonia stem cell population. Genome-wide CHD4 chromatin localization and transcriptomic analyses revealed that CHD4 binds the promoters and regulates the expression of genes involved in spermatogonia cell survival and differentiation. These results uncover the requirements of CHD4 in mammalian gonad development, and point to unique roles for the NURD complex with respect to other chromatin remodelers during gamete development.Significance StatementGametogenesis is a fundamental developmental program required for sustained fertility and survival of all sexually reproducing species. The developing male gamete undergoes numerous cell divisions and developmental stage transitions that are carefully monitored by epigenetic mechanisms. One prominent mechanism is directed by chromatin remodeling complexes, which modify chromatin structure and thereby control fundamental cellular processes such as gene transcription. In this work, we focused in understanding the role of CHD4 and CHD3 proteins, catalytic subunits of the NURD chromatin-remodeling complex, in mouse gametogenesis. We find that CHD4 has an essential function in gametogenesis, with an absolute requirement for survival of spermatogonia populations in the developing testis. This is accompanied by CHD4-mediated transcriptional regulation of genes important for spermatogonia survival, and differentiation.


2012 ◽  
Vol 11 (8) ◽  
pp. 4326-4337 ◽  
Author(s):  
Matthew A. M. Todd ◽  
David J. Picketts

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