31 research outputs found
PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis
The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes
Molecular structure and developmental expression of zebrafish atp2a genes
[[abstract]]We isolated two atp2a genes, atp2a1 and atp2a2a, from embryonic zebrafish. Amino acid sequences deduced from zebrafish atp2a genes are aligned with orthologue proteins from other species, the results showed that they share high percentage of identities (82%ā94%) and acidic pIs (5.03ā5.33). Whole mount in situ hybridization experiments showed that atp2a1 and atp2a2a are maternal inherited genes which can be detected at 1-cell stage embryos and express in the entire animal pole from 6 hours post-fertilization (hpf) to 12 hpf. At the later stages (48ā96 hpf), expression of atp2a1 was restricted in head and trunk muscles as well as in some neurons. In contrast to the strongly expression of atp2a1 in head muscle, expression of atp2a2a was detected in head muscle in a fainter manner. In addition, transcripts of atp2a2a were observed in the developing heart during early cardiogenesis. The present studies not only help us to comparatively analyze atp2a genes across species, but also provide useful information about expressions during early embryogenesis that will help in further investigations of functional studies of Atp2a in the future.[[incitationindex]]SCI[[booktype]]ē“
MicroRNA profiling identifies miR-7-5p and miR-26b-5p as differentially expressed in hypertensive patients with left ventricular hypertrophy
Chromatin fine structure profiles for a developmentally regulated gene: reorganization of the lysozyme locus before trans-activator binding and gene expression
Terminal transferase-dependent PCR (TDPCR) for in vivo UV photofootprinting of eukaryotic cells
Circulating microRNAs and hypertensionāfrom new insights into blood pressure regulation to biomarkers of cardiovascular risk
Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible
The murine c-fms (Csf1r) gene encodes the macrophage colony-stimulating factor receptor, which is essential for macrophage development. It is expressed at a low level in haematopoietic stem cells and is switched off in all non-macrophage cell types. To examine the role of chromatin structure in this process we studied epigenetic silencing of c-fms during B-lymphopoiesis. c-fms chromatin in stem cells and multipotent progenitors is in the active conformation and bound by transcription factors. A similar result was obtained with specified common myeloid and lymphoid progenitor cells. In developing B cells, c-fms chromatin is silenced in distinct steps, whereby first the binding of transcription factors and RNA expression is lost, followed by a loss of nuclease accessibility. Interestingly, regions of de novo DNA methylation in B cells overlap with an intronic antisense transcription unit that is differently regulated during lymphopoiesis. However, even at mature B cell stages, c-fms chromatin is still in a poised conformation and c-fms expression can be re-activated by conditional deletion of the transcription factor Pax5
Relationship of cardiovascular disease risk factors and noncoding RNAs with hypertension: a case-control study
Embryonic but Not Postnatal Reexpression of Hepatocyte Nuclear Factor 1Ī± (HNF1Ī±) Can Reactivate the Silent Phenylalanine Hydroxylase Gene in HNF1Ī±-Deficient Hepatocytes
The failure to transcribe the phenylalanine hydroxylase (PAH) gene in the liver of hepatocyte nuclear factor 1Ī± (HNF1Ī±)-deficient mice correlated with DNA hypermethylation and the presence of an inactive chromatin structure (M. Pontoglio, D. M. Faust, A. Doyen, M. Yaniv, and M. C. Weiss, Mol. Cell. Biol. 17:4948ā4956, 1997). To evaluate the precise role played by HNF1Ī±, DNA methylation, or histone acetylation in PAH gene silencing, we examined conditions that could restore PAH gene expression in HNF1Ī±-deficient hepatocytes. We show that reactivation of PAH transcription can be achieved by reexpression of HNF1Ī± in embryonic (i.e., embryonic day 12.5 [e12.5] to e13.5) hepatocytes but not in fetal (e17.5), newborn, and adult HNF1Ī±-deficient hepatocytes. This defines a temporal competence window during which HNF1Ī± can act to (re)program PAH gene transcription. We also show that PAH gene silencing can be partially relieved in HNF1Ī±-deficient hepatocytes by treatment with the demethylating agent 5-azacytidine, even in the absence of HNF1Ī±. Treatment using 5-azacytidine combined with trichostatin, a histone deacetylase inhibitor, resulted in a synergistic reactivation of the silenced PAH gene in adult hepatocytes, but this activity was not further increased by HNF1Ī± reexpression. These results suggest that the HNF1Ī± homeoprotein is involved in stage-specific developmental control of the methylation state and chromatin remodeling of the PAH gene