Localization Of RNA Polymerase Research Articles

Cycles of gene expression and genome response during mammalian tissue regeneration

BackgroundCompensatory liver hyperplasia—or regeneration—induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries.ResultsDuring the first 10–20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10–20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first internal exon, was preceded 5′ by H3K36me2; 3′ of the first internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription.ConclusionsPH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH.

Abstract 255: Laminar Shear Stress Enhances the Activity and Localization of RNA Polymerase II on the eNOS Gene

We have previously found that laminar shear enhances eNOS mRNA stability and translation by altering endothelial NO synthase (eNOS) mRNA 3′ polyadenylation. Transcription is tightly coupled to pre-mRNA processing, and is coordinated by RNA polymerase (RNAP) II. We assessed whether laminar shear stress alters the activity and localization of RNAP II on the eNOS gene. We found that endothelial cells exposed to laminar shear stress had a 2-fold (n=3, p < 0.05) increase in total RNAP II protein levels compared to control; this effect was dose-dependent (0–15 dynes/cm2). Since three different RNAP II phosphoisoforms are associated with different stages of mRNA synthesis, we examined whether shear stress affected protein expression of these particular phosphoisoforms. We found that cells exposed to laminar shear had a 3-fold increase (p<0.05) in expression of RNAP II phosphorylated at serine 2, which is associated with transcription elongation and 3′ polyadenylation. In contrast, shear stress did not alter expression of the other phosphoisoforms. We performed chromatin immunoprecipitation (ChIP) analysis to examine shear-induced changes in RNAP II localization on the eNOS gene. Using antibody against total RNAP II, eNOS sequences along the entire gene were amplified. Using the phosphoserine 2 specific RNAP II antibody, we found active RNAP II predominantly bound to the 3′UTR (exon 26) and downstream sequence of eNOS in sheared cells. These findings were confirmed by quantitative ChIP (3 fold increase, n = 8, p = 0.0378). This suggests that shear-induced changes in RNAP II phosphorylation enhance its ability to polyadenylate eNOS mRNA. Serine 2 phosphorylation is dependent on cyclin-dependent kinase 9 (CDK 9), and shear-induced recruitment of CDK 9 to the eNOS gene was examined. We found that endothelial cells exposed to laminar shear had enhanced localization of CDK 9 to the eNOS promoter and exons 8 and 22, and diminished localizationto exon 26 and in the downstream sequence. This indicates that shear recruits CDK 9 while RNAP II is bound to the promoter and early exons to activate RNAP by phosphorylating serine 2. In conclusion, laminar shear stress enhances eNOS mRNA processing and increases gene expression through recruitment of CDK 9 and activation of RNAP II.

Immunogold localization of RNA polymerase II and pre-mRNA splicing factors in Tenebrio molitor oocyte nuclei with special emphasis on karyosphere development

Ultrastructural and immunomorphological characteristics of the developing karyosphere and extrachromosomal nuclear bodies (NBs) in Tenebrio molitor oocytes are presented. Three consecutive stages of karyosphere development were identified: reticular, compact and ring-shaped. At the beginning of the karyosphere development (reticular and compact stages), condensed chromosomes are associated with a fibrogranular material (FGM). The successive karyosphere development is accompanied by the reorganization of FGM into fibrogranular NBs. Special attention was given to the nuclear distribution of hyperphosphorylated and non-phosphorylated forms of RNA polymerase II (pol II) and pre-mRNA splicing factors (snRNPs and SC35 protein) during karyosphere development and NB formation. The immunoelectron microscopy revealed that two forms of pol II and splicing factors being assembled in FGM are deposited in appropriate NBs. Some NBs were also shown to contain coilin, a marker protein for Cajal (coiled) bodies. We suggest that different types of NBs appearing in T. molitor oocyte nuclei along with the cessation of transcriptional activity during the karyosphere development represent storage domains for inactive RNA transcription/processing machinery to later usage in early embryogenesis.

Lipopolysaccharide-mediated induction of RNA polymerase I activity and amount in murine B lymphocytes.

The effect of lipopolysaccharide on RNA polymerase I activity in primary cultures of murine B lymphocytes has been examined. In cells treated with mitogen for 48 h, the activity of RNA polymerase I was approximately 15 times greater than in control cells. In situ localization of RNA polymerase I using indirect immunofluorescence indicated that there was at least a 10-fold increase in the amount of this enzyme associated with nucleoli of 48 h mitogen-treated cells relative to control cells. Immunoblotting experiments demonstrated a similar increase in the concentration of the 190-kDa subunit bound to DNA; the concentrations of the other polymerase I-associated polypeptides did not correlate with rRNA synthesis. Assuming 1 mol of the 190-kDa polypeptide/mol of polymerase I, it was estimated that 2,300 and 30,000 molecules of enzyme were associated with rDNA in the unstimulated and stimulated B cell, respectively. Thus, an increased cellular concentration of the 190-kDa subunit of RNA polymerase I and its association with ribosomal DNA may be a crucial step in rRNA synthesis.

Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry.

Rabbit antibodies to RNA polymerase I from a rat hepatoma have been used to localize the enzyme in a variety of cells at the light and electron microscopic level. In interphase cells the immunofluorescence pattern indicated that polymerase I is contained exclusively within the nucleolus. That this fluorescence, which appeared punctated rather than uniform, represented transcriptional complexes of RNA polymerase I and rRNA genes was suggested by the observation that it was enhanced in regenerating liver and in a hepatoma and was markedly diminished in cells treated with actinomycin D. Electron microscopic immunolocalization using gold-coupled second antibodies showed that transcribed rRNA genes are located in, and probably confined to, the fibrillar centers of the nucleolus. In contrast, the surrounding dense fibrillar component, previously thought to be the site of nascent pre-rRNA, did not contain detectable amounts of polymerase I. During mitosis, polymerase I molecules were detected by immunofluorescence microscopy at the chromosomal nucleolus organizer region, indicating that a considerable quantity of the enzyme remains bound to the rRNA genes. From this we conclude that rRNA genes loaded with polymerase I molecules are transmitted from one cell generation to the next one and that factors other than the polymerase itself are involved in the modulation of transcription of DNA containing rRNA genes during the cell cycle.

Immunoelectron microscopic localization of RNA polymerase B on isolated polytene chromosomes of Chironomus tentans.

RNA polymerase B (or II) was localized by immunoelectron microscopy in ultrathin sections of polytene chromosomes isolated from larval salivary glands of Chironomus tentans. The enzyme was found at decondensed sites (puffs and interbands), whereas no detectable RNA polymerase B was present in condensed loci (bands). Within each of the large puffs the highest enzyme concentration was observed wherever the chromatin was in the most decondensed state. Otherwise the enzyme appeared homogeneously distributed within puffs and interbands. This immunoelectron microscopic study, along with the recently published immunofluorescent and autoradiographic analysis of isolated Chironomus chromosomes (Sass, 1982) unequivocally demonstrates that RNA polymerase B is present in most, if not all interbands.

Localization of RNA polymerase B and histones in the nucleus of primary spermatocytes of Drosophila hydei, studied by immunofluorescence microscopy.

By means of indirect immunofluorescence microscopy, we have studied the distribution of RNA polymerase B, of the nucleosomal histones H2b, H3, and H4 and of histone H1, in nuclei of primary spermatocytes of Drosophila hydei. RNA polymerase B and histones, including H1, are found to be present on the loop structures of the Y chromosome. The nucleolus stains only for the histones, but not for RNA polymerase B. Various mutants deficient for some of the loops or altering their morphology, were used to identify the individual chromosomal segments. In growing spermatocytes of the genetic constitution X/0, autosomes and the chromosome X react strongly with antibodies against RNA polymerase B, but not with antibodies against histones. The results suggest that the autosomes, the chromosome X and the Y chromosomal loop structures, with the exception of the nucleolus, are transcribed mostly by RNA polymerase B.

Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster.

RNA polymerase (RNA nucleotidyltransferase) B (or II) and histone H1 of Drosophila melanogster were localized on salivary gland polytene chromosomes using the indirect immunofluorescence technique. RNA polymerase B is present almost exclusively in puffs and interband regions, whereas histone H1 is found primarily in bands. The puff at region 3C, known to be transcriptionally active in larval salivary glands, gives a bright fluorescence with antibodies against RNA polymerase B. This fluorescence disappears after exposure of the larvae to 37 degrees for 45 min. The heat shock treatment results in a general reduction of fluorescence intensity with the appearance of brightly staining heat shock puffs. Heat-induced removal of RNA polymerase molecules from a puff does not immediately alter its morphology. We propose than an interband represents that fraction of the total number of gene copies in a band that are active, the inactive copies being present in a condensed form in the adjacent band. Large puffs would originate through the decondensation and activation of most or all gene copies in a band.