Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 7 de 7
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Genesis ; 58(3-4): e23351, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31838787

RESUMO

Analysis of the human and murine transcriptomes has identified long noncoding RNAs (lncRNAs) as major functional components in both species. Transcriptional profiling of the murine limb led to our discovery of lncRNA-HIT, which our previous in vitro analyses suggested a potential role for this lncRNA in the development of limb, craniofacial, and genitourinary tissues (Carlson et al., 2015). To test this hypothesis, we developed a conditional lncRNA-HIT loss of function allele which uses Cre recombinase to activate an shRNA specific for lncRNA-HIT. Activation of the lncRNA-HIT shRNA allele resulted in a robust knock-down of lncRNA-HIT as well as co-activation of a mCherry reporter, confirming the efficacy of the shRNA allele to reduce endogenous lncRNA levels in a tissue- and cell-type specific manner. Developmental analyses of embryos expressing the activated shRNA and mCherry co-reporter revealed multiple malformations corresponding to the sites of shRNA activation, affecting craniofacial, limb, and genitourinary tissue development. These results confirm the efficacy of lncRNA-HIT shRNA allele to knock-down endogenous transcripts in tissue- and cell type specific manner and indicate a requirement for lncRNA-HIT in the development of these tissues.


Assuntos
Alelos , Perfilação da Expressão Gênica , Inativação Gênica , RNA Longo não Codificante/genética , Transcriptoma , Animais , Biologia Computacional/métodos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Humanos , Camundongos , Especificidade de Órgãos , Fenótipo , RNA Interferente Pequeno/genética , Ativação Transcricional
2.
Cell Rep ; 17(11): 2913-2926, 2016 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-27974206

RESUMO

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.


Assuntos
Padronização Corporal/genética , Extremidades/crescimento & desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Knockout , Ligação Proteica , Fatores de Transcrição/metabolismo
3.
PLoS Genet ; 11(12): e1005680, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26633036

RESUMO

Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.


Assuntos
Diferenciação Celular/genética , Condrogênese/genética , RNA Longo não Codificante/genética , Proteína p120 Ativadora de GTPase/genética , Animais , Epigênese Genética/genética , Extremidades/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Botões de Extremidades/crescimento & desenvolvimento , Mesoderma/crescimento & desenvolvimento , Camundongos , RNA Longo não Codificante/biossíntese , Proteína p120 Ativadora de GTPase/biossíntese
4.
Biomol NMR Assign ; 9(2): 267-70, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25491407

RESUMO

The homeobox gene (Hoxd13) codes for a transcription factor protein that binds to AT-rich DNA sequences and controls expression of proteins that control embryonic morphogenesis. We report NMR chemical shift assignments of mouse Hoxd13 DNA binding domain bound to an 11-residue DNA duplex (BMRB No. 25133).


Assuntos
DNA/metabolismo , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectroscopia de Prótons por Ressonância Magnética
5.
Dev Dyn ; 242(6): 687-98, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23553814

RESUMO

BACKGROUND: Retinoic acid (RA), plays an essential role in the growth and patterning of vertebrate limb. While the developmental processes regulated by RA are well understood, little is known about the transcriptional mechanisms required to precisely control limb RA synthesis. Here, Aldh1a2 functions as the primary enzyme necessary for RA production which regulates forelimb outgrowth and hindlimb digit separation. Because mice lacking HOXA13 exhibit similar defects in digit separation as Aldh1a2 mutants, we hypothesized that HOXA13 regulates Aldh1a2 to facilitate RA-mediated interdigital programmed cell death (IPCD) and digit separation. RESULTS: In this report, we identify Aldh1a2 as a direct target of HOXA13. In absence of HOXA13 function, Aldh1a2 expression, RA signaling, and IPCD are reduced. In the limb, HOXA13 binds a conserved cis-regulatory element in the Aldh1a2 locus that can be regulated by HOXA13 to promote gene expression. Finally, decreased RA signaling and IPCD can be partially rescued in the Hoxa13 mutant hindlimb by maternal RA supplementation. CONCLUSIONS: Defects in IPCD and digit separation in Hoxa13 mutant mice may be caused in part by reduced levels of RA signaling stemming from a loss in the direct regulation of Aldh1a2. These findings provide new insights into the transcriptional regulation of RA signaling necessary for limb morphogenesis.


Assuntos
Aldeído Desidrogenase/metabolismo , Apoptose , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/fisiologia , Família Aldeído Desidrogenase 1 , Animais , Sequência de Bases , Padronização Corporal , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Retinal Desidrogenase , Ácido Retinoico 4 Hidroxilase , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Transgenes , Tretinoína/metabolismo
6.
J Nutr Biochem ; 18(7): 443-8, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16997540

RESUMO

Dietary copper (Cu) deficiency leads to cardiac morphological and functional defects suggestive of heart failure. However, simultaneous cytoprotective events also appear to occur. The molecular mechanisms responsible for this complex alteration of cardiac function by Cu deficiency have not been elucidated. Because prior work has implicated altered nitric oxide (NO) metabolism in this altered function, we have examined this pathway in further detail. Male Sprague-Dawley rats were fed diets that were either Cu adequate (6 mg Cu/kg diet) or Cu deficient (<0.5 mg Cu/kg diet) for 5 weeks. Endothelial NO synthase (NOS) and inducible NOS (iNOS) protein expressions, as measured by Western blot analysis, were 58% and 40% higher, respectively, in Cu-deficient than in Cu-adequate rat hearts. Cardiac NOS activity, as measured by conversion of (3)H-arginine to (3)H-citrulline, was 130% higher in Cu-deficient than in Cu-adequate rats. NFkappaB is a known transcription factor for iNOS. Activation of NFkappaB, determined by an ELISA for the p65 subunit, was found to be 33% higher in Cu-deficient than in Cu-adequate rats. Coupled with prior evidence of elevated cardiac nitrate/nitrite production in Cu-deficient rats, these data suggest multiple pathways for enhanced NO production that may contribute to altered cardiac function under dietary Cu deficiency.


Assuntos
Cobre/deficiência , Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Arginina/metabolismo , Citrulina/metabolismo , Dieta , Masculino , Modelos Animais , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Sprague-Dawley
7.
Proc Natl Acad Sci U S A ; 100(16): 9500-5, 2003 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-12874382

RESUMO

The mechanisms by which the hereditary hemochromatosis protein, HFE, decreases transferrin-mediated iron uptake were examined. Coimmunoprecipitation studies using solubilized cell extracts demonstrated that transferrin (Tf) competed with HFE for binding to the transferrin receptor (TfR) similar to previous in vitro studies using soluble truncated forms of HFE and the TfR. At concentrations of Tf approaching those found in the blood, no differences in Tf binding to cells were detected, which is consistent with the lower binding constant of HFE for TfR versus Tf. However, cells expressing HFE still showed a decrease in Tf-mediated iron uptake at concentrations of Tf sufficient to dissociate HFE from the TfR. These results indicate that the association of HFE with TfR is not essential for its ability to lower intracellular iron stores. To test the effect of HFE on lowering intracellular iron levels independently of its association with TfR, a mutated HFE (fW81AHFE) that shows greatly reduced affinity for the TfR was transfected into tetracycline-controlled transactivator HeLa cells. HeLa cells expressing fW81AHFE behaved in a similar manner to cells expressing wild-type HFE with respect to decreased intracellular iron levels measured by iron regulatory protein gel-shift assays and ferritin levels. The results indicate that HFE can lower intracellular iron levels independently of its interaction with the TfR.


Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/fisiologia , Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Mutação , Ligação Competitiva , Western Blotting , Linhagem Celular , Regulação para Baixo , Células HeLa , Proteína da Hemocromatose , Humanos , Microscopia de Fluorescência , Testes de Precipitina , Ligação Proteica , Receptores da Transferrina/metabolismo , Fatores de Tempo , Ativação Transcricional , Transferrina/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...