RESUMO
We present a method, named Mx-TOP, for profiling of three epigenetic regulatory layers-chromatin accessibility, general DNA modification, and DNA hydroxymethylation-from a single library. The approach is based on chemo-enzymatic covalent tagging of unmodified CG sites and hydroxymethylated cytosine (5hmC) along with GC sites in chromatin, which are then mapped using tag-selective base-resolution TOP-seq sequencing. Our in-depth validation of the approach revealed its sensitivity and informativity in evaluating chromatin accessibility and DNA modification interactions that drive transcriptional regulation. We employed the technology in a study of chromatin and DNA demethylation dynamics during in vitro neuronal differentiation. The study highlighted the involvement of gene body 5hmC in modulating an extensive decoupling between promoter accessibility and transcription. The importance of 5hmC in chromatin remodeling was further demonstrated by the observed resistance of the developmentally acquired open loci to the global 5hmC erasure in neuronal progenitors.
Assuntos
Cromatina , Metilação de DNA , Cromatina/genética , Citosina , Regulação da Expressão Gênica , DNA/metabolismo , 5-MetilcitosinaRESUMO
All living organisms must respond to, and defend against, environmental stresses. Depending on the extent and severity of stress, cells try to alter their metabolism and adapt to a new state. Changes in alternative splicing of pre-mRNA are a crucial regulation mechanism through which cells are able to respond to a decrease in oxygen tension in the cellular environment. Currently, only limited data are available in the literature on how short-term hypoxia influences mRNA isoform formation. In this work, we discovered that expressions of the same genes that are activated during cellular stress are also activated in cells under short-term hypoxic conditions. Our results demonstrate that short-term hypoxia influences the splicing of genes associated with cell stress and apoptosis; however, the mRNA isoform formation patterns from the same pre-mRNAs in cells under short-term hypoxic conditions and prolonged hypoxia are different. Obtained data also show that short-term cellular hypoxia increases protein phosphatase but not protein kinase expression. Enhanced levels of protein phosphatase expression in cells are clearly important for changing mRNA isoform formation.
Assuntos
Isoformas de RNA , Precursores de RNA , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Oxigênio/metabolismo , Fosfoproteínas Fosfatases , Isoformas de Proteínas/genética , Precursores de RNA/genéticaRESUMO
The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many disease states. Recent studies have revealed that tumorigenesis and hypoxia are involved in large-scale alterations in alternative pre-mRNA splicing. Fas pre-mRNA is alternatively spliced by excluding exon 6 to produce soluble Fas (sFas) protein that lacks a transmembrane domain and acts by inhibiting Fas mediated apoptosis. In the present study we show that U2AF is involved in hypoxia dependent anti-apoptotic Fas mRNA isoform formation. Our performed studies show that U2AF-RNA interaction is reduced in hypoxic cells, leading to reduction of Fas and increased sFas mRNAs formation. Efficient U2AF-RNA interactions of both subunits are important for Fas exon 6 inclusion into forming mRNA in normoxic and hypoxic cells.
Assuntos
Hipóxia/genética , Fator de Processamento U2AF/fisiologia , Receptor fas/genética , Processamento Alternativo/genética , Células HCT116 , Humanos , Hipóxia/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Processamento U2AF/genética , Receptor fas/metabolismoRESUMO
The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many diseases, including Alzheimer's disease (AD). In the presented study, we have examined the influence of cellular hypoxia on mRNA splice variant formation from Alzheimer's disease-related Tau and APP genes in brain cells. We have shown that the hypoxic microenvironment influenced the formation of Tau mRNA splice variants, but had no effect on APP mRNA splice variant formation. Additionally, our presented results indicate that splicing factor SRSF1 but not SRSF5 alters the formation of Tau cellular mRNA splice variants in hypoxic cells. Obtained results have also shown that hypoxic brain cells possess enhanced CLK1-4 kinase mRNA levels. This study underlines that cellular hypoxia can influence disease development through changing pre-mRNA splicing.
Assuntos
Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Hipóxia Celular/genética , RNA Mensageiro/genética , Proteínas tau/genética , Processamento Alternativo/genética , Encéfalo/metabolismo , Linhagem Celular Tumoral , Humanos , Íntrons/genética , Precursores de RNA/genética , Transcrição Gênica/genéticaRESUMO
The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many disease states. Recent studies have revealed that tumorigenesis and hypoxia involve large-scale alterations in alternative pre-mRNA splicing. Cancer associated Fas protein plays a central role in the physiological regulation of programmed cell death and has been implicated in the pathogenesis of various malignancies and diseases of the immune system. Fas pre-mRNA is alternatively spliced by excluding exon 6 to produce soluble Fas (sFas) protein that lacks a transmembrane domain and acts by inhibiting Fas mediated apoptosis. Another cancer related protein Rac1 plays an important regulatory role specifically in cells' motility, growth and survival. Rac pre-mRNA is alternatively spliced to produce Rac1b protein, which is upregulated in metastatic diseases. In the present study we, for the first time, show that anti-apoptotic Fas mRNA isoform formation is regulated by cellular microenvironment - hypoxia. Hypoxic microenvironment, however, does not influence Rac1 pre-mRNAs alternative splicing. Also our presented results indicate that splicing factors hnRNP A1 and SPF45, previously shown to regulate Fas alternative splicing in normoxic cells, are not involved in hypoxia dependent alternative Fas pre-mRNA splicing regulation in an amount dependent manner. Our observations on hypoxia dependent alternative Fas pre-mRNA splicing regulation indicate a probable involvement of other, yet unidentified splicing factors. Presented data also shows the contribution of pre-mRNA splicing to cell survival under unfavorable conditions.
Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/genética , Neoplasias/genética , Fatores de Processamento de RNA/genética , Receptor fas/genética , Apoptose/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Íntrons/genética , Neoplasias/patologia , Precursores de RNA/genética , Splicing de RNA/genética , Hipóxia Tumoral/genética , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
BACKGROUND: Cell lines derived from human tumors have been extensively used as experimental models of neoplastic disease. Although such cell lines differ from both normal and cancerous tissue. OBJECTIVE: The data obtained used DNA and RNA microarray systems does not give full information about protein expression levels in cells and tissues. We present experimental evidence that splicing factor SRSF1, SRSF2, U2AF35, U2AF65 and KHSRP expression levels in gastrointestinal tract (colon, gastric and pancreatic) tumors differ compare to healthy tissues and in cell lines, derived from corresponding organs. METHODS: Protein expression was analyzed using Western blots. RT-PCR method was used for Fas and Rac splicing analysis. RESULTS: Obtained results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumors in vivo. Expression levels of individual splicing factors in tumors might serve as tumor markers. Not all experimental results obtained from cell lines reflect changes that occur in tumors. Also Fas and Rac, cancer associated genes, tumor associated sFas and Rac1b mRNA isoform profiles in cell lines do not correspond to profiles that are observed in tumors. CONCLUSIONS: Not all experimental results obtained in cell lines reflect changes that occur in real tumors.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Neoplasias Gastrointestinais/genética , Isoformas de RNA/genética , Fatores de Transcrição/biossíntese , Transcrição Gênica , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Neoplasias Gastrointestinais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Isoformas de RNA/biossíntese , Fatores de Processamento de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Monoamine oxidase B (MAO-B) plays an important role in the metabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues. Increased levels of MAO-B mRNA and enzymatic activity have been reported in platelets from patients with Parkinson's and Alzheimer's diseases, however the triggers of enhanced mRNA levels are unknown. Our results demonstrate for the first time that G/A dimorphism in intron 13 sequence creates splicing enhancer thus stimulating intron 13 removal efficiency. The increased MAO-B protein levels might serve as a surrogate marker for - Parkinson disease.