Occupancy of RNA Polymerase II Phosphorylated on Serine 5 (RNAP S5 ^P ) and RNAP S2 ^P on Varicella-Zoster Virus Genes 9, 51, and 66 Is Independent of Transcript Abundance and Polymerase Location within the Gene

Abstract

Regulation of gene transcription in varicella-zoster virus (VZV), a ubiquitous human neurotropic alphaherpesvirus, requires coordinated binding of multiple host and virus proteins onto specific regions of the virus genome. Chromatin immunoprecipitation (ChIP) is widely used to determine the location of specific proteins along a genomic region. Since the size range of sheared virus DNA fragments governs the limit of accurate protein localization, particularly for compact herpesvirus genomes, we used a quantitative PCR (qPCR)-based assay to determine the efficiency of VZV DNA shearing before ChIP, after which the assay was used to determine the relationship between transcript abundance and the occupancy of phosphorylated RNA polymerase II (RNAP) on the gene promoter, body, and terminus of VZV genes 9, 51, and 66. The abundance of VZV gene 9, 51, and 66 transcripts in VZV-infected human fetal lung fibroblasts was determined by reverse transcription-linked quantitative PCR. Our results showed that the C-terminal domain of RNAP is hyperphosphorylated at serine 5 (S5 P ) on VZV genes 9, 51, and 66 independently of transcript abundance and the location within the virus gene at both 1 and 3 days postinfection (dpi). In contrast, phosphorylated serine 2 (S2 P )-modified RNAP was not detected at any virus gene location at 3 dpi and was detected at levels only slightly above background levels at 1 dpi. IMPORTANCERegulation of herpesvirus gene transcription is an elaborate choreography between proteins and DNA that is revealed by chromatin immunoprecipitation (ChIP). We used a quantitative PCR-based assay to determine fragment size after DNA shearing, a critical parameter in ChIP assays, and exposed a basic difference in the mechanism of transcription between mammalian cells and VZV. We found that hyperphosphorylation at serine 5 of the C-terminal domain of RNAP along the lengths of VZV genes (the promoter, body, and transcription termination site) was independent of mRNA abundance. In contrast, little to no enrichment of serine 3 phosphorylation of RNAP was detected at these virus gene regions. This is distinct from the findings for RNAP at highly regulated host genes, where RNAP S5 P occupancy decreased and S2 P levels increased as the polymerase transited through the gene. Overall, these results suggest that RNAP associates with human and virus transcriptional units through different mechanisms. C hromatin immunoprecipitation (ChIP) has been used to determine the association of proteins with DNA in cells of humans (1), mice (2), Drosophila melanogaster (3), Caenorhabditis elegans (4), and Saccharomyces cerevisiae yeast (5) and has shown that gene regulation requires the interaction of multiple nuclear proteins, including RNA polymerase II (RNAP), with various transcription factors across the genome. ChIP has also been used to explore the phosphorylation status of the C-terminal domain (CTD) of RNAP at different positions along a transcription unit and showed that in eukaryotes, phosphorylation of serine 5 (S5 P )...

The Jumonji-domain histone demethylase inhibitor JIB-04 deregulates oncogenic programs and increases DNA damage in Ewing Sarcoma, resulting in impaired cell proliferation and survival, and reduced tumor growth

“…Samples were submitted to the University of Colorado Cancer Center Microarray and Genomics shared resource for analysis of RNA quality, library preparation, and directional mRNA next-generation sequencing at 50 cycles of single-end reads on an Illumina Hi-Seq 4000 instrument. Sequencing data were processed through a custom computational pipeline consisting of the open-source gSNAP, Cufflinks, and R for alignment and discovery of differential gene expression [ 58 , 59 ]. Fragments per kilobase of exon per million mapped reads (FPKM) were used for comparison of transcript levels, and significant differences in gene expression were calculated using ANOVA in R. Functional annotation analysis was performed using the National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery (DAVID) public on-line tool ( http://david.abcc.ncifcrf.gov/ ) using Biological Process Gene Ontology (GO) terms.…”

AP-2α and AP-2β cooperatively orchestrate homeobox gene expression during branchial arch patterning

“…1. Pipeline 1: For an initial 'pipeline' to identify mRNA-based gene expression changes between control and DCM mandibular prominences, derived fastq sequences were analyzed by applying a custom computational workflow (Baird et al, 2014;Bradford et al, 2015;Henderson et al, 2015;Maycotte et al, 2015) consisting of the open-source gSNAP (Wu and Nacu, 2010), Cufflinks (Trapnell et al, 2010), and R for sequence alignment and ascertainment of differential gene expression. In short, reads generated were mapped to the mouse genome (Mm10) by gSNAP, expression (FPKM) derived by Cufflinks and differential expression analyzed with ANOVA in R.…”

Rna Sequencing and Pathway Analysis Identify Tumor Necrosis Factor Alpha Driven Small Proline-Rich Protein Dysregulation in Chronic Rhinosinusitis

“…After RNA-seq, derived sequences were analyzed by applying a custom computational pipeline that consisted of the opensource gSNAP, Cufflinks, and R for sequence alignment and ascertainment of differential gene expression. [12][13][14][15] In short, the reads generated were mapped to the human genome (hg10) by gSNAP, expression (fragments per kilobase of transcript per million mapped reads; FPKM) derived by Cufflinks, and differential expression was analyzed with analysis of variance in R. 16,17 Analyses of variance were performed to compare the expression levels of all four control subjects and all four subjects with CRS on a gene-by-gene basis. Once the gene list was formed, data were analyzed by using Ingenuity Pathway Analysis (Qiagen, Redwood City, CA).…”