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

Angiotensin II and the ERK pathway mediate the induction of myocardin by hypoxia in cultured rat neonatal cardiomyocytes

Hypoxic injury to cardiomyocytes is a stress that causes cardiac pathology through cardiac-restricted gene expression. SRF (serum-response factor) and myocardin are important for cardiomyocyte growth and differentiation in response to myocardial injuries. Previous studies have indicated that AngII (angiotensin II) stimulates both myocardin expression and cardiomyocyte hypertrophy. In the present study, we evaluated the expression of myocardin and AngII after hypoxia in regulating gene transcription in neonatal cardiomyocytes. Cultured rat neonatal cardiomyocytes were subjected to hypoxia, and the expression of myocardin and AngII were evaluated. Different signal transduction pathway inhibitors were used to identify the pathway(s) responsible for myocardin expression. An EMSA (electrophoretic mobility-shift assay) was used to identify myocardin/SRF binding, and a luciferase assay was used to identify transcriptional activity of myocardin/SRF in neonatal cardiomyocytes. Both myocardin and AngII expression increased after hypoxia, with AngII appearing at an earlier time point than myocardin. Myocardin expression was stimulated by AngII and ERK (extracellular-signal-regulated kinase) phosphorylation, but was suppressed by an ARB (AngII type 1 receptor blocker), an ERK pathway inhibitor and myocardin siRNA (small interfering RNA). AngII increased both myocardin expression and transcription in neonatal cardiomyocytes. Binding of myocardin/SRF was identified using an EMSA, and a luciferase assay indicated the transcription of myocardin/SRF in neonatal cardiomyocytes. Increased BNP (B-type natriuretic peptide), MHC (myosin heavy chain) and [3H]proline incorporation into cardiomyocytes was identified after hypoxia with the presence of myocardin in hypertrophic cardiomyocytes. In conclusion, hypoxia in cardiomyocytes increased myocardin expression, which is mediated by the induction of AngII and the ERK pathway, to cause cardiomyocyte hypertrophy. Myocardial hypertrophy was identified as an increase in transcriptional activities, elevated hypertrophic and cardiomyocyte phenotype markers, and morphological hypertrophic changes in cardiomyocytes

TSA downregulates Wilms tumor gene 1 (Wt1) expression at multiple levels

The Wilms tumor gene WT1 encodes a zinc-finger transcription factor that is inactivated in a subset of pediatric kidney cancers. During embryogenesis, WT1 is expressed in a time- and tissue-specific manner in various organs including gonads and kidney but also in the hematopoietic system. Although widely regarded as a tumor suppressor gene, wild-type WT1 is overexpressed in a variety of hematologic malignancies, most notably in acute lymphoblastic leukemia as well as myelodysplastic syndromes. Reduction of WT1 expression levels leads to decrease of proliferation and apoptosis of leukemic cells, suggesting that in certain contexts WT1 might act as an oncogene. We show here that histone deacetylase inhibitors like Trichostatin A (TSA) can promptly and dramatically downregulate Wt1 expression levels in different cell lines. This effect was mostly due to the cessation of transcription and was mediated by sequences located in intron 3 of Wt1. In addition, TSA also caused enhanced degradation of the Wt1 protein by the proteasome. This was at least in part due to induction of the ubiquitin-conjugating enzyme UBCH8. Thus, downregulation of Wt1 expression might contribute to the beneficial effects of histone deacetylase inhibitors that are currently used in clinical trials as cancer therapeutics

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%–94%) and acidic pIs (5.03–5.33). 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 (48–96 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]]紙

Identification of factors mediating the developmental regulation of the early acting –3.9 kb chicken lysozyme enhancer element

The chicken lysozyme gene –3.9 kb enhancer forms a DNase I hypersensitive site (DHS) early in macrophage differentiation, but not in more primitive multipotent myeloid precursor cells. A nucleosome becomes precisely positioned across the enhancer in parallel with DHS formation. In transfection assays, the 5′-part of the –3.9 kb element has ubiquitous enhancer activity. The 3′-part has no stimulatory activity, but is necessary for enhancer repression in lysozyme non-expressing cells. Recent studies have shown that the chromatin fine structure of this region is affected by inhibition of histone deacetylase activity after Trichostatin A (TSA) treatment, but only in lysozyme non-expressing cells. These results indicated a developmental modification of chromatin structure from a dynamic, but inactive, to a stabilised, possibly hyperacetylated, active state. Here we have identified positively and negatively acting transcription factors binding to the –3.9 kb enhancer and determined their contribution to enhancer activity. Furthermore, we examined the influence of TSA treatment on enhancer activity in macrophage cells and lysozyme non-expressing cells, including multipotent macrophage precursors. Interestingly, TSA treatment was able to restore enhancer activity fully in macrophage precursor cells, but not in non-macrophage lineage cells. These results suggest (i) that the transcription factor complement of multipotent progenitor cells is similar to that of lysozyme-expressing cells and (ii) that developmental regulation of the –3.9 kb enhancer is mediated by the interplay of repressing and activating factors that respond to or initiate changes in the chromatin acetylation state

A Functional Chromatin Domain Does Not Resist X Chromosome Inactivation: Silencing of cLys Correlates with Methylation of a Dual Promoter-Replication Origin

To investigate the molecular mechanism(s) involved in the propagation and maintenance of X chromosome inactivation (XCI), the 21.4-kb chicken lysozyme (cLys) chromatin domain was inserted into the Hprt locus on the mouse X chromosome. The inserted fragment includes flanking matrix attachment regions (MARs), an origin of bidirectional replication (OBR), and all the cis-regulatory elements required for correct tissue-specific expression of cLys. It also contains a recently identified and widely expressed second gene, cGas41. The cLys domain is known to function as an autonomous unit resistant to chromosomal position effects, as evidenced by numerous transgenic mouse lines showing copy-number-dependent and development-specific expression of cLys in the myeloid lineage. We asked the questions whether this functional chromatin domain was resistant to XCI and whether the X inactivation signal could spread across an extended region of avian DNA. A generally useful method was devised to generate pure populations of macrophages with the transgene either on the active (Xa) or the inactive (Xi) chromosome. We found that (i) cLys and cGas41 are expressed normally from the Xa; (ii) the cLys chromatin domain, even when bracketed by MARs, is not resistant to XCI; (iii) transcription factors are excluded from lysozyme enhancers on the Xi; and (iv) inactivation correlates with methylation of a CpG island that is both an OBR and a promoter of the cGas41 gene