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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(9): 861-864, 2021 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-34487531

RESUMO

OBJECTIVE: To explore the genotype-phenotype correlation in a child with Kabuki syndrome type 1 (KS1) caused by a mosaic frameshift variant of KMT2D gene. METHODS: Trio-based whole exome sequencing (WES) was carried for the patient and her parents. Candidate variant was verified by Sanger sequencing. RESULTS: The proband, a 3-year-and-2-month-old Chinese girl, presented with distinctive facial features, cognitive impairment, mild developmental delay, dermatoglyphic abnormalities, minor skeletal anomalies, ventricular septal defect, and autistic behavior. Trio-based WES revealed that the proband has carried a de novo mosaic frameshit variant of the KMT2D gene, namely NM_003482.3:c.13058delG (p.Pro4353Argfs*31) (GRCh37/hg19), for which the mosaicism rate was close to 21%. The variant was unreported previously and was confirmed by Sanger sequencing. Chromosomal microarray analysis (CMA) has revealed no pathogenic or likely pathogenic copy number variations. Compared with previously reported cases, our patient has presented obvious behavior anomalies including autism, anxiety and sleep problems, which were rarely reported. CONCLUSION: This study has expanded the spectrum of KMT2D gene variants, enriched the clinical phenotypes of KS1, and facilitated genetic counseling for the family.


Assuntos
Variações do Número de Cópias de DNA , Proteínas de Ligação a DNA , Anormalidades Múltiplas , China , Proteínas de Ligação a DNA/genética , Face/anormalidades , Feminino , Doenças Hematológicas , Humanos , Lactente , Proteínas de Neoplasias/genética , Fenótipo , Doenças Vestibulares
2.
Nat Commun ; 12(1): 5240, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475390

RESUMO

ß-actin is a crucial component of several chromatin remodeling complexes that control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes (PRCs). While previous work has shown that ß-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in ß-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of ß-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that ß-actin levels can induce changes in chromatin structure by affecting the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2. Our results show that changes in ß-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversible changes in 3D genome architecture. Our findings reveal that ß-actin-dependent chromatin remodeling plays a role in shaping the chromatin landscape and influences the regulation of genes involved in development and differentiation.


Assuntos
Actinas/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Actinas/genética , Animais , Cromatina/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Fibroblastos , Dosagem de Genes , Técnicas de Inativação de Genes , Histonas/metabolismo , Camundongos , Proteínas Nucleares/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Ligação Proteica , Fatores de Transcrição/metabolismo
3.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(9): 912-916, 2021 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-34487543

RESUMO

MAMLD1 gene has been implicated in 46,XY disorders of sex development (DSD) in recent years. Patients carrying MAMLD1 gene variants showed a "continuous spectrum" of simple micropenis, mild, moderate and severe hypospadias with micropenis, cryptorchidism, split scrotum and even complete gonadal dysplasia. The function of MAMLD1 gene in sexual development has not been fully elucidated, and its role in DSD has remained controversial. This article has reviewed recent findings on the role of the MAMLD1 gene in DSD, including the MAMLD1 gene, its encoded protein, genetic variants, clinical phenotype and possible pathogenic mechanism in DSD.


Assuntos
Proteínas de Ligação a DNA , Transtornos do Desenvolvimento Sexual , Proteínas de Ligação a DNA/genética , Transtornos do Desenvolvimento Sexual/genética , Humanos , Masculino , Mutação , Proteínas Nucleares/genética , Fenótipo , Desenvolvimento Sexual , Fatores de Transcrição/genética
4.
Nat Commun ; 12(1): 5091, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429415

RESUMO

Ten-eleven translocation (TET) proteins, the dioxygenase for DNA hydroxymethylation, are important players in nervous system development and diseases. However, their role in myelination and remyelination after injury remains elusive. Here, we identify a genome-wide and locus-specific DNA hydroxymethylation landscape shift during differentiation of oligodendrocyte-progenitor cells (OPC). Ablation of Tet1 results in stage-dependent defects in oligodendrocyte (OL) development and myelination in the mouse brain. The mice lacking Tet1 in the oligodendrocyte lineage develop behavioral deficiency. We also show that TET1 is required for remyelination in adulthood. Transcriptomic, genomic occupancy, and 5-hydroxymethylcytosine (5hmC) profiling reveal a critical TET1-regulated epigenetic program for oligodendrocyte differentiation that includes genes associated with myelination, cell division, and calcium transport. Tet1-deficient OPCs exhibit reduced calcium activity, increasing calcium activity rescues the differentiation defects in vitro. Deletion of a TET1-5hmC target gene, Itpr2, impairs the onset of OPC differentiation. Together, our results suggest that stage-specific TET1-mediated epigenetic programming and intracellular signaling are important for proper myelination and remyelination in mice.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Camundongos Mutantes Neurológicos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Remielinização/fisiologia , 5-Metilcitosina/análogos & derivados , Animais , Ciclo Celular , Diferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA/genética , Genoma , Camundongos , Camundongos Knockout , Oligodendroglia/metabolismo , Organogênese , Proteínas Proto-Oncogênicas/genética
5.
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
6.
BMJ Case Rep ; 14(8)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446510

RESUMO

A 55-year-old woman presented with a 3-month history of right groin swelling, discomfort and impaired mobility. On examination, a palpable mass was noted both to the right of midline in the lower abdomen and in the right groin. MRI of the pelvis showed two masses involving the anterior abdominal wall and right groin, as well as lymph node involvement. CT imaging revealed multiple bilateral pulmonary metastases. Pathology demonstrated a myxohayline stroma morphology. Tumour was also notable for NR4A3 gene region rearrangement and mutation in KIT exon 11 at position c.1669 T>G. Based on these findings, she was diagnosed with extraskeletal myxoid chondrosarcoma (EMC). The patient has been on imatinib, a tyrosine kinase inhibitor with activity against KIT, for 3 years with stable disease. Metastatic EMC is generally treated with surgical resection and perioperative radiation therapy with adjuvant chemotherapy and is associated with poor prognosis.


Assuntos
Condrossarcoma , Receptores de Esteroides , Condrossarcoma/diagnóstico por imagem , Condrossarcoma/tratamento farmacológico , Condrossarcoma/genética , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Mesilato de Imatinib/uso terapêutico , Pessoa de Meia-Idade , Mutação , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética
7.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360855

RESUMO

The FMS-like tyrosine kinase 3 (FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor S63845. AML patient samples with a strong response to AC-4-130 and S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Pirimidinas/uso terapêutico , Fator de Transcrição STAT5/antagonistas & inibidores , Tiofenos/uso terapêutico , Proteínas Supressoras de Tumor/antagonistas & inibidores , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Proto-Oncogênicas/genética , Tirosina Quinase 3 Semelhante a fms/genética
9.
J Transl Med ; 19(1): 332, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34353343

RESUMO

BACKGROUND: Pancreatic cancer (PC) is one of the most fatal digestive system cancers. tripartite motif-29 (TRIM29) has been reported as oncogene in several human cancers. However, the precise role and underlying signal cascade of TRIM29 in PC progression remain unclear. METHODS: Western blot, qRT-PCR and immunohistochemistry were used to analyze TRIM29 and Yes-associated protein 1 (YAP1) levels. CCK8 assays, EdU assays and flow cytometry were designed to explore the function and potential mechanism of TRIM29 and YAP1 in the proliferation of PC. Next, a nude mouse model of PC was established for validating the roles of TRIM29 and YAP1 in vivo. The relationship among TRIM29 and YAP1 was explored by co-immunoprecipitation and in vitro ubiquitination assay. RESULTS: TRIM29 and YAP1 was significantly upregulated in PC patient samples, and TRIM29 expression was closely related to a malignant phenotype and poorer overall survival (OS) of PC patients. Functional assays revealed that TRIM29 knockdown suppresses cell growth, arrests cell cycle progression and promotes cell apoptosis of PC cells in vivo and in vitro. Furthermore, the rescue experiments demonstrated that TRIM29-induced proliferation is dependent on YAP1 in PC cells. Mechanistically, TRIM29 regulates YAP1 expression by directly binding to YAP1, and reduced its ubiquitination and degradation. CONCLUSION: Taken together, these results identify a novel mechanism used by PC growth, and provide insight regarding the role of TRIM29 in PC.


Assuntos
Proteínas de Ligação a DNA , Neoplasias Pancreáticas , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Fatores de Transcrição , Ubiquitina-Proteína Ligases
10.
Zool Res ; 42(5): 562-573, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34355875

RESUMO

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Blastocisto/fisiologia , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mórula/fisiologia , Suínos , ATPases Associadas a Diversas Atividades Celulares/genética , Animais , Proteínas de Ligação a DNA/genética , Técnicas de Cultura Embrionária/veterinária , Fertilização In Vitro , Regulação da Expressão Gênica/fisiologia , Oócitos/fisiologia , Permeabilidade
11.
Nat Commun ; 12(1): 4877, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385434

RESUMO

Chronically elevated intraocular pressure (IOP) is the major risk factor of primary open-angle glaucoma, a leading cause of blindness. Dysfunction of the trabecular meshwork (TM), which controls the outflow of aqueous humor (AqH) from the anterior chamber, is the major cause of elevated IOP. Here, we demonstrate that mice deficient in the Krüppel-like zinc finger transcriptional factor GLI-similar-1 (GLIS1) develop chronically elevated IOP. Magnetic resonance imaging and histopathological analysis reveal that deficiency in GLIS1 expression induces progressive degeneration of the TM, leading to inefficient AqH drainage from the anterior chamber and elevated IOP. Transcriptome and cistrome analyses identified several glaucoma- and extracellular matrix-associated genes as direct transcriptional targets of GLIS1. We also identified a significant association between GLIS1 variant rs941125 and glaucoma in humans (P = 4.73 × 10-6), further supporting a role for GLIS1 into glaucoma etiology. Our study identifies GLIS1 as a critical regulator of TM function and maintenance, AqH dynamics, and IOP.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Glaucoma/fisiopatologia , Pressão Intraocular/fisiologia , Malha Trabecular/fisiopatologia , Fatores de Transcrição/metabolismo , Animais , Humor Aquoso/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Glaucoma/genética , Glaucoma/metabolismo , Células HEK293 , Humanos , Pressão Intraocular/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA-Seq/métodos , Malha Trabecular/metabolismo , Fatores de Transcrição/genética
12.
Medicine (Baltimore) ; 100(33): e26850, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34414935

RESUMO

BACKGROUND: Gastric cancer (GC) is a strong cause of global cancer mortality. Nucleotide excision repair (NER) can modulate platinum-based chemotherapeutic efficacy by removing drug-produced DNA damage. Some studies have found a link between excision repair cross complementation group 1 (ERCC1) rs2298881, one gene in NER pathway, and response to chemotherapy. However, the results have been disputed. METHODS: We conducted a meta-analysis to reevaluate the association between polymorphisms of NER gene (ERCC1 rs2298881) and the clinical outcomes in gastric cancer patients receiving platinum-based chemotherapy. Searching PubMed, Web of Science, EMBASE, Google Scholar, and China National Knowledge Infrastructure, 2 independent searchers found all pertinent literatures up to May 1, 2021. We enrolled studies according to consistent selection criteria, extracted and vitrified data. Crude odds ratios (ORs) and hazard ratios (HRs) with 95% confidence interval (CI) were applied to evaluate the effect of ERCC1 rs2298881 on patients treated by platinum-based chemotherapy. RESULTS: By the data gathered from 6 independent studies, 1940 cases diagnosed with gastric cancer and treated with chemotherapy were included, containing 1208 Good-Responders and 732 Poor-Responders. With a comprehensive meta-analysis, we found that the patients with ERCC1 rs2298881A allele had a worse response to chemotherapy than those who with rs2298881C allele under allelic model (A vs C), with the pooled OR of 0.780 (95% CI: 0.611-0.996, P = .046). And our analysis indicated that AA genotype was associated with unfavorable overall survival (HR = 1.540, 95% CI = 1.106-2.144, P = .011) compared with CC genotype. CONCLUSIONS: ERCC1 rs2298881 is suggested as a marker of clinical outcome in gastric cancer patients treated by platinum-based chemotherapy.


Assuntos
Antineoplásicos/uso terapêutico , Cisplatino/uso terapêutico , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Oxaliplatina/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Humanos , Polimorfismo de Nucleotídeo Único , Prognóstico , Neoplasias Gástricas/mortalidade
13.
Nat Commun ; 12(1): 4843, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376693

RESUMO

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Repetições de Microssatélites/genética , Proteínas de Ligação a Telômeros/metabolismo , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Reparo do DNA por Junção de Extremidades , DNA Polimerase I/genética , DNA Primase/genética , DNA de Cadeia Simples , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
14.
Nat Commun ; 12(1): 4813, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376664

RESUMO

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c- effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis.


Assuntos
Envelhecimento/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Fatores Etários , Envelhecimento/genética , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Feminino , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Estimativa de Kaplan-Meier , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Fatores Sexuais , Proteínas com Domínio T/imunologia , Proteínas com Domínio T/metabolismo
15.
Nat Commun ; 12(1): 5056, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417458

RESUMO

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.


Assuntos
Epigênese Genética , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Micrometástase de Neoplasia , Ligação Proteica , Carga Tumoral
16.
Nat Commun ; 12(1): 5074, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417463

RESUMO

ß cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and human ß cells with inflammation but its expression is reduced in surviving ß cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate ß cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO ß cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between ß cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in ß cells may reduce activating pathologic immune cells and killing of ß cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 1/patologia , Inflamação/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Citotoxicidade Imunológica , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Feminino , Humanos , Imunidade , Inflamação/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Transcrição Genética
17.
Mol Cell ; 81(16): 3400-3409.e3, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352203

RESUMO

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.


Assuntos
DNA Ligase Dependente de ATP/ultraestrutura , Enzimas Reparadoras do DNA/ultraestrutura , Proteína Quinase Ativada por DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Apoptose/genética , Microscopia Crioeletrônica , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/ultraestrutura , Complexos Multiproteicos/genética , Fosforilação/genética
18.
Methods Mol Biol ; 2351: 251-274, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382194

RESUMO

In this chapter, we describe the proteomic approach named "Native Chromatin Proteomics" (N-ChroP) that couples a modified Chromatin ImmunoPrecipitation (ChIP) protocol with the mass spectrometry (MS) analysis of immunoprecipitated proteins to study the combinatorial enrichment or exclusion of histone post-translational modifications (PTMs) at specific genomic regions, such as promoters or enhancers. We describe the protocol steps from the digestion of chromatin and nucleosome immunoprecipitation to histone digestion and peptide enrichment prior to MS analysis, up to the MS raw data analysis. We also discuss current challenges and offer suggestions based on the direct hands-on experience acquired during the method setup.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Genômica , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Cromatografia Líquida , Proteínas de Ligação a DNA/metabolismo , Análise de Dados , Genômica/métodos , Nucleossomos/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem
19.
Methods Mol Biol ; 2351: 275-288, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382195

RESUMO

Functionalization of the genome is carried out by proteins that bind to DNA to regulate gene expression. Since this process is highly dynamic, context-dependent, and rarely performed by single proteins alone, we here describe ChIP-SICAP to identify proteins that co-localize with a protein of interest on the genome. Benefiting from its nature as a dual purification approach via ChIP and DNA biotinylation, ChIP-SICAP distinguishes genuine chromatin-binders and is uniquely placed to identify novel players in genome regulation.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Biotinilação , DNA/genética , DNA/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Peptídeo Hidrolases , Ligação Proteica , Proteômica/métodos
20.
Methods Mol Biol ; 2351: 289-303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382196

RESUMO

Interactions between regulatory proteins and specific genomic regions are critical for all chromatin-based processes, including transcription, DNA replication, and DNA repair. Genome-wide mapping of such interactions is most commonly performed with chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq), but a number of orthogonal methods employing targeted enzymatic activity have also been introduced. We previously described a genome-wide implementation of chromatin endogenous cleavage (ChEC-Seq), wherein a protein of interest is fused to micrococcal nuclease (MNase) to enable targeted, calcium-dependent genomic cleavage. Here, we describe the ChEC-Seq protocol for use in budding yeast though it can be used in other organisms in conjunction with appropriate methods for introduction of an MNase fusion protein.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação/métodos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Estudo de Associação Genômica Ampla , Estudo de Associação Genômica Ampla/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo
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