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1.
Methods Mol Biol ; 2416: 201-211, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34870838

RESUMO

Regulatory elements, such as promoters and enhancers, typically show reduced nucleosome occupancy, which is a feature that is commonly referred to as "open chromatin". The distribution of open chromatin sites can provide important clues about the transcription factors and regulatory networks that drive gene expression in a given cell. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is a rapid and robust method for mapping open chromatin sites. ATAC-seq data can also discern the binding sites of nucleosomes and transcription factors. In this chapter, we describe how to produce and assess the quality of ATAC-seq libraries that are generated from naïve human pluripotent stem cells.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Células-Tronco Pluripotentes , Cromatina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nucleossomos/genética , Análise de Sequência de DNA , Fatores de Transcrição/genética
2.
Cell Rep ; 36(2): 109337, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34260915

RESUMO

RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.


Assuntos
Diferenciação Celular , Ribonucleoproteínas Nucleares Heterogêneas/química , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Neurônios/metabolismo , Neurônios/patologia , Deleção de Sequência , Proteína Supressora de Tumor p53/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Processamento Alternativo/genética , Motivos de Aminoácidos , Arginina/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Metilação , Células-Tronco Neurais/metabolismo , Neurogênese , Ligação Proteica , Estabilidade Proteica , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas/genética , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Processamento de Serina-Arginina/metabolismo
3.
Stem Cell Reports ; 15(1): 198-213, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32619492

RESUMO

Human embryonic stem cells (hESCs) readily differentiate to somatic or germ lineages but have impaired ability to form extra-embryonic lineages such as placenta or yolk sac. Here, we demonstrate that naive hESCs can be converted into cells that exhibit the cellular and molecular phenotypes of human trophoblast stem cells (hTSCs) derived from human placenta or blastocyst. The resulting "transdifferentiated" hTSCs show reactivation of core placental genes, acquisition of a placenta-like methylome, and the ability to differentiate to extravillous trophoblasts and syncytiotrophoblasts. Modest differences are observed between transdifferentiated and placental hTSCs, most notably in the expression of certain imprinted loci. These results suggest that naive hESCs can differentiate to extra-embryonic lineage and demonstrate a new way of modeling human trophoblast specification and placental methylome establishment.


Assuntos
Metilação de DNA/genética , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Transcriptoma/genética , Trofoblastos/citologia , Transdiferenciação Celular/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Feminino , Impressão Genômica , Humanos , Integrina alfa2/metabolismo , Placenta/citologia , Gravidez , Primeiro Trimestre da Gravidez/fisiologia , Reprodutibilidade dos Testes , Trofoblastos/metabolismo
4.
Elife ; 32014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25317948

RESUMO

Nematodes and insects are the two most speciose animal phyla and nematode-insect associations encompass widespread biological interactions. To dissect the chemical signals and the genes mediating this association, we investigated the effect of an oriental beetle sex pheromone on the development and behavior of the nematode Pristionchus pacificus. We found that while the beetle pheromone is attractive to P. pacificus adults, the pheromone arrests embryo development, paralyzes J2 larva, and inhibits exit of dauer larvae. To uncover the mechanism that regulates insect pheromone sensitivity, a newly identified mutant, Ppa-obi-1, is used to reveal the molecular links between altered attraction towards the beetle pheromone, as well as hypersensitivity to its paralyzing effects. Ppa-obi-1 encodes lipid-binding domains and reaches its highest expression in various cell types, including the amphid neuron sheath and excretory cells. Our data suggest that the beetle host pheromone may be a species-specific volatile synomone that co-evolved with necromeny.


Assuntos
Comportamento Animal/efeitos dos fármacos , Besouros/parasitologia , Interações Hospedeiro-Parasita/efeitos dos fármacos , Nematoides/crescimento & desenvolvimento , Feromônios/farmacologia , Animais , Clonagem Molecular , Embrião não Mamífero/efeitos dos fármacos , Genes de Helmintos , Cetonas/farmacologia , Larva/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Nematoides/efeitos dos fármacos , Nematoides/embriologia , Nematoides/genética , Neuroglia/metabolismo
5.
J Vis Exp ; (56): e3270, 2011 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-22025167

RESUMO

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis and Pristionchus pacificus. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F(1) progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.


Assuntos
Técnicas de Silenciamento de Genes/métodos , Nematoides/genética , Interferência de RNA , Transgenes , Animais , Microinjeções/métodos
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