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1.
Nat Commun ; 12(1): 4674, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344879

RESUMO

In most organisms, the number and distribution of crossovers that occur during meiosis are tightly controlled. All chromosomes must receive at least one 'obligatory crossover' and crossovers are prevented from occurring near one another by 'crossover interference'. However, the mechanistic basis of this phenomenon of crossover interference has remained mostly mysterious. Using quantitative super-resolution cytogenetics and mathematical modelling, we investigate crossover positioning in the Arabidopsis thaliana wild-type, an over-expressor of the conserved E3 ligase HEI10, and a hei10 heterozygous line. We show that crossover positions can be explained by a predictive, diffusion-mediated coarsening model, in which large, approximately evenly-spaced HEI10 foci grow at the expense of smaller, closely-spaced clusters. We propose this coarsening process explains many aspects of Arabidopsis crossover positioning, including crossover interference. Consistent with this model, we also demonstrate that crossover positioning can be predictably modified in vivo simply by altering HEI10 dosage, with higher and lower dosage leading to weaker and stronger crossover interference, respectively. As HEI10 is a conserved member of the RING finger protein family that functions in the interference-sensitive pathway for crossover formation, we anticipate that similar mechanisms may regulate crossover positioning in diverse eukaryotes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas Cromossômicas não Histona/metabolismo , Troca Genética/genética , Meiose/genética , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Simulação por Computador , Dosagem de Genes , Estágio Paquíteno/genética , Complexo Sinaptonêmico/genética , Complexo Sinaptonêmico/metabolismo
2.
Mol Cell ; 81(15): 3082-3095.e6, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34197738

RESUMO

To understand how chromatin domains coordinate gene expression, we dissected select genetic elements organizing topology and transcription around the Prdm14 super enhancer in mouse embryonic stem cells. Taking advantage of allelic polymorphisms, we developed methods to sensitively analyze changes in chromatin topology, gene expression, and protein recruitment. We show that enhancer insulation does not rely strictly on loop formation between its flanking boundaries, that the enhancer activates the Slco5a1 gene beyond its prominent domain boundary, and that it recruits cohesin for loop extrusion. Upon boundary inversion, we find that oppositely oriented CTCF terminates extrusion trajectories but does not stall cohesin, while deleted or mutated CTCF sites allow cohesin to extend its trajectory. Enhancer-mediated gene activation occurs independent of paused loop extrusion near the gene promoter. We expand upon the loop extrusion model to propose that cohesin loading and extrusion trajectories originating at an enhancer contribute to gene activation.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos , Animais , Fator de Ligação a CCCTC/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas , Coativador 2 de Receptor Nuclear/genética , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
3.
Mol Cell ; 81(15): 3065-3081.e12, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34297911

RESUMO

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Transcrição Genética/fisiologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Núcleo Celular/genética , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Laminas/genética , Laminas/metabolismo , RNA Polimerase II/metabolismo , Imagem Individual de Molécula/métodos
4.
Int J Mol Sci ; 22(13)2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34203408

RESUMO

TENT4A (PAPD7) is a non-canonical poly(A) polymerase, of which little is known. Here, we show that TENT4A regulates multiple biological pathways and focuses on its multilayer regulation of translesion DNA synthesis (TLS), in which error-prone DNA polymerases bypass unrepaired DNA lesions. We show that TENT4A regulates mRNA stability and/or translation of DNA polymerase η and RAD18 E3 ligase, which guides the polymerase to replication stalling sites and monoubiquitinates PCNA, thereby enabling recruitment of error-prone DNA polymerases to damaged DNA sites. Remarkably, in addition to the effect on RAD18 mRNA stability via controlling its poly(A) tail, TENT4A indirectly regulates RAD18 via the tumor suppressor CYLD and via the long non-coding antisense RNA PAXIP1-AS2, which had no known function. Knocking down the expression of TENT4A or CYLD, or overexpression of PAXIP1-AS2 led each to reduced amounts of the RAD18 protein and DNA polymerase η, leading to reduced TLS, highlighting PAXIP1-AS2 as a new TLS regulator. Bioinformatics analysis revealed that TLS error-prone DNA polymerase genes and their TENT4A-related regulators are frequently mutated in endometrial cancer genomes, suggesting that TLS is dysregulated in this cancer.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias do Endométrio/metabolismo , Mutação/genética , Polinucleotídeo Adenililtransferase/metabolismo , RNA Mensageiro/metabolismo , Western Blotting , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Biologia Computacional , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Replicação do DNA/genética , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Neoplasias do Endométrio/genética , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Células MCF-7 , Reação em Cadeia da Polimerase , Polinucleotídeo Adenililtransferase/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Ubiquitinação/fisiologia
6.
Nucleic Acids Res ; 49(13): 7406-7423, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34214177

RESUMO

Heterochromatin binding protein HP1ß plays an important role in chromatin organization and cell differentiation, however the underlying mechanisms remain unclear. Here, we generated HP1ß-/- embryonic stem cells and observed reduced heterochromatin clustering and impaired differentiation. We found that during stem cell differentiation, HP1ß is phosphorylated at serine 89 by CK2, which creates a binding site for the pluripotency regulator KAP1. This phosphorylation dependent sequestration of KAP1 in heterochromatin compartments causes a downregulation of pluripotency factors and triggers pluripotency exit. Accordingly, HP1ß-/- and phospho-mutant cells exhibited impaired differentiation, while ubiquitination-deficient KAP1-/- cells had the opposite phenotype with enhanced differentiation. These results suggest that KAP1 regulates pluripotency via its ubiquitination activity. We propose that the formation of subnuclear membraneless heterochromatin compartments may serve as a dynamic reservoir to trap or release cellular factors. The sequestration of essential regulators defines a novel and active role of heterochromatin in gene regulation and represents a dynamic mode of remote control to regulate cellular processes like cell fate decisions.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Embrionárias/metabolismo , Heterocromatina/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Animais , Caseína Quinase II/metabolismo , Diferenciação Celular , Linhagem Celular , Células Cultivadas , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Cricetinae , Células-Tronco Embrionárias/citologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Fosforilação , Serina/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/fisiologia
7.
Nat Commun ; 12(1): 4551, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315879

RESUMO

Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Genoma Humano , Transcriptoma/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Estabilidade Proteica
8.
Nat Commun ; 12(1): 4360, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272384

RESUMO

The glucocorticoid receptor (GR) regulates gene expression, governing aspects of homeostasis, but is also involved in cancer. Pharmacological GR activation is frequently used to alleviate therapy-related side-effects. While prior studies have shown GR activation might also have anti-proliferative action on tumours, the underpinnings of glucocorticoid action and its direct effectors in non-lymphoid solid cancers remain elusive. Here, we study the mechanisms of glucocorticoid response, focusing on lung cancer. We show that GR activation induces reversible cancer cell dormancy characterised by anticancer drug tolerance, and activation of growth factor survival signalling accompanied by vulnerability to inhibitors. GR-induced dormancy is dependent on a single GR-target gene, CDKN1C, regulated through chromatin looping of a GR-occupied upstream distal enhancer in a SWI/SNF-dependent fashion. These insights illustrate the importance of GR signalling in non-lymphoid solid cancer biology, particularly in lung cancer, and warrant caution for use of glucocorticoids in treatment of anticancer therapy related side-effects.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cromatina/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Glucocorticoides/farmacologia , Neoplasias Pulmonares/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Imidazóis/farmacologia , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Camundongos , Proteômica , Pirazinas/farmacologia , RNA Interferente Pequeno , RNA-Seq , Receptor IGF Tipo 1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202641

RESUMO

The cohesin complex is crucial for mediating sister chromatid cohesion and for hierarchal three-dimensional organization of the genome. Mutations in cohesin genes are present in a range of cancers. Extensive research over the last few years has shown that cohesin mutations are key events that contribute to neoplastic transformation. Cohesin is involved in a range of cellular processes; therefore, the impact of cohesin mutations in cancer is complex and can be cell context dependent. Candidate targets with therapeutic potential in cohesin mutant cells are emerging from functional studies. Here, we review emerging targets and pharmacological agents that have therapeutic potential in cohesin mutant cells.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Predisposição Genética para Doença , Mutação , Neoplasias/genética , Biomarcadores Tumorais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Gerenciamento Clínico , Regulação da Expressão Gênica , Humanos , Terapia de Alvo Molecular/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/terapia , Especificidade de Órgãos , Ligação Proteica , Relação Estrutura-Atividade
10.
Cancer Sci ; 112(9): 3711-3721, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34107118

RESUMO

Antimitotic drugs such as vinca alkaloids and taxanes cause mitotic cell death after prolonged mitotic arrest. However, a fraction of cells escape from mitotic arrest by undergoing mitotic slippage, which is related to resistance to antimitotic drugs. Tipping the balance to mitotic cell death thus can be a way to overcome the drug resistance. Here we found that depletion of a mitotic regulator, CHAMP1 (chromosome alignment-maintaining phosphoprotein, CAMP), accelerates the timing of mitotic cell death after mitotic arrest. Live cell imaging revealed that CHAMP1-depleted cells died earlier than mock-treated cells in the presence of antimitotic drugs that resulted in the reduction of cells undergoing mitotic slippage. Depletion CHAMP1 reduces the expression of antiapoptotic Bcl-2 family proteins, especially Mcl-1. We found that CHAMP1 maintains Mcl-1 expression both at protein and mRNA levels independently of the cell cycle. At the protein level, CHAMP1 maintains Mcl-1 stability by suppressing proteasome-dependent degradation. Depletion of CHAMP1 reduces cell viability, and exhibits synergistic effects with antimitotic drugs. Our data suggest that CHAMP1 plays a role in the maintenance of Mcl-1 expression, implying that CHAMP1 can be a target to overcome the resistance to antimitotic drugs.


Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais/genética , Células A549 , Animais , Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitose/efeitos dos fármacos , Mitose/genética , Neoplasias/genética , Neoplasias/patologia , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Nucleic Acids Res ; 49(12): 6788-6803, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133717

RESUMO

Speckle-type Poz protein (SPOP), an E3 ubiquitin ligase adaptor, is the most frequently mutated gene in prostate cancer. The SPOP-mutated subtype of prostate cancer shows high genomic instability, but the underlying mechanisms causing this phenotype are still largely unknown. Here, we report that upon DNA damage, SPOP is phosphorylated at Ser119 by the ATM serine/threonine kinase, which potentiates the binding of SPOP to homeodomain-interacting protein kinase 2 (HIPK2), resulting in a nondegradative ubiquitination of HIPK2. This modification subsequently increases the phosphorylation activity of HIPK2 toward HP1γ, and then promotes the dissociation of HP1γ from trimethylated (Lys9) histone H3 (H3K9me3) to initiate DNA damage repair. Moreover, the effect of SPOP on the HIPK2-HP1γ axis is abrogated by prostate cancer-associated SPOP mutations. Our findings provide new insights into the molecular mechanism of SPOP mutations-driven genomic instability in prostate cancer.


Assuntos
Proteínas de Transporte/metabolismo , Instabilidade Genômica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/química , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Histonas/metabolismo , Humanos , Masculino , Mutação , Fosforilação , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/química , Serina/metabolismo , Ubiquitinação
12.
Nature ; 595(7865): 125-129, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34108683

RESUMO

In higher eukaryotes, many genes are regulated by enhancers that are 104-106 base pairs (bp) away from the promoter. Enhancers contain transcription-factor-binding sites (which are typically around 7-22 bp), and physical contact between the promoters and enhancers is thought to be required to modulate gene expression. Although chromatin architecture has been mapped extensively at resolutions of 1 kilobase and above; it has not been possible to define physical contacts at the scale of the proteins that determine gene expression. Here we define these interactions in detail using a chromosome conformation capture method (Micro-Capture-C) that enables the physical contacts between different classes of regulatory elements to be determined at base-pair resolution. We find that highly punctate contacts occur between enhancers, promoters and CCCTC-binding factor (CTCF) sites and we show that transcription factors have an important role in the maintenance of the contacts between enhancers and promoters. Our data show that interactions between CTCF sites are increased when active promoters and enhancers are located within the intervening chromatin. This supports a model in which chromatin loop extrusion1 is dependent on cohesin loading at active promoters and enhancers, which explains the formation of tissue-specific chromatin domains without changes in CTCF binding.


Assuntos
Pareamento de Bases/genética , Genoma/genética , Animais , Sítios de Ligação , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Elementos Facilitadores Genéticos/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , alfa-Globinas/genética
13.
Nat Commun ; 12(1): 3184, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075040

RESUMO

During spermatogenesis, meiosis is accompanied by a robust alteration in gene expression and chromatin status. However, it remains elusive how the meiotic transcriptional program is established to ensure completion of meiotic prophase. Here, we identify a protein complex that consists of germ-cell-specific zinc-finger protein ZFP541 and its interactor KCTD19 as the key transcriptional regulators in mouse meiotic prophase progression. Our genetic study shows that ZFP541 and KCTD19 are co-expressed from pachytene onward and play an essential role in the completion of the meiotic prophase program in the testis. Furthermore, our ChIP-seq and transcriptome analyses identify that ZFP541 binds to and suppresses a broad range of genes whose function is associated with biological processes of transcriptional regulation and covalent chromatin modification. The present study demonstrates that a germ-cell specific complex that contains ZFP541 and KCTD19 promotes the progression of meiotic prophase towards completion in male mice, and triggers the reconstruction of the transcriptional network and chromatin organization leading to post-meiotic development.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Nucleares/metabolismo , Estágio Paquíteno/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Espermátides/citologia , Espermatogênese/genética , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas Cromossômicas não Histona/genética , Modelos Animais de Doenças , Feminino , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Infertilidade Masculina/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Oócitos/citologia , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA-Seq , Espermátides/metabolismo , Fatores de Transcrição/genética , Transcrição Genética
14.
Nucleic Acids Res ; 49(11): 6213-6237, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086943

RESUMO

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.


Assuntos
Processamento Alternativo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Éxons , Feminino , Células HeLa , Código das Histonas , Humanos , Receptores de Hialuronatos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
15.
Nat Genet ; 53(7): 1050-1063, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33986538

RESUMO

ATP-dependent chromatin remodelers are commonly mutated in human cancer. Mammalian SWI/SNF complexes comprise three conserved multisubunit chromatin remodelers (cBAF, ncBAF and PBAF) that share the BRG1 (also known as SMARCA4) subunit responsible for the main ATPase activity. BRG1 is the most frequently mutated Snf2-like ATPase in cancer. In the present study, we have investigated the role of SWI/SNF in genome instability, a hallmark of cancer cells, given its role in transcription, DNA replication and DNA-damage repair. We show that depletion of BRG1 increases R-loops and R-loop-dependent DNA breaks, as well as transcription-replication (T-R) conflicts. BRG1 colocalizes with R-loops and replication fork blocks, as determined by FANCD2 foci, with BRG1 depletion being epistatic to FANCD2 silencing. Our study, extended to other components of SWI/SNF, uncovers a key role of the SWI/SNF complex, in particular cBAF, in helping resolve R-loop-mediated T-R conflicts, thus, unveiling a new mechanism by which chromatin remodeling protects genome integrity.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA , Estruturas R-Loop , Fatores de Transcrição/metabolismo , Transcrição Genética , Adenosina Trifosfatases/metabolismo , Cromatina/genética , Cromatina/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Loci Gênicos , Instabilidade Genômica , Humanos
16.
Mol Cell Biol ; 41(7): e0004421, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33941618

RESUMO

Prions are self-perpetuating, alternative protein conformations associated with neurological diseases and normal cellular functions. Saccharomyces cerevisiae contains many endogenous prions, providing a powerful system to study prionization. Previously, we demonstrated that Swi1, a component of the SWI/SNF chromatin-remodeling complex, can form the prion [SWI+]. A small region, Swi11-38, with a unique amino acid composition of low complexity, acts as a prion domain and supports [SWI+] propagation. Here, we further examine Swi11-38 through site-directed mutagenesis. We found that mutations of the two phenylalanine residues or the threonine tract inhibit Swi11-38 aggregation. In addition, mutating both phenylalanines can abolish de novo prion formation by Swi11-38, whereas mutating only one phenylalanine does not. Replacement of half of or the entire eight-threonine tract with alanines has the same effect, possibly disrupting a core region of Swi11-38 aggregates. We also show that Swi11-38 and its prion-fold-maintaining mutants form high-molecular-weight, SDS-resistant aggregates, whereas the double-phenylalanine mutants eliminate these protein species. These results indicate the necessity of the large hydrophobic residues and threonine tract in Swi11-38 in prionogenesis, possibly acting as important aggregable regions. Our findings thus highlight the importance of specific amino acid residues in the Swi1 prion domain in prion formation and maintenance.


Assuntos
Proteínas Cromossômicas não Histona/genética , Regulação Fúngica da Expressão Gênica/genética , Mutação/genética , Príons/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/metabolismo , Príons/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
18.
Nat Commun ; 12(1): 2919, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006846

RESUMO

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterize the transcriptional profile of cortical neurons from CdLS patients and find deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupts 3D genome organization and transcriptional control in post-mitotic cortical mouse neurons, demonstrating that cohesin is continuously required for neuronal gene expression. The genes affected by acute depletion of cohesin belong to similar gene ontology classes and show significant numerical overlap with genes deregulated in CdLS. Interestingly, reconstitution of cohesin function largely rescues altered gene expression, including the expression of genes deregulated in CdLS.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Síndrome de Cornélia de Lange/genética , Regulação da Expressão Gênica , Mutação , Neurônios/metabolismo , Adulto , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Adulto Jovem
19.
Toxicol Appl Pharmacol ; 423: 115568, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33965371

RESUMO

N-methyl pyrrolidone (NMP) is an FDA approved molecule used as an excipient in pharmaceutical industry. Besides having a central role in formulation of drugs, the most important function of any excipient is to guarantee the safety of the medicine during and after its administration. Several studies have shown that exposure to NMP and especially in rats produce a gonadotoxic effect leading to infertility. However, the mechanisms underlying the effect of NMP on male reproduction are unknown. The aim of this study was to assess the reproductive toxicity of NMP in male rats and to elucidate the underlying mechanism. Male Sprague Dawley rats were injected intraperitoneally, twice/ week, at a dose of 108 mg/ 100 g of body weight with NMP. Analysis of reproductive parameters revealed testicular atrophy in NMP treated animals compared to control animals. Germ cell composition within the seminiferous tubules was disturbed and manifested in an increase in number of cells with fragmented DNA. A subsequent decrease in number of spermatocytes and spermatids was observed. Alpha screen assay shows that NMP acts at the concentrations we applied in vivo as a low affinity inhibitor for BRDT (testis specific bromodomain protein). BRDT inhibition is mirrored by a significant decrease in the expression of early stage spermatocyte markers (lmna, aurkc and ccna1), during which BRDT expression predominates. A significant decrease in testosterone levels was also observed. Since NMP interferes with spermatogenesis on various levels, its use in humans must be carefully monitored.


Assuntos
Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/metabolismo , Pirrolidinonas/toxicidade , Espermatogênese/efeitos dos fármacos , Teratógenos/toxicidade , Animais , Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante/sangue , Masculino , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Espermatogênese/fisiologia , Testosterona/sangue
20.
Nat Commun ; 12(1): 2576, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33958593

RESUMO

Nitric oxide (NO) is a diffusible signaling molecule that modulates animal and plant immune responses. In addition, reactive nitrogen species derived from NO can display antimicrobial activities by reacting with microbial cellular components, leading to nitrosative stress (NS) in pathogens. Here, we identify FgAreB as a regulator of the NS response in Fusarium graminearum, a fungal pathogen of cereal crops. FgAreB serves as a pioneer transcription factor for recruitment of the chromatin-remodeling complex SWI/SNF at the promoters of genes involved in the NS response, thus promoting their transcription. FgAreB plays important roles in fungal infection and growth. Furthermore, we show that a transcription repressor (FgIxr1) competes with the SWI/SNF complex for FgAreB binding, and negatively regulates the NS response. NS, in turn, promotes the degradation of FgIxr1, thus enhancing the recruitment of the SWI/SNF complex by FgAreB.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/metabolismo , Fusarium/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Doenças das Plantas/microbiologia , Proteína SMARCB1/metabolismo , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Fusarium/genética , Fusarium/patogenicidade , Fatores de Transcrição GATA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Óxido Nítrico/metabolismo , Estresse Nitrosativo , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Triticum/microbiologia , Zea mays/microbiologia
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