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1.
Science ; 370(6513): 227-231, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033220

RESUMO

Stem cells in plants constantly supply daughter cells to form new organs and are expected to safeguard the integrity of the cells from biological invasion. Here, we show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV). The stem cell regulator WUSCHEL responds to CMV infection and represses virus accumulation in the meristem central and peripheral zones. WUSCHEL inhibits viral protein synthesis by repressing the expression of plant S-adenosyl-l-methionine-dependent methyltransferases, which are involved in ribosomal RNA processing and ribosome stability. Our results reveal a conserved strategy in plants to protect stem cells against viral intrusion and provide a molecular basis for WUSCHEL-mediated broad-spectrum innate antiviral immunity in plants.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Arabidopsis/virologia , Cucumovirus , Proteínas de Homeodomínio/fisiologia , Imunidade Inata , Doenças das Plantas/virologia , Imunidade Vegetal , Proteínas de Arabidopsis/genética , Proteínas de Homeodomínio/genética , Meristema/citologia , Meristema/imunologia , Meristema/virologia , Metiltransferases/metabolismo , RNA Ribossômico/metabolismo , Células-Tronco/imunologia , Células-Tronco/virologia
2.
Nucleic Acids Res ; 48(9): 4915-4927, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232336

RESUMO

Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.


Assuntos
Quebras de DNA de Cadeia Dupla , Histona Desmetilases/fisiologia , Proteínas de Homeodomínio/fisiologia , Reparo de DNA por Recombinação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Instabilidade Genômica , Células HeLa , Histona Desmetilases/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos
3.
Breast Cancer Res ; 22(1): 34, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32272947

RESUMO

BACKGROUND: Osteoclast activation is a hallmark of breast cancer-induced bone disease while little is known about the role of osteoblasts in this process. Recently, we identified the homeodomain protein TG-interacting factor-1 (Tgif1) as a crucial regulator of osteoblast function. In this study, we demonstrate that lack of Tgif1 also restricts the progression of breast cancer bone metastases. METHODS: Transwell migration assays were used to investigate the osteoblast-breast cancer cell interaction in vitro. Molecular analyses included RNA sequencing, immunoblotting, and qRT-PCR. To determine the role of Tgif1 in metastatic bone disease, 4T1 breast cancer cells were injected intracardially into mice with a germ line deletion of Tgif1 (Tgif1-/-) or control littermates (Tgif1+/+). Progression of bone metastases and alterations in the bone microenvironment were assessed using bioluminescence imaging, immunofluorescence staining, confocal microscopy, and histomorphometry. RESULTS: Medium conditioned by osteoblasts stimulated breast cancer cell migration, indicating a potential role of osteoblasts during bone metastasis progression. Tgif1 expression was strongly increased in osteoblasts upon stimulation by breast cancer cells, demonstrating the implication of Tgif1 in the osteoblast-breast cancer cell interaction. Indeed, conditioned medium from osteoblasts of Tgif1-/- mice failed to induce breast cancer cell migration compared to control, suggesting that Tgif1 in osteoblasts augments cancer cell motility. Semaphorin 3E (Sema3E), which is abundantly secreted by Tgif1-/- osteoblasts, dose-dependently reduced breast cancer cell migration while silencing of Sema3E expression in Tgif1-/- osteoblasts partially restored the impaired migration. In vivo, we observed a decreased number of breast cancer bone metastases in Tgif1-/- mice compared to control littermates. Consistently, the presence of single breast cancer cells or micro-metastases in the tibiae was reduced in Tgif1-/- mice. Breast cancer cells localized in close proximity to Endomucin-positive vascular cells as well as to osteoblasts. Although Tgif1 deficiency did not affect the bone marrow vasculature, the number and activity of osteoblasts were reduced compared to control. This suggests that the protective effect on bone metastases might be mediated by osteoblasts rather than by the bone marrow vasculature. CONCLUSION: We propose that the lack of Tgif1 in osteoblasts increases Sema3E expression and attenuates breast cancer cell migration as well as metastases formation.


Assuntos
Neoplasias Ósseas/prevenção & controle , Osso e Ossos/patologia , Neoplasias da Mama/prevenção & controle , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/fisiologia , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/fisiologia , Semaforinas/genética , Microambiente Tumoral , Animais , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Osso e Ossos/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoblastos/metabolismo , Osteoblastos/patologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(12): 6741-6751, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32152117

RESUMO

Neurodegenerative diseases feature specific misfolded or misassembled proteins associated with neurotoxicity. The precise mechanisms by which protein aggregates first arise in the majority of sporadic cases have remained unclear. Likely, a first critical mass of misfolded proteins starts a vicious cycle of a prion-like expansion. We hypothesize that viruses, having evolved to hijack the host cellular machinery for catalyzing their replication, lead to profound disturbances of cellular proteostasis, resulting in such a critical mass of protein aggregates. Here, we investigated the effect of influenza virus (H1N1) strains on proteostasis of proteins associated with neurodegenerative diseases in Lund human mesencephalic dopaminergic cells in vitro and infection of Rag knockout mice in vivo. We demonstrate that acute H1N1 infection leads to the formation of α-synuclein and Disrupted-in-Schizophrenia 1 (DISC1) aggregates, but not of tau or TDP-43 aggregates, indicating a selective effect on proteostasis. Oseltamivir phosphate, an antiinfluenza drug, prevented H1N1-induced α-synuclein aggregation. As a cell pathobiological mechanism, we identified H1N1-induced blocking of autophagosome formation and inhibition of autophagic flux. In addition, α-synuclein aggregates appeared in infected cell populations connected to the olfactory bulbs following intranasal instillation of H1N1 in Rag knockout mice. We propose that H1N1 virus replication in neuronal cells can induce seeds of aggregated α-synuclein or DISC1 that may be able to initiate further detrimental downstream events and should thus be considered a risk factor in the pathogenesis of synucleinopathies or a subset of mental disorders. More generally, aberrant proteostasis induced by viruses may be an underappreciated factor in initiating protein misfolding.


Assuntos
Proteínas de Homeodomínio/fisiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/complicações , Infecções por Orthomyxoviridae/complicações , Proteostase , Sinucleinopatias/etiologia , alfa-Sinucleína/química , Animais , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Influenza Humana/virologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo
5.
Development ; 147(4)2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32001441

RESUMO

In several model animals, the earliest phases of embryogenesis are regulated by lineage-specific genes, such as Drosophila bicoid Sea urchin (echinoid) embryogenesis is initiated by zygotic expression of pmar1, a paired-class homeobox gene that has been considered to be present only in the lineage of modern urchins (euechinoids). In euechinoids, Pmar1 promotes endomesoderm specification by repressing the hairy and enhancer of split C (hesC) gene. Here, we have identified the basal echinoid (cidaroid) pmar1 gene, which also promotes endomesoderm specification but not by repressing hesC A further search for related genes demonstrated that other echinoderms have pmar1-related genes named phb Functional analyses of starfish Phb proteins indicated that, similar to cidaroid Pmar1, they promote activation of endomesoderm regulatory gene orthologs via an unknown repressor that is not HesC. Based on these results, we propose that Pmar1 may have recapitulated the regulatory function of Phb during the early diversification of echinoids and that the additional repressor HesC was placed under the control of Pmar1 in the euechinoid lineage. This case provides an exceptional model for understanding how early developmental processes diverge.


Assuntos
Endoderma/fisiologia , Proteínas de Homeodomínio/fisiologia , Mesoderma/fisiologia , Ouriços-do-Mar/embriologia , Animais , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Larva/fisiologia , Fenótipo , Filogenia , Receptores Notch/fisiologia , Ouriços-do-Mar/genética
6.
Leukemia ; 34(8): 2051-2063, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32076119

RESUMO

Blast crisis of chronic myeloid leukemia is associated with poor survival and the accumulation of genomic lesions. Using whole-exome and/or RNA sequencing of patients at chronic phase (CP, n = 49), myeloid blast crisis (MBC, n = 19), and lymphoid blast crisis (LBC, n = 20), we found 25 focal gene deletions and 14 fusions in 24 patients in BC. Deletions predominated in LBC (83% of structural variants). Transcriptional analysis identified the upregulation of genes involved in V(D)J recombination, including RAG1/2 and DNTT in LBC. RAG recombination is a reported mediator of IKZF1 deletion. We investigated the extent of RAG-mediated genomic lesions in BC. Molecular hallmarks of RAG activity; DNTT-mediated nucleotide insertions and a RAG-binding motif at structural variants were exclusively found in patients with high RAG expression. Structural variants in 65% of patients in LBC displayed these hallmarks compared with only 5% in MBC. RAG-mediated events included focal deletion and novel fusion of genes associated with hematologic cancer: IKZF1, RUNX1, CDKN2A/B, and RB1. Importantly, 8/8 patients with elevated DNTT at CP diagnosis progressed to LBC by 12 months, potentially enabling early prediction of LBC. This work confirms the central mutagenic role of RAG in LBC and describes potential clinical utility in CML management.


Assuntos
Crise Blástica/genética , Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Nucleares/fisiologia , Recombinação Genética , Biologia Computacional , DNA Nucleotidilexotransferase/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Proteínas de Homeodomínio/genética , Humanos , Proteínas Nucleares/genética
7.
Life Sci ; 243: 117230, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31923422

RESUMO

AIMS: Accumulating evidence has confirmed the involvement of the homeobox (HOX) gene family in carcinogenesis. HOXC11, belongs to the homeobox-C (HOXC) gene cluster, has been reported to play important roles in the development of several cancers. However, its expression and clinical value in pan-cancer remain elusive. MATERIALS AND METHODS: Bioinformatics analysis, CCK-8 assay, Flow cytometry and Western blot were used to analyze gene expression and patient survival, cell proliferation, cell apoptosis and protein level, respectively. KEY FINDINGS: In this study, we comprehensively analyzed the expression profile and prognostic value of HOXC11 in human pan-cancer using online The Cancer Genome Atlas (TCGA) databases. HOXC11 was widely up-regulated in tumor tissues when compared with the normal tissues in pan-cancer across nine cancer types. In addition, high mRNA level of HOXC11 predicted poor overall survival (OS) of patients with adrenocortical carcinoma (ACC), colon adenocarcinoma (COAD), kidney renal clear cell carcinoma (KIRC), mesothelioma (MESO) and pancreatic adenocarcinoma (PAAD), respectively. By comparative analysis, we found that HOXC11 was up-regulated and closely correlated patient OS in COAD and KIRC. Functionally, down-regulation of HOXC11 inhibited cell proliferation but promoted apoptosis of COAD and KIRC in vitro. Mechanistically, HOXC11 promoted cell proliferation of COAD and KIRC might by inactivating the peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathway. SIGNIFICANCE: Our findings suggest that HOXC11 may act as a tumor driving gene in COAD and KIRC.


Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células Renais/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Homeodomínio/fisiologia , Neoplasias Renais/metabolismo , Oncogenes , Adenocarcinoma/patologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias do Colo/patologia , Regulação para Baixo/fisiologia , Proteínas de Homeodomínio/genética , Humanos , Neoplasias Renais/patologia , PPAR gama/metabolismo , Análise de Sobrevida
8.
Chin Med J (Engl) ; 133(3): 344-350, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31904730

RESUMO

Diabetes mellitus and pancreatic ductal adenocarcinoma are two common diseases worldwidely which are both derived from different components of pancreas. The pancreatic and duodenal homeobox-1 (PDX1) is an essential transcription factor for the early development of pancreas that is required for the differentiation of all pancreatic cell lineages. Current evidence suggests an important role of PDX1 in both the origin and progression of pancreatic diseases. In this review, we discussed recent studies of PDX1 in diabetes mellitus and pancreatic cancer, and the therapeutic strategies derived from this transcription factor.


Assuntos
Carcinoma Ductal Pancreático/etiologia , Diabetes Mellitus/etiologia , Proteínas de Homeodomínio/fisiologia , Neoplasias Pancreáticas/etiologia , Transativadores/fisiologia , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Diabetes Mellitus/tratamento farmacológico , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Células Secretoras de Insulina/fisiologia , Neoplasias Pancreáticas/tratamento farmacológico , Transativadores/antagonistas & inibidores
9.
Metabolism ; 103: 154014, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31751577

RESUMO

BACKGROUND: Inhibition of Irx3 and Irx5 has been shown to reduce body weight and white adipose tissue (WAT) mass through cell-autonomous and sympathetic-induced increases in adipocyte beiging and thermogenesis in mice and humans. However, the underlying mechanisms of the Irx control over beiging are still largely unknown, as illustrated by recent reports showing divergent effects of Irx3 on adipocyte metabolism and function. Here, we investigated the role of Irx3 in controlling beige preadipocyte function and differentiation. METHODS: Stable knock out of Irx3 in ME3 mouse preadipocytes capable of beiging was performed using a CRISPR-Cas9 system, and the effect on cell differentiation was assessed by qPCR, RNA-seq, Oil-red-O lipid staining and Alcian Blue staining of proteoglycans. Changes in cell identities were validated using cell type enrichment analysis from RNA-seq data. Proliferation and cell cycle progression in undifferentiated cells were measured by WST-1 and flow cytometry, reactive oxygen species (ROS) generation was determined by fluorescence spectrometry and mitochondrial respiration was investigated by Seahorse assay. RESULTS: Irx3 was found to be essential for the identity, function and adipogenic differentiation of beige adipocyte precursors. Irx3-KO impaired proliferation, ROS generation and mitochondrial respiration in the preadipocytes. We further observed profound changes in numerous genes during both early and late stages of adipogenic differentiation, including genes important for adipocyte differentiation, cell cycle progression, oxidative phosphorylation (OXPHOS) and morphogenesis. Irx3-KO cells failed to accumulate lipids following adipogenic stimuli, and cell enrichment analysis revealed a loss of preadipocyte identity and a gain of chondrocyte-like identity in Irx3-KO cells during early differentiation. Finally, unlike the control cells, the Irx3-KO cells readily responded to chondrogenic stimuli. CONCLUSIONS: Irx3 is required for preadipocyte identity and differentiation capacity. Our findings suggest that, while inhibition of Irx3 may be beneficial during later developmental stages to modulate adipogenesis in the beige direction, constitutive and complete absence of Irx3 in the embryonic fibroblast stage leads to detrimental loss of adipogenic differentiation capacity.


Assuntos
Adipogenia/genética , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Adipócitos Bege/fisiologia , Animais , Diferenciação Celular/genética , Linhagem da Célula/genética , Células Cultivadas , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Fatores de Transcrição/genética
10.
Life Sci ; 242: 117229, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31887298

RESUMO

AIMS: Neutrophil elastase (NE) is a critical proteolytic enzyme that is involved in cancer. We previously reported high NE expression in peripheral blood neutrophils from acute promyelocytic leukemia (APL) patients. The present study aimed to elucidate the specific role and mechanisms of NE in APL development. MATERIALS AND METHODS: NE expression was detected in APL bone marrow samples and analyzed in the BloodSpot database. CCK-8 assay and flow cytometry were used to assess cell proliferation and cell cycle distribution, respectively. The expression levels of proliferation and differentiation markers were measured by Western blotting and quantitative real-time PCR. The co-expression and interaction of NE and p200 cut-like homeobox 1 (CUX1) were evaluated by indirect immunofluorescence, co-immunoprecipitation, and in situ proximity ligation assay. KEY FINDINGS: NE was highly expressed in APL bone marrow and blood neutrophils. NE overexpression promoted the proliferation and inhibited the differentiation of NB4 cells, whereas NE downregulation achieved the opposite results in U937 cells. Mechanistically, NE interacted with and effectively hydrolyzed the tumor suppressor p200 CUX1. Rescue experiments revealed that p200 CUX1 upregulation reversed the functional influence of NE on APL cells. SIGNIFICANCE: NE-mediated proteolysis of the tumor suppressor p200 CUX1 promotes APL progression. NE/p200 CUX1 axis is a novel and promising therapeutic target for APL treatment.


Assuntos
Proteínas de Homeodomínio/metabolismo , Leucemia Promielocítica Aguda/enzimologia , Elastase de Leucócito/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Adolescente , Adulto , Western Blotting , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Células HL-60 , Proteínas de Homeodomínio/fisiologia , Humanos , Imunoprecipitação , Leucemia Promielocítica Aguda/metabolismo , Elastase de Leucócito/fisiologia , Masculino , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sincalida/metabolismo , Fatores de Transcrição/fisiologia , Células U937
11.
PLoS Biol ; 17(11): e3000499, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31675356

RESUMO

The onset of sexual maturity involves dramatic changes in physiology and gene expression in many animals. These include abundant yolk protein production in egg-laying species, an energetically costly process under extensive transcriptional control. Here, we used the model organism Caenorhabditis elegans to provide evidence for the spatiotemporally defined interaction of two evolutionarily conserved transcription factors, CEH-60/PBX and UNC-62/MEIS, acting as a gateway to yolk protein production. Via proteomics, bimolecular fluorescence complementation (BiFC), and biochemical and functional readouts, we show that this interaction occurs in the intestine of animals at the onset of sexual maturity and suffices to support the reproductive program. Our electron micrographs and functional assays provide evidence that intestinal PBX/MEIS cooperation drives another process that depends on lipid mobilization: the formation of an impermeable epicuticle. Without this lipid-rich protective layer, mutant animals are hypersensitive to exogenous oxidative stress and are poor partners for mating. Dedicated communication between the hypodermis and intestine in C. elegans likely supports these physiological outcomes, and we propose a fundamental role for the conserved PBX/MEIS interaction in multicellular signaling networks that rely on lipid homeostasis.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores Genéricos de Transcrição/fisiologia , Vitelogênese/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Metabolismo dos Lipídeos , Estresse Oxidativo , Permeabilidade , Fatores de Transcrição , Fatores Genéricos de Transcrição/genética , Fatores Genéricos de Transcrição/metabolismo
12.
Proc Natl Acad Sci U S A ; 116(47): 23636-23642, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685615

RESUMO

Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.


Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Prosencéfalo/embriologia , Animais , Sistemas CRISPR-Cas , Proteínas do Olho/fisiologia , Técnicas de Inativação de Genes , Genes Reporter , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/genética , Holoprosencefalia/genética , Proteínas de Homeodomínio/fisiologia , Hipotálamo/anormalidades , Hipotálamo/embriologia , Hipotálamo/metabolismo , Óperon Lac , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Prosencéfalo/anormalidades , Prosencéfalo/metabolismo , Transdução de Sinais , Transgenes
13.
J Clin Invest ; 129(12): 5489-5500, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31710307

RESUMO

Deep venous thrombosis (DVT) and secondary pulmonary embolism cause approximately 100,000 deaths per year in the United States. Physical immobility is the most significant risk factor for DVT, but a molecular and cellular basis for this link has not been defined. We found that the endothelial cells surrounding the venous valve, where DVTs originate, express high levels of FOXC2 and PROX1, transcription factors known to be activated by oscillatory shear stress. The perivalvular venous endothelial cells exhibited a powerful antithrombotic phenotype characterized by low levels of the prothrombotic proteins vWF, P-selectin, and ICAM1 and high levels of the antithrombotic proteins thrombomodulin (THBD), endothelial protein C receptor (EPCR), and tissue factor pathway inhibitor (TFPI). The perivalvular antithrombotic phenotype was lost following genetic deletion of FOXC2 or femoral artery ligation to reduce venous flow in mice, and at the site of origin of human DVT associated with fatal pulmonary embolism. Oscillatory blood flow was detected at perivalvular sites in human veins following muscular activity, but not in the immobile state or after activation of an intermittent compression device designed to prevent DVT. These findings support a mechanism of DVT pathogenesis in which loss of muscular activity results in loss of oscillatory shear-dependent transcriptional and antithrombotic phenotypes in perivalvular venous endothelial cells, and suggest that prevention of DVT and pulmonary embolism may be improved by mechanical devices specifically designed to restore perivalvular oscillatory flow.


Assuntos
Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Hemodinâmica/fisiologia , Trombose Venosa/prevenção & controle , Adulto , Animais , Feminino , Fatores de Transcrição Forkhead/fisiologia , Proteínas de Homeodomínio/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Pessoa de Meia-Idade , Fluxo Sanguíneo Regional , Proteínas Supressoras de Tumor/fisiologia
15.
Nat Commun ; 10(1): 4671, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604922

RESUMO

Mitochondrial trifunctional protein deficiency, due to mutations in hydratase subunit A (HADHA), results in sudden infant death syndrome with no cure. To reveal the disease etiology, we generated stem cell-derived cardiomyocytes from HADHA-deficient hiPSCs and accelerated their maturation via an engineered microRNA maturation cocktail that upregulated the epigenetic regulator, HOPX.  Here we report, matured HADHA mutant cardiomyocytes treated with an endogenous mixture of fatty acids manifest the disease phenotype: defective calcium dynamics and repolarization kinetics which results in a pro-arrhythmic state. Single cell RNA-seq reveals a cardiomyocyte developmental intermediate, based on metabolic gene expression. This intermediate gives rise to mature-like cardiomyocytes in control cells but, mutant cells transition to a pathological state with reduced fatty acid beta-oxidation, reduced mitochondrial proton gradient, disrupted cristae structure and defective cardiolipin remodeling. This study reveals that HADHA (tri-functional protein alpha), a monolysocardiolipin acyltransferase-like enzyme, is required for fatty acid beta-oxidation and cardiolipin remodeling, essential for functional mitochondria in human cardiomyocytes.


Assuntos
Cardiolipinas/metabolismo , Ácidos Graxos/metabolismo , Subunidade alfa da Proteína Mitocondrial Trifuncional/fisiologia , Miócitos Cardíacos/metabolismo , Cálcio/metabolismo , Linhagem Celular , Eletrofisiologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Células-Tronco Embrionárias Humanas , Humanos , MicroRNAs/fisiologia , Mitocôndrias/fisiologia , Proteína Mitocondrial Trifuncional/deficiência , Subunidade alfa da Proteína Mitocondrial Trifuncional/genética , Subunidade alfa da Proteína Mitocondrial Trifuncional/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Oxirredução , Técnicas de Patch-Clamp , RNA-Seq , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/fisiologia
16.
J Immunol ; 203(9): 2472-2484, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562212

RESUMO

Innate lymphoid cells (ILCs) are strategically positioned at mucosal barrier surfaces where they respond quickly to infection or injury. Therefore, we hypothesized that ILCs are key contributors to the early immune response in the intestine against Listeria monocytogenes Using a modified strain of L. monocytogenes that mimics human gastrointestinal listeriosis in mice, we find ILCs to be essential for control of early replication of L. monocytogenes in the intestine as well as for restricted dissemination of bacteria to peripheral tissues. Specifically, group 1 ILCs (ILC1s) and group 3 ILCs (ILC3s) respond to infection with proliferation and IFN-γ and IL-22 production. Mechanistically, we show that the transcription factor STAT4 is required for the proliferative and IFN-γ effector response by ILC1s and ILC3s, and loss of STAT4 signaling in the innate immune compartment results in an inability to control bacterial growth and dissemination. Interestingly, STAT4 acts acutely as a transcription factor to promote IFN-γ production. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with L. monocytogenes and identify STAT4 as a central modulator of ILC-mediated protection.


Assuntos
Gastroenteropatias/imunologia , Listeriose/imunologia , Linfócitos/imunologia , Fator de Transcrição STAT4/fisiologia , Animais , Proteínas de Homeodomínio/fisiologia , Imunidade Inata , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia
17.
DNA Cell Biol ; 38(11): 1313-1322, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545082

RESUMO

This study investigated whether overexpression of paired-related homeobox 1 (prrx1) can successfully induce differentiation of brown adipose-derived stem cells (BADSCs) into sinus node-like cells. The experiments were performed in two groups: adenovirus-green fluorescent protein (Ad-GFP) group and Ad-prrx1 group. After 5-7 days of adenoviral transfection, the expression levels of sinus node cell-associated pacing protein (hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 [HCN4]) and ion channel (calcium channel, voltage-dependent, T type, alpha 1G subunit [Cacna1g]), as well as transcription factors (T-box 18 [TBX18], insulin gene enhancer binding protein 1 [ISL-1], paired-like homeodomain transcription factor 2 [pitx2], short stature homeobox 2 [shox2]), were detected by western blot and reverse transcription-quantitative polymerase chain reaction. Immunofluorescence assay was carried out to detect whether prrx1 was coexpressed with HCN4, TBX18, and ISL-1. Finally, whole-cell patch-clamp technique was used to record pacing current hyperpolarization-activated inward current (If). The isolated cells were CD90+, CD29+, and CD45-, indicating that pure BADSCs were successfully isolated. After 5-7 days of Ad transfection into cells, the mRNA levels and protein levels of pacing-related factors (TBX18, ISL-1, HCN4, shox2, and Cacna1g) in Ad-prrx1 group were significantly higher than those in Ad-GFP group. However, the expression level of pitx2 was decreased. Immunofluorescence analysis showed that prrx1 was coexpressed with TBX18, ISL-1, and HCN4 in the Ad-prrx1 group, which did not appear in the Ad-GFP group. Whole-cell patch clamps were able to record the If current in the experimental group rather than in the Ad-GFP group. Overexpression of prrx1 can successfully induce sinus node-like cells.


Assuntos
Tecido Adiposo Marrom/fisiologia , Células-Tronco Adultas/fisiologia , Diferenciação Celular/genética , Proteínas de Homeodomínio/fisiologia , Nó Sinoatrial/fisiologia , Tecido Adiposo Marrom/citologia , Células-Tronco Adultas/citologia , Animais , Transdiferenciação Celular/genética , Células Cultivadas , Masculino , Ratos , Ratos Sprague-Dawley , Nó Sinoatrial/citologia , Transfecção
18.
Gynecol Oncol ; 155(2): 340-348, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31477279

RESUMO

OBJECTIVE: To determine the involvement of homeobox D9 (HOXD9) in the survival, proliferation, and metastasis of cervical cancer cells through regulating the expression of human papillomavirus (HPV) 16 E6/E7 genes using the P97 promoter. METHODS: One hundred cases of cervical cancer (CC), CC cell lines SKG-I, SKG-II, SKG-IIIa, SKG-IIIb, HeLa, and SiHa, and a human tumor xenograft mouse model were used to examine the roles of HOXD9 in CC. Knockdown experiments employed RNA interference of HOXD9. qPCR, functional assays, western blotting, DNA microarray, and luciferase and ChIP assays were applied for assessments. RESULTS: All CC cell lines expressed HOXD9 mRNA and protein. In uterine CC, HOXD9 gene expression was significantly higher than in normal cervical tissues. A positive correlation of lymphovascular space invasion and lymph node metastasis with high levels of HOXD9 expression was found in patient samples. HOXD9-knockdown cells in the mouse xenograft model only formed small or no tumors. Knockdown of HOXD9 markedly reduced CC cell proliferation, migration and invasion, induced apoptosis, increased P53 protein expression, and suppressed HPV E6/E7 expression by directly binding to the P97 promoter of HPV16 E6/E7 genes. A positive correlation between HOXD9 and HPV16 E6 expression was found in CC patients. CONCLUSIONS: HOXD9 promotes HPV16 E6 and E7 expression by direct binding to the P97 promoter, which enhances proliferation, migration, and metastasis of CCr cells. Our results suggest that HOXD9 could be a prognostic biomarker and potential therapeutic target in CC.


Assuntos
Proteínas de Homeodomínio/fisiologia , Proteínas de Neoplasias/fisiologia , Infecções por Papillomavirus/genética , Regiões Promotoras Genéticas/genética , Neoplasias do Colo do Útero/virologia , Animais , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Papillomavirus Humano 16/genética , Humanos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas Oncogênicas Virais/metabolismo , Oncogenes , Proteínas E7 de Papillomavirus/metabolismo , Fenótipo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/genética
19.
Development ; 146(16)2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31371379

RESUMO

Highly conserved DM domain-containing transcription factors (Doublesex/MAB-3/DMRT1) are responsible for generating sexually dimorphic features. In the Drosophila central nervous system, a set of Doublesex (Dsx)-expressing neuroblasts undergo apoptosis in females whereas their male counterparts proliferate and give rise to serotonergic neurons crucial for adult mating behaviour. Our study demonstrates that the female-specific isoform of Dsx collaborates with Hox gene Abdominal-B (Abd-B) to bring about this apoptosis. Biochemical results suggest that proteins AbdB and Dsx interact through their highly conserved homeodomain and DM domain, respectively. This interaction is translated into a cooperative binding of the two proteins on the apoptotic enhancer in the case of females but not in the case of males, resulting in female-specific activation of apoptotic genes. The capacity of AbdB to use the sex-specific isoform of Dsx as a cofactor underlines the possibility that these two classes of protein are capable of cooperating in selection and regulation of target genes in a tissue- and sex-specific manner. We propose that this interaction could be a common theme in generating sexual dimorphism in different tissues across different species.


Assuntos
Apoptose , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/citologia , Drosophila/genética , Genes Homeobox , Proteínas de Homeodomínio/fisiologia , Células-Tronco Neurais/citologia , Animais , Apoptose/genética , Proteínas de Ligação a DNA/genética , Drosophila/embriologia , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Masculino , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Caracteres Sexuais
20.
Curr Diab Rep ; 19(9): 76, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375924

RESUMO

PURPOSE OF THE REVIEW: Here, we review recent findings in the field of generating insulin-producing cells by pancreatic transcription factor (pTF)-induced liver transdifferentiation (TD). TD is the direct conversion of functional cell types from one lineage to another without passing through an intermediate stage of pluripotency. We address potential reasons for the restricted efficiency of TD and suggest modalities to overcome these challenges, to bring TD closer to its clinical implementation in autologous cell replacement therapy for insulin-dependent diabetes. RECENT FINDINGS: Liver to pancreas TD is restricted to cells that are a priori predisposed to undergo the developmental process. In vivo, the predisposition of liver cells is affected by liver zonation and hepatic regeneration. The TD propensity of liver cells is related to permissive epigenome which could be extended to TD-resistant cells by specific soluble factors. An obligatory role for active Wnt signaling in continuously maintaining a "permissive" epigenome is suggested. Moreover, the restoration of the pancreatic niche and vasculature promotes the maturation of TD cells along the ß cell function. Future studies on liver to pancreas TD should include the maturation of TD cells by 3D culture, the restoration of vasculature and the pancreatic niche, and the extension of TD propensity to TD-resistant cells by epigenetic modifications. Liver to pancreas TD is expected to result in the generation of custom-made "self" surrogate ß cells for curing diabetes.


Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Fígado/citologia , Pâncreas/citologia , Transdiferenciação Celular/fisiologia , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/terapia , Proteínas de Homeodomínio/fisiologia , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/fisiologia , Fígado/fisiologia , Pâncreas/fisiologia , Transativadores/fisiologia
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