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1.
Hum Mol Genet ; 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34605909

RESUMO

The 10q24.33 locus is known to be associated with susceptibility to cutaneous malignant melanoma (CMM), but the mechanisms underlying this association have been not extensively investigated. We carried out an integrative genomic analysis of 10q24.33 using epigenomic annotations and in vitro reporter gene assays to identify regulatory variants. We found two putative functional single nucleotide polymorphisms (SNPs) in an enhancer and in the promoter of OBFC1, respectively, in neural crest and CMM cells, one, rs2995264, altering enhancer activity. The minor allele G of rs2995264 correlated with lower OBFC1 expression in 470 CMM tumors and was confirmed to increase the CMM risk in a cohort of 484 CMM cases and 1801 controls of Italian origin. Hi-C and chromosome conformation capture (3C) experiments showed the interaction between the enhancer-SNP region and the promoter of OBFC1 and an isogenic model characterized by CRISPR-Cas9 deletion of the enhancer-SNP region confirmed the potential regulatory effect of rs2995264 on OBFC1 transcription. Moreover, the presence of G-rs2995264 risk allele reduced the binding affinity of the transcription factor MEOX2. Biologic investigations showed significant cell viability upon depletion of OBFC1, specifically in CMM cells that were homozygous for the protective allele. Clinically, high levels of OBFC1 expression associated with histologically favorable CMM tumors. Finally, preliminary results suggested the potential effect of decreased OBFC1 expression on telomerase activity in tumorigenic conditions. Our results support the hypothesis that reduced expression of OBFC1 gene through functional heritable DNA variation can contribute to malignant transformation of normal melanocytes.

2.
Mol Cell ; 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34551281

RESUMO

ADP-ribose (ADPr) readers are essential components of ADP-ribosylation signaling, which regulates genome maintenance and immunity. The identification and discrimination between monoADPr (MAR) and polyADPr (PAR) readers is difficult because of a lack of suitable affinity-enrichment reagents. We synthesized well-defined ADPr probes and used these for affinity purifications combined with relative and absolute quantitative mass spectrometry to generate proteome-wide MAR and PAR interactomes, including determination of apparent binding affinities. Among the main findings, MAR and PAR readers regulate various common and distinct processes, such as the DNA-damage response, cellular metabolism, RNA trafficking, and transcription. We monitored the dynamics of PAR interactions upon induction of oxidative DNA damage and uncovered the mechanistic connections between ubiquitin signaling and ADP-ribosylation. Taken together, chemical biology enables exploration of MAR and PAR readers using interaction proteomics. Furthermore, the generated MAR and PAR interaction maps significantly expand our current understanding of ADPr signaling.

3.
Nat Commun ; 12(1): 5015, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408139

RESUMO

Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas/metabolismo , Biotina/metabolismo , Biotinilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Espectrometria de Massas , Ligação Proteica , Proteínas/química , Proteômica
4.
Biochim Biophys Acta Mol Basis Dis ; 1867(12): 166259, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34450246

RESUMO

A genomic locus 8 kb downstream of the transcription factor GFI1B (Growth Factor Independence 1B) predisposes to clonal hematopoiesis and myeloproliferative neoplasms. One of the most significantly associated polymorphisms in this region is rs621940-G. GFI1B auto-represses GFI1B, and altered GFI1B expression contributes to myeloid neoplasms. We studied whether rs621940-G affects GFI1B expression and growth of immature cells. GFI1B ChIP-seq showed clear binding to the rs621940 locus. Preferential binding of various hematopoietic transcription factors to either the rs621940-C or -G allele was observed, but GFI1B showed no preference. In gene reporter assays the rs621940 region inhibited GFI1B promoter activity with the G-allele having less suppressive effects compared to the C-allele. However, CRISPR-Cas9 mediated deletion of the locus in K562 cells did not alter GFI1B expression nor auto-repression. In healthy peripheral blood mononuclear cells GFI1B expression did not differ consistently between the rs621940 alleles. Long range and targeted deep sequencing did not detect consistent effects of rs621940-G on allelic GFI1B expression either. Finally, we observed that myeloid colony formation was not significantly affected by either rs621940 allele in 193 healthy donors. Together, these findings show no evidence that rs621940 or its locus affect GFI1B expression, auto-repression or growth of immature myeloid cells.

5.
Gut Microbes ; 13(1): 1966278, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34455931

RESUMO

The human gut microbiota plays a central role in intestinal health and disease. Yet, many of its bacterial constituents are functionally still largely unexplored. A crucial prerequisite for bacterial survival and proliferation is the creation and/or exploitation of an own niche. For many bacterial species that are linked to human disease, the inner mucus layer was found to be an important niche. Allobaculum mucolyticum is a newly identified, IBD-associated species that is thought be closely associated with the host epithelium. To explore how this bacterium is able to effectively colonize this niche, we screened its genome for factors that may contribute to mucosal colonization. Up to 60 genes encoding putative Carbohydrate Active Enzymes (CAZymes) were identified in the genome of A. mucolyticum. Mass spectrometry revealed 49 CAZymes of which 26 were significantly enriched in its secretome. Functional assays demonstrated the presence of CAZyme activity in A. mucolyticum conditioned medium, degradation of human mucin O-glycans, and utilization of liberated non-terminal monosaccharides for bacterial growth. The results support a model in which sialidases and fucosidases remove terminal O-glycan sugars enabling subsequent degradation and utilization of carbohydrates for A. mucolyticum growth. A. mucolyticum CAZyme secretion may thus facilitate bacterial colonization and degradation of the mucus layer and may pose an interesting target for future therapeutic intervention.

6.
Nature ; 596(7870): 133-137, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34234345

RESUMO

The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Cromatina/genética , Cromatina/metabolismo , Ilhas de CpG/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Cromatina/química , Montagem e Desmontagem da Cromatina/genética , Metilação de DNA , Regulação da Expressão Gênica , Genes Essenciais , Humanos , Camundongos , Imagem Individual de Molécula
7.
8.
Trends Genet ; 2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34304914

RESUMO

DNA methylation has long been considered the primary epigenetic mediator of genomic imprinting in mammals. Recent epigenetic profiling during early mouse development revealed the presence of domains of trimethylation of lysine 27 on histone H3 (H3K27me3) and chromatin compaction specifically at the maternally derived allele, independent of DNA methylation. Within these domains, genes are exclusively expressed from the paternally derived allele. This novel mechanism of noncanonical imprinting plays a key role in the development of mouse extraembryonic tissues and in the regulation of imprinted X-chromosome inactivation, highlighting the importance of parentally inherited epigenetic histone modifications. Here, we discuss the mechanisms underlying H3K27me3-mediated noncanonical imprinting in perspective of the dynamic chromatin landscape during early mouse development and explore evolutionary origins of noncanonical imprinting.

9.
Methods Mol Biol ; 2272: 209-224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34009616

RESUMO

In recent years, workflows coupling DNA affinity purifications from crude nuclear extracts with quantitative mass spectrometry-based proteomics have enabled comprehensive mapping of protein-DNA interactions in an unbiased manner. Here, we describe a detailed protocol for one such method in which affinity purifications with extracts from cells or tissues of interest are combined with a chemical stable isotope labeling method, dimethyl labeling, to identify specific interaction partners for (hydroxy)methylated and non-methylated DNA sequences of interest.


Assuntos
5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Metilação de DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Marcação por Isótopo/métodos , Proteoma/metabolismo , Biologia Computacional/métodos , DNA/análise , Epigênese Genética , Humanos , Oxirredução , Proteoma/análise
10.
Cell Rep ; 35(5): 109073, 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33951430

RESUMO

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

11.
EMBO J ; 40(14): e106536, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34009673

RESUMO

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ˜ 100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing and suggest a link between minor spliceosome defects and constitutional aneuploidy in humans.

12.
Mol Oncol ; 15(7): 1882-1900, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33797847

RESUMO

Bladder urothelial cell carcinoma (UCC) incidence is about three times higher in men compared with women. There are several indications for the involvement of hormonal factors in the aetiology of UCC. Here, we provide evidence of androgen signalling in UCC progression. Microarray and qPCR analysis revealed that the androgen receptor (AR) mRNA level is upregulated in a subset of UCC cases. In an AR-positive UCC-derived cell line model, UM-UC-3-AR, androgen treatment increased clonogenic capacity inducing the formation of big stem cell-like holoclones, while AR knockdown or treatment with the AR antagonist enzalutamide abrogated this clonogenic advantage. Additionally, blockage of AR signalling reduced the cell migration potential of androgen-stimulated UM-UC-3-AR cells. These phenotypic changes were accompanied by a rewiring of the transcriptome with almost 300 genes being differentially regulated by androgens, some of which correlated with AR expression in UCC patients in two independent data sets. Our results demonstrate that AR signals in UCC favouring the development of an aggressive phenotype and highlights its potential as a therapeutic target for bladder cancer.

13.
FASEB J ; 35(4): e21366, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33749890

RESUMO

Hepatocyte nuclear factor 1ß (HNF1ß) is an essential transcription factor in development of the kidney, liver, and pancreas. HNF1ß-mediated transcription of target genes is dependent on the cell type and the development stage. Nevertheless, the regulation of HNF1ß function by enhancers and co-factors that allow this cell-specific transcription is largely unknown. To map the HNF1ß interactome we performed mass spectrometry in a mouse kidney inner medullary collecting duct cell line. Pterin-4a-carbinolamine dehydratase 2 (PCBD2) was identified as a novel interaction partner of HNF1ß. PCBD2 and its close homolog PCBD1 shuttle between the cytoplasm and nucleus to exert their enzymatic and transcriptional activities. Although both PCBD proteins share high sequence identity (48% and 88% in HNF1 recognition helix), their tissue expression patterns are unique. PCBD1 is most abundant in kidney and liver while PCBD2 is also abundant in lung, spleen, and adipose tissue. Using immunolocalization studies and biochemical analysis we show that in presence of HNF1ß the nuclear localization of PCBD1 and PCBD2 increases significantly. Promoter luciferase assays demonstrate that co-factors PCBD1 and PCBD2 differentially regulate the ability of HNF1ß to activate the promoters of transcriptional targets important in renal electrolyte homeostasis. Deleting the N-terminal sequence of PCBD2, not found in PCBD1, diminished the differential effects of the co-factors on HNF1ß activity. All together these results indicate that PCBD1 and PCBD2 can exert different effects on HNF1ß-mediated transcription. Future studies should confirm whether these unique co-factor activities also apply to HNF1ß-target genes involved in additional processes besides ion transport in the kidney.


Assuntos
Fator 1-beta Nuclear de Hepatócito/metabolismo , Hidroliases/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Células HEK293 , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Hidroliases/genética , Espectrometria de Massas , Camundongos , Modelos Moleculares , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Regiões Promotoras Genéticas , Conformação Proteica , Transporte Proteico , Transcrição Genética
14.
Mol Cell Proteomics ; 20: 100056, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33556626

RESUMO

Regulation of gene expression is essential for the functioning of all eukaryotic organisms. Understanding gene expression regulation requires determining which proteins interact with regulatory elements in chromatin. MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation, as it allows studying protein function and protein complex formation in their in vivo chromatin-bound context. Total chromatin isolated from cells can be directly analyzed using MS or further fractionated into transcriptionally active and inactive chromatin prior to MS-based analysis. Newly formed chromatin that is assembled during DNA replication can also be specifically isolated and analyzed. Furthermore, capturing specific chromatin domains facilitates the identification of previously unknown transcription factors interacting with these domains. Finally, in recent years, advances have been made toward identifying proteins that interact with a single genomic locus of interest. In this review, we highlight the power of chromatin proteomics approaches and how these provide complementary alternatives compared with conventional affinity purification methods. Furthermore, we discuss the biochemical challenges that should be addressed to consolidate and expand the role of chromatin proteomics as a key technology in the context of gene expression regulation and epigenetics research in health and disease.

15.
Cell Rep ; 34(5): 108705, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33535034

RESUMO

Membraneless organelles are liquid condensates, which form through liquid-liquid phase separation. Recent advances show that phase separation is essential for cellular homeostasis by regulating basic cellular processes, including transcription and signal transduction. The reported number of proteins with the capacity to mediate protein phase separation (PPS) is continuously growing. While computational tools for predicting PPS have been developed, obtaining a proteome-wide overview of PPS probabilities has remained challenging. Here, we present a phase separation analysis and prediction (PSAP) machine-learning classifier that, based solely on the amino acid content of a training set of known PPS proteins, can determine the phase separation likelihood for each protein in a given proteome. Through comparison with PPS databases, existing predictors, and experimental evidence, we demonstrate the validity and advantages of the PSAP classifier. We anticipate that the PSAP predictor provides a useful tool for future research aimed at identifying phase separating proteins in health and disease.

16.
Nat Commun ; 12(1): 1342, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637760

RESUMO

Bulky DNA lesions in transcribed strands block RNA polymerase II (RNAPII) elongation and induce a genome-wide transcriptional arrest. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions, but how transcription is restored in the genome following DNA repair remains unresolved. Here, we find that the TCR-specific CSB protein loads the PAF1 complex (PAF1C) onto RNAPII in promoter-proximal regions in response to DNA damage. Although dispensable for TCR-mediated repair, PAF1C is essential for transcription recovery after UV irradiation. We find that PAF1C promotes RNAPII pause release in promoter-proximal regions and subsequently acts as a processivity factor that stimulates transcription elongation throughout genes. Our findings expose the molecular basis for a non-canonical PAF1C-dependent pathway that restores transcription throughout the human genome after genotoxic stress.


Assuntos
Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Celular , DNA/efeitos da radiação , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Mapas de Interação de Proteínas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Transcrição Genética , Raios Ultravioleta
17.
EMBO J ; 40(4): e104975, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33428246

RESUMO

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

18.
Mol Cell ; 81(4): 845-858.e8, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33406384

RESUMO

Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.


Assuntos
Composição de Bases , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Camundongos , Camundongos Mutantes , Células-Tronco Embrionárias Murinas/citologia , Mutação , Neurônios/citologia , Fatores de Transcrição/genética , Regulação para Cima , Dedos de Zinco
19.
Eur J Immunol ; 51(2): 471-482, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33065764

RESUMO

RasGRP1 is a Ras guanine nucleotide exchange factor, and an essential regulator of lymphocyte receptor signaling. In mice, Rasgrp1 deletion results in defective T lymphocyte development. RASGRP1-deficient patients suffer from immune deficiency, and the RASGRP1 gene has been linked to autoimmunity. However, how RasGRP1 levels are regulated, and if RasGRP1 dosage alterations contribute to autoimmunity remains unknown. We demonstrate that diminished Rasgrp1 expression caused defective T lymphocyte selection in C57BL/6 mice, and that the severity of inflammatory disease inversely correlates with Rasgrp1 expression levels. In patients with autoimmunity, active inflammation correlated with decreased RASGRP1 levels in CD4+ T cells. By analyzing H3K27 acetylation profiles in human T cells, we identified a RASGRP1 enhancer that harbors autoimmunity-associated SNPs. CRISPR-Cas9 disruption of this enhancer caused lower RasGRP1 expression, and decreased binding of RUNX1 and CBFB transcription factors. Analyzing patients with autoimmunity, we detected reduced RUNX1 expression in CD4+ T cells. Lastly, we mechanistically link RUNX1 to transcriptional regulation of RASGRP1 to reveal a key circuit regulating RasGRP1 expression, which is vital to prevent inflammatory disease.


Assuntos
Autoimunidade/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Transcrição Genética/genética , Animais , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/imunologia , Subunidade alfa 2 de Fator de Ligação ao Core/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Fatores de Troca do Nucleotídeo Guanina/imunologia , Histonas/genética , Histonas/imunologia , Humanos , Inflamação/genética , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transcrição Genética/imunologia
20.
FEBS J ; 288(10): 3231-3245, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33283408

RESUMO

The multi-subunit nucleosome remodeling and deacetylase (NuRD) complex consists of seven subunits, each of which comprises two or three paralogs in vertebrates. These paralogs define mutually exclusive and functionally distinct complexes. In addition, several proteins in the complex are multimeric, which complicates structural studies. Attempts to purify sufficient amounts of endogenous complex or recombinantly reconstitute the complex for structural studies have proven quite challenging. Until now, only substructures of individual domains or proteins and low-resolution densities of (partial) complexes have been reported. In this study, we comprehensively investigated the relative orientation of different subunits within the NuRD complex using multiple cross-link IP mass spectrometry (xIP-MS) experiments. Our results confirm that the core of the complex is formed by MTA, RBBP, and HDAC proteins. Assembly of a copy of MBD and GATAD2 onto this core enables binding of the peripheral CHD and CDK2AP proteins. Furthermore, our experiments reveal that not only CDK2AP1 but also CDK2AP2 interacts with the NuRD complex. This interaction requires the C terminus of CHD proteins. Our data provide a more detailed understanding of the topology of the peripheral NuRD subunits relative to the core complex. DATABASE: Proteomics data are available in the PRIDE database under the accession numbers PXD017244 and PXD017378.

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