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1.
Nucleic Acids Res ; 49(11): 6281-6295, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107030

RESUMO

Epigenetics, especially histone marks, functions beyond the DNA sequences to regulate gene expression. Depletion of NSD1, which catalyzes H3K36me2, leads to both up- and down-regulation of gene expression, indicating NSD1 is associated with both active and repressed gene expression. It's known that NSD1 regulates the deposition and expansion of H3K27me3, a repressive mark for gene expression, to keep active gene transcription. However, how NSD1 functions to repress gene expression is largely unknown. Here, we find that, when NSD1 is knocked out in mouse embryonic stem cells (mESCs), H3K27ac increases correlatively with the decrease of H3K36me2 at active enhancers, which is associated with mesoderm differentiation genes, leading to elevated gene expression. Mechanistically, NSD1 recruits HDAC1, the deacetylase of H3K27ac, to chromatin. Moreover, HDAC1 knockout (KO) recapitulates the increase of H3K27ac at active enhancers as the NSD1 depletion. Together, we propose that NSD1 deposits H3K36me2 and recruits HDAC1 at active enhancers to serve as a 'safeguard', preventing further activation of active enhancer-associated genes.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Código das Histonas , Histona-Lisina N-Metiltransferase/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Técnicas de Inativação de Genes , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Mesoderma/metabolismo , Camundongos
2.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064673

RESUMO

Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group (SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar 'Fuding Dabaicha'. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes (BASL, SBT1.2(SDD1), EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.


Assuntos
Arabidopsis/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Pressão Osmótica , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Chá/enzimologia , Regulação da Expressão Gênica de Plantas , Histona-Lisina N-Metiltransferase/genética , Proteínas de Plantas/genética , Chá/química
3.
Nat Commun ; 12(1): 2792, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33990599

RESUMO

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Animais , Antineoplásicos/química , Domínio Catalítico/efeitos dos fármacos , Domínio Catalítico/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Desenho de Fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Feminino , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Moleculares , Proteína de Leucina Linfoide-Mieloide/genética , Oncogenes , Domínios Proteicos , Proteínas Recombinantes de Fusão/genética
4.
J Med Case Rep ; 15(1): 228, 2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-33957966

RESUMO

BACKGROUND: Acute myeloid leukemia (AML) is a disease with a significant amount of cytogenetic heterogeneity including mixed-lineage leukemia (MLL) gene rearrangements. Pediatric AML commonly has genetic rearrangements which involve chromosome 11q23 in 15-20% of cases, and these genetic abnormalities have been associated with a poorer prognosis (Grimwade et al. in Blood 92:2322-2333, 1998; Raimondi et al. in Blood 94:3707-3716, 1999; Lie et al. in Br J Haematol 122: 217-225). MLL rearrangements in AML have been shown to have multiple different fusion partners (Meyer et al. in Leukemia 23:1490-1499). Heterogeneity of these cytogenetic abnormalities makes it difficult to determine how to approach patients from a treatment standpoint. This difficulty is further complicated when patients have more than a single MLL rearrangement. CASE PRESENTATION: A 10-year-old Caucasian girl presented with easy bruising and was found to have acute myeloid leukemia. Her cytogenetics showed two different MLL rearrangements, t(9;11)(p22;q23) and t(11;19)(q23;p13.3). At initial presentation she had no other cytogenetic findings. She responded well to initial therapy and achieved remission following the first induction cycle and completed four rounds of chemotherapy. She subsequently had a relapse of her AML, and her cytogenetics were consistent with a single MLL rearrangement, t(9;11)(p22;q23), in addition to monosomy 7. She was treated with reduction therapy and a haplo-identical bone marrow transplant but ultimately succumbed to her disease. CONCLUSION: MLL rearrangements are common in AML, but clinical significance continues to be elusive, and there is conflicting data on the prognostic significance. In the setting of multiple MLL rearrangements, there is concern for reduced survival, although treatment modifications are not currently done in this setting. This report details a case with multiple MLL rearrangements that initially responded to therapy but ultimately had disease progression with a selection of a leukemic clone containing a single MLL rearrangement.


Assuntos
Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Criança , Feminino , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Translocação Genética
5.
Cell Prolif ; 54(6): e13045, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33949020

RESUMO

OBJECTIVES: Cutaneous wound healing is one of the major medical problems worldwide. Epigenetic modifiers have been identified as important players in skin development, homeostasis and wound repair. SET domain-containing 2 (SETD2) is the only known histone H3K36 tri-methylase; however, its role in skin wound healing remains unclear. MATERIALS AND METHODS: To elucidate the biological role of SETD2 in wound healing, conditional gene targeting was used to generate epidermis-specific Setd2-deficient mice. Wound-healing experiments were performed on the backs of mice, and injured skin tissues were collected and analysed by haematoxylin and eosin (H&E) and immunohistochemical staining. In vitro, CCK8 and scratch wound-healing assays were performed on Setd2-knockdown and Setd2-overexpression human immortalized keratinocyte cell line (HaCaT). In addition, RNA-seq and H3K36me3 ChIP-seq analyses were performed to identify the dysregulated genes modulated by SETD2. Finally, the results were validated in functional rescue experiments using AKT and mTOR inhibitors (MK2206 and rapamycin). RESULTS: Epidermis-specific Setd2-deficient mice were successfully established, and SETD2 deficiency resulted in accelerated re-epithelialization during cutaneous wound healing by promoting keratinocyte proliferation and migration. Furthermore, the loss of SETD2 enhanced the scratch closure and proliferation of keratinocytes in vitro. Mechanistically, the deletion of Setd2 resulted in the activation of AKT/mTOR signalling pathway, while the pharmacological inhibition of AKT and mTOR with MK2206 and rapamycin, respectively, delayed wound closure. CONCLUSIONS: Our results showed that SETD2 loss promoted cutaneous wound healing via the activation of AKT/mTOR signalling.


Assuntos
Histona-Lisina N-Metiltransferase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pele/lesões , Serina-Treonina Quinases TOR/metabolismo , Cicatrização , Animais , Linhagem Celular , Células Cultivadas , Deleção de Genes , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Transdução de Sinais , Pele/metabolismo , Pele/patologia , Regulação para Cima
6.
Nat Commun ; 12(1): 2901, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006870

RESUMO

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.


Assuntos
Proteínas de Ciclo Celular/genética , GTP Fosfo-Hidrolases/genética , Histona-Lisina N-Metiltransferase/genética , Leucemia Mielomonocítica Crônica/genética , Proteínas de Membrana/genética , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Animais , Proteínas de Ciclo Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Estimativa de Kaplan-Meier , Leucemia Mielomonocítica Crônica/metabolismo , Leucemia Mielomonocítica Crônica/terapia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Transplante de Células-Tronco/métodos , Transplante Homólogo , Sequenciamento Completo do Exoma/métodos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Blood Adv ; 5(9): 2325-2338, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33938943

RESUMO

Multiple myeloma (MM) is an (epi)genetic highly heterogeneous plasma cell malignancy that remains mostly incurable. Deregulated expression and/or genetic defects in epigenetic-modifying enzymes contribute to high-risk disease and MM progression. Overexpression of the histone methyltransferase G9a was reported in several cancers, including MM, correlating with disease progression, metastasis, and poor prognosis. However, the exact role of G9a and its interaction partner G9a-like protein (GLP) in MM biology and the underlying mechanisms of action remain poorly understood. Here, we report that high G9a RNA levels are associated with a worse disease outcome in newly diagnosed and relapsed MM patients. G9a/GLP targeting using the specific G9a/GLP inhibitors BIX01294 and UNC0638 induces a G1-phase arrest and apoptosis in MM cell lines and reduces primary MM cell viability. Mechanistic studies revealed that G9a/GLP targeting promotes autophagy-associated apoptosis by inactivating the mTOR/4EBP1 pathway and reducing c-MYC levels. Moreover, genes deregulated by G9a/GLP targeting are associated with repressive histone marks. G9a/GLP targeting sensitizes MM cells to the proteasome inhibitors (PIs) bortezomib and carfilzomib, by (further) reducing mTOR signaling and c-MYC levels and activating p-38 and SAPK/JNK signaling. Therapeutic treatment of 5TGM1 mice with BIX01294 delayed in vivo MM tumor growth, and cotreatment with bortezomib resulted in a further reduction in tumor burden and a significantly prolonged survival. In conclusion, we provide evidence that the histone methyltransferases G9a/GLP support MM cell growth and survival by blocking basal autophagy and sustaining high c-MYC levels. G9a/GLP targeting represents a promising strategy to improve PI-based treatment in patients with high G9a/GLP levels.


Assuntos
Histona-Lisina N-Metiltransferase , Mieloma Múltiplo , Animais , Apoptose , Autofagia , Morte Celular , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Inibidores de Proteassoma/farmacologia
8.
Redox Biol ; 43: 102004, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34020310

RESUMO

Epigenetic regulation disorder is important in the onset and pathogenesis of inflammatory bowel disease (IBD). SETD2, a trimethyltransferase of histone H3K36, is frequently mutated in IBD samples with a high risk of developing colorectal cancer (CRC). However, functions of SETD2 in IBD and colitis-associated CRC remain largely undefined. Here, we found that SETD2 modulates oxidative stress to attenuate colonic inflammation and tumorigenesis in mice. SETD2 expression became decreased in IBD patients and dextran sodium sulfate (DSS)-induced colitic mice. Setd2Vil-KO mice showed increased susceptibility to DSS-induced colitis, accompanied by more severe epithelial barrier disruption and markedly increased intestinal permeability that subsequently facilitated inflammation-associated CRC. Mechanistically, we found that Setd2 depletion resulted in excess reactive oxygen species (ROS) by directly down-regulating antioxidant genes, which led to defects in barrier integrity and subsequently inflammatory damage. Moreover, overexpression of antioxidant PRDX6 in Setd2Vil-KO intestinal epithelial cells (IECs) largely alleviated the overproductions of ROS and improved the cellular survival. Together, our findings highlight an epigenetic mechanism by which SETD2 modulates oxidative stress to regulate intestinal epithelial homeostasis and attenuate colonic inflammation and tumorigenesis. SETD2 might therefore be a pivotal regulator that maintains the homeostasis of the intestinal mucosal barrier.


Assuntos
Colite , Epigênese Genética , Animais , Colite/genética , Colo/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo
9.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925480

RESUMO

Conventional chemotherapy for acute myeloid leukemia regimens generally encompass an intensive induction phase, in order to achieve a morphological remission in terms of bone marrow blasts (<5%). The majority of cases are classified as Primary Induction Response (PIR); unfortunately, 15% of children do not achieve remission and are defined Primary Induction Failure (PIF). This study aims to characterize the gene expression profile of PIF in children with Acute Myeloid Leukemia (AML), in order to detect molecular pathways dysfunctions and identify potential biomarkers. Given that NUP98-rearrangements are enriched in PIF-AML patients, we investigated the association of NUP98-driven genes in primary chemoresistance. Therefore, 85 expression arrays, deposited on GEO database, and 358 RNAseq AML samples, from TARGET program, were analyzed for "Differentially Expressed Genes" (DEGs) between NUP98+ and NUP98-, identifying 110 highly confident NUP98/PIF-associated DEGs. We confirmed, by qRT-PCR, the overexpression of nine DEGs, selected on the bases of the diagnostic accuracy, in a local cohort of PIF patients: SPINK2, TMA7, SPCS2, CDCP1, CAPZA1, FGFR1OP2, MAN1A2, NT5C3A and SRP54. In conclusion, the integrated analysis of NUP98 mutational analysis and transcriptome profiles allowed the identification of novel putative biomarkers for the prediction of PIF in AML.


Assuntos
Biomarcadores Tumorais/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Quinase 6 Dependente de Ciclina/genética , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Humanos , Lactente , Recém-Nascido , Masculino , Família Multigênica , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Falha de Tratamento
10.
Int J Mol Sci ; 22(7)2021 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-33916664

RESUMO

DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans.


Assuntos
Metilação de DNA/genética , Doenças Genéticas Inatas/genética , Histonas/genética , Mutação , Doenças Raras/genética , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Doenças Genéticas Inatas/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/genética , Doenças Raras/metabolismo
11.
Nat Commun ; 12(1): 2428, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893291

RESUMO

Heterochromatin is a critical chromatin compartment, whose integrity governs genome stability and cell fate transitions. How heterochromatin features, including higher-order chromatin folding and histone modifications associated with transcriptional silencing, are maintained following a genotoxic stress challenge is unknown. Here, we establish a system for targeting UV damage to pericentric heterochromatin in mammalian cells and for tracking the heterochromatin response to UV in real time. We uncover profound heterochromatin compaction changes during repair, orchestrated by the UV damage sensor DDB2, which stimulates linker histone displacement from chromatin. Despite massive heterochromatin unfolding, heterochromatin-specific histone modifications and transcriptional silencing are maintained. We unveil a central role for the methyltransferase SETDB1 in the maintenance of heterochromatic histone marks after UV. SETDB1 coordinates histone methylation with new histone deposition in damaged heterochromatin, thus protecting cells from genome instability. Our data shed light on fundamental molecular mechanisms safeguarding higher-order chromatin integrity following DNA damage.


Assuntos
Dano ao DNA , Reparo do DNA , DNA/genética , Heterocromatina/genética , Animais , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/efeitos da radiação , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Células MCF-7 , Metilação , Camundongos , Células NIH 3T3 , Raios Ultravioleta
12.
Breast Cancer Res Treat ; 187(2): 339-347, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33844099

RESUMO

PURPOSE: SETD2 is one of the key epigenetic regulatory genes involved in histone modifications. Its alterations were potentially oncogenic and commonly found in cancers. Interestingly, SETD2 is one of the most frequent mutated genes found exclusively in phyllodes tumor of the breast (PT). However, little has been done to further characterize SETD2 alterations in PT. METHODS: In this study, we examined the alterations of SETD2 gene and protein expression in a large cohort of PTs. Their correlations with SETD2 downstream target, H3K36me3 expression, and clinicopathologic features in PT were also assessed. RESULTS: SETD2 mutation was found in 15.9% of our cases and was mostly predicted to be damaging mutations. Interestingly, SETD2 mutations were associated with lower H3K36me3 expression, particularly those with damaging mutations (p = .041). Neither SETD2 mutations nor H3K36me3 expression was associated with PT grading and other clinicopathological features. By contrast, the SETD2 protein expression cannot reflect its mutation status and showed a different trend of clinicopathological correlations from H3K36me3. CONCLUSIONS: Our findings may suggest a potential involvement of epigenetic regulation via SETD2 alterations and downstream H3K36me3 on PT development. SETD2 mutations may occur early in the pathogenic process of PTs and its loss per se may not be sufficient for progression to malignancy. Exclusive alterations of SETD2 in PT can be used as markers for the diagnosis of fibroepithelial lesions. The association of H3K36me3 with SETD2 mutations may also indicate the value of evaluation of H3K36me3 expression in the diagnosis of fibroepithelial lesions.


Assuntos
Neoplasias da Mama , Tumor Filoide , Neoplasias da Mama/genética , Epigênese Genética , Feminino , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Histonas/metabolismo , Humanos , Tumor Filoide/genética
13.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33809237

RESUMO

Recent developments in tissue clearing methods have significantly advanced the three-dimensional analysis of biological structures in whole, intact tissue, providing a greater understanding of spatial relationships and biological circuits. Nonetheless, studies have reported issues with maintaining structural integrity and preventing tissue disintegration, limiting the wide application of these techniques to fragile tissues such as developing embryos. Here, we present an optimized passive tissue clearing technique (PACT)-based embryo clearing method, initial embedding PACT (IMPACT)-Basic, that improves tissue rigidity without compromising optical transparency. We also present IMPACT-Advance, which is specifically optimized for thin slices of mouse embryos past E13.5. We demonstrate proof-of-concept by investigating the expression of two relatively understudied PR domain (PRDM) proteins, PRDM10 and PRDM13, in intact cleared mouse embryos at various stages of development. We observed strong PRDM10 and PRDM13 expression in the developing nervous system and skeletal cartilage, suggesting a functional role for these proteins in these tissues throughout embryogenesis.


Assuntos
Desenvolvimento Embrionário/genética , Histona-Lisina N-Metiltransferase/genética , Imageamento Tridimensional/métodos , Fatores de Transcrição/genética , Animais , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos
14.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33803994

RESUMO

We have determined the effect of glyphosate and aminomethylphosphonic acid (AMPA) on expression of genes involved in chromatin architecture in human peripheral blood mononuclear cells (PBMCs). The cells were incubated with glyphosate and AMPA in the concentrations ranging from 0.5 to 100 µM and from 0.5, to 250 µM, respectively. The expression profile of the following genes by quantitative Real-Time PCR was evaluated: Genes involved in the DNA methylation (DNMT1, DNMT3A) and DNA demethylation process (TET3) and those involved in chromatin remodeling: genes involved in the modification of histone methylation (EHMT1, EHMT2) and genes involved in the modification of histone deacetylation (HDAC3, HDAC5). Gene profiling showed that glyphosate changed the expression of DNMT1, DMNT3A, and HDAC3, while AMPA changed the expression of DNMT1 and HDAC3. The results also revealed that glyphosate at lower concentrations than AMPA upregulated the expression of the tested genes. Both compounds studied altered expression of genes, which are characteristic for the regulation of transcriptionally inactive chromatin. However, the unknown activity of many other proteins involved in chromatin structure regulation prevents to carry out an unambiguous evaluation of the effect of tested xenobiotics on the studied process. Undoubtedly, we have observed that glyphosate and AMPA affect epigenetic processes that regulate chromatin architecture.


Assuntos
Cromatina/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Dioxigenases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Cromatina/genética , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/genética , Glicina/análogos & derivados , Glicina/farmacologia , Herbicidas , Antígenos de Histocompatibilidade/genética , Histona Desacetilases/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
15.
Cancer Med ; 10(7): 2216-2231, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33655698

RESUMO

OBJECTIVES: This study aimed to explore the novel biomarkers for immune checkpoint inhibitor (ICI) responses in non-small cell lung cancer (NSCLC) by integrating genomic profiling, tumor mutational burden (TMB), and expression of programmed death receptor 1 ligand (PD-L1). MATERIALS AND METHODS: Tumor and blood samples from 637 Chinese patients with NSCLC were collected for targeted panel sequencing. Genomic alterations, including single nucleotide variations, insertions/deletions, copy number variations, and gene rearrangements, were assessed and TMB was computed. TMB-high (TMB-H) was defined as ≥10 mutations/Mb. PD-L1 positivity was defined as ≥1% tumor cells with membranous staining. Genomic data and ICI outcomes of 240 patients with NSCLC were derived from cBioPortal. RESULTS: EGFR-sensitizing mutations, ALK, RET, and ROS1 rearrangements were associated with lower TMB and PD-L1+/TMB-H proportions, whereas KRAS, ALK, RET, and ROS1 substitutions/indels correlated with higher TMB and PD-L1+/TMB-H proportions than wild-type genotypes. Histone-lysine N-methyltransferase 2 (KMT2) family members (KMT2A, KMT2C, and KMT2D) were frequently mutated in NSCLC tumors, and these mutations were associated with higher TMB and PD-L1 expression, as well as higher PD-L1+/TMB-H proportions. Specifically, patients with KMT2C mutations had higher TMB and PD-L1+/TMB-H proportions than wild-type patients. The median progression-free survival (PFS) was 5.47 months (95% CI 2.5-NA) in patients with KMT2C mutations versus 3.17 months (95% CI 2.6-4.27) in wild-type patients (p = 0.058). Furthermore, in patients with NSCLC who underwent ICI treatment, patients with TP53/KMT2C co-mutations had significantly longer PFS and greater durable clinical benefit (HR: 0.48, 95% CI: 0.24-0.94, p = 0.033). TP53 mutation combined with KMT2C or KRAS mutation was a better biomarker with expanded population benefit from ICIs therapy and increased the predictive power (HR: 0.46, 95% CI: 0.26-0.81, p = 0.007). CONCLUSION: We found that tumors with different alterations in actionable target genes had variable expression of PD-L1 and TMB in NSCLC. TP53/KMT2C co-mutation might serve as a predictive biomarker for ICI responses in NSCLC. IMPLICATIONS FOR PRACTICE: Cancer immunotherapies, especially immune checkpoint inhibitors (ICIs), have revolutionized the treatment of non-small cell lung cancer (NSCLC); however, only a proportion of patients derive durable responses to this treatment. Biomarkers with greater accuracy are highly needed. In total, 637 Chinese patients with NSCLC were analyzed using next-generation sequencing and IHC to characterize the unique features of genomic alterations and TMB and PD-L1 expression. Our study demonstrated that KMT2C/TP53 co-mutation might be an accurate, cost-effective, and reliable biomarker to predict responses to PD-1 blockade therapy in NSCLC patients and that adding KRAS to the biomarker combination creates a more robust parameter to identify the best responders to ICI therapy.


Assuntos
Antígeno B7-H1/análise , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/genética , Perfil Genético , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/genética , Mutação , Idoso , Quinase do Linfoma Anaplásico/genética , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/terapia , China , Feminino , Rearranjo Gênico , Genes erbB-1 , Genes p53 , Genes ras , Marcadores Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Histona-Lisina N-Metiltransferase/genética , Humanos , Imunoterapia Adotiva , Neoplasias Pulmonares/química , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret/genética , Estudos Retrospectivos
16.
Aging (Albany NY) ; 13(5): 7397-7415, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33658396

RESUMO

In this study, we used public databases to investigate the prognostic significance of epigenetic regulatory gene expression in patients with non small-cell lung cancer (NSCLC). Oncomine database analysis showed that the mRNA levels of seven epigenetic regulatory genes, UHRF1, EZH2, TTF2, SUV39H2, PCNA, WHSC1 and RAD54L, genes were significantly upregulated in NSCLC patients as compared to normal lung tissues. Functional enrichment analysis of these seven genes showed that the most enriched GO terms were DNA repair and rhythmic process, whereas, the most enriched KEGG pathway was lysine degradation pathway. The mRNA and protein expression levels of UHRF1, EZH2, TTF2, WHSC1 and RAD54L significantly correlated with tumor stage in NSCLC patients. Moreover, NSCLC patients exhibiting higher UHRF1, EZH2, WHSC1 and RAD54L mRNA and protein expression levels had poorer progression-free survival and overall survival. These findings demonstrate that UHRF1, EZH2, WHSC1 and RAD54L are potential prognostic biomarkers to distinguish high-risk from low-risk NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/genética , Adenosina Trifosfatases/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , DNA Helicases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Predisposição Genética para Doença/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Prognóstico , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Repressoras/genética , Análise de Sobrevida , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
17.
Nat Commun ; 12(1): 1956, 2021 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-33782403

RESUMO

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.


Assuntos
Carcinogênese/genética , Regulação Leucêmica da Expressão Gênica , Proteínas de Homeodomínio/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Animais , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Perfilação da Expressão Gênica , Xenoenxertos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Transgênicos , Família Multigênica , Mutação , Proteína Meis1/genética , Proteína Meis1/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Mielopoese/genética , Proteínas Nucleares/deficiência , Regiões Promotoras Genéticas , RNA Longo não Codificante/agonistas , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Transcrição Genética
18.
Phytomedicine ; 84: 153499, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33667841

RESUMO

BACKGROUND: There have been many researches on the effects of flavonoids on tumor treatment or adjuvant therapy, but there are few studies revealing their epigenetic effect on tumors. Hesperetin is a common citrus flavanone widely distributed among citrus fruits. The role of hesperetin in gastric cancer metastasis is unclear. PURPOSE: To investigate the effect of hesperetin on gastric cancer metastasis and its underlying mechanism. METHODS: We used cancer cell lines cultured in medium and nude mice implantation as in vitro and in vivo models to investigate the impact of hesperetin treatment on the migration and invasion of gastric cancer cells. The molecular biological experiments such as transwell assay, western blotting, qPCR, ChIP-qPCR, immunostaining and transfection were conducted to explore the molecular mechanisms. RESULTS: We found that hesperetin obviously reduced the protein abundance of DOT1L and the methylation of histone H3K79 in a variety of cells. In gastric cancer cells, the treatment of hesperetin decreased cell migration and invasion and the expression of genes closely related to the metastatic capability. Mechanistically, hesperetin affected the stability of DOT1L protein by regulating the activity of CBP. CONCLUSION: These findings highlight the epigenetic effect of hesperetin and provide a new perspective to understand the tumor suppressive effect of flavonoids.


Assuntos
Hesperidina/farmacologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Neoplasias Gástricas/patologia , Animais , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Camundongos , Camundongos Nus
19.
Biochem Biophys Res Commun ; 552: 98-105, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33743353

RESUMO

SET domain-containing 2 (SETD2), the primary methyltransferase for histone 3 lysine-36 trimethylation (H3K36me3) in mammals, is associated with many hematopoietic diseases when mutated. Previous works have emphasized its role in maintaining adult hematopoietic stem cells or tumorigenesis, however, whether and how SETD2 regulates erythropoiesis during embryonic development is relatively unexplored. In this study, using a conditional SETD2 knockout (KO) mouse model, we reveal that SETD2 plays an essential role in fetal erythropoiesis. Loss of Setd2 in hematopoietic cells ablates H3K36me3, and leads to anemia with a significant decrease in erythroid cells in the peripheral blood at E18.5. This is due to impaired erythroblast differentiation in both spleen and liver. We also find increased proportions of nucleated erythrocytes in the blood of Setd2 KO embryos. Lastly, we ascribe embryonic erythropoiesis-related genes Vegfc, Vegfr3, and Prox1, as likely downstream targets of SETD2 regulation. Our study reveals a critical role of SETD2 in fetal erythropoiesis that precedes adult hematopoiesis, and provide unique insights into the defects in erythroid lineages, such as anemia.


Assuntos
Diferenciação Celular/genética , Eritroblastos/metabolismo , Eritropoese/genética , Feto/metabolismo , Histona-Lisina N-Metiltransferase/genética , Animais , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Eritroblastos/citologia , Eritrócitos/citologia , Eritrócitos/metabolismo , Feto/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos Knockout , Camundongos Transgênicos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Life Sci ; 273: 119286, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33662429

RESUMO

AIMS: Hepatic ischemia/reperfusion (I/R) injury is a critical factor affecting the prognosis of liver surgery. The aim of this study is to explore the effects of SET8 on hepatic I/R injury and the putative mechanisms. MAIN METHODS: The expression of SET8 and MARK4 in I/R group and sham group were detected both in vivo and in vitro. In addition, mouse and RAW 264.7 cells were transfected with MARK4 siRNA and SET8 siRNA knockdown of MARK4 and SET8, respectively. The expression of SET8, MARK4 and NLRP3-associated proteins were detected after different treatments. The pathology of liver and the serologic detection were detected after different treatments. KEY FINDINGS: Our present study identified SET domain-containing protein 8 (SET8) as an efficient protein, which can negatively regulate hepatic I/R-mediated inflammatory response and ameliorate hepatic I/R injury by suppressing microtubule affinity-regulating kinase 4 (MARK4)/ NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway. The data showed that MARK4 deficiency inhibited hypoxia/reoxygenation (H/R)-induced NLRP3 inflammasome activation, while SET8 deficiency showed the opposite effect. We further demonstrated that SET8 restrained NLRP3 inflammasome activation by inhibiting MARK4. Moreover, we verified SET8 made protective effect on hepatic I/R injury. SIGNIFICANCE: SET8 plays an essential role in hepatic ischemia/reperfusion injury in mice by suppressing MARK4/NLRP3 inflammasome pathway. Our results may offer a new strategy to mitigate hepatic I/R injury.


Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Histona-Lisina N-Metiltransferase/metabolismo , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Traumatismo por Reperfusão/prevenção & controle , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Células Cultivadas , Histona-Lisina N-Metiltransferase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
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