Browsing by Subject "DNA transcription"

Now showing 1 - 5 of 5
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    Dynamics of Co-Transcriptional Pre-mRNA Folding Influences the Induction of Dystrophin Exon Skipping by Antisense Oligonucleotides
    (PLOS, 2008-03-26) Wee, Keng Boon; Dwi Pramono, Zacharias Aloysius; Wang, Jian Li; MacDorman, Karl F.; Lai, Poh San; Yee, Woon Chee; Human-Centered Computing, School of Informatics and Computing
    Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a “window of analysis” that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered “engaged” if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2) engaged nucleotides at 3′ or 5′ ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotides at 3′ or 5′ ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3′ end of a short target site attenuates AON efficiency more than at 5′ end.
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    Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination
    (Elsevier, 2019-03-29) Frossi, Barbara; Antoniali, Giulia; Yu, Kefei; Akhtar, Nahid; Kaplan, Mark H.; Kelley, Mark R.; Tell, Gianluca; Pucillo, Carlo E.; Pediatrics, School of Medicine
    The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.
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    RNA Polymerase II Binding Patterns Reveal Genomic Regions Involved in MicroRNA Gene Regulation
    (Public Library of Science, 2010-11-02) Wang, Guohua; Wang, Yadong; Shen, Changyu; Huangn, Yi-wen; Huang, Kun; Huang, Tim H. M.; Nephew, Kenneth P.; Li, Lang; Liu, Yunlong; Medicine, School of Medicine
    MicroRNAs are small non-coding RNAs involved in post-transcriptional regulation of gene expression. Due to the poor annotation of primary microRNA (pri-microRNA) transcripts, the precise location of promoter regions driving expression of many microRNA genes is enigmatic. This deficiency hinders our understanding of microRNA-mediated regulatory networks. In this study, we develop a computational approach to identify the promoter region and transcription start site (TSS) of pri-microRNAs actively transcribed using genome-wide RNA Polymerase II (RPol II) binding patterns derived from ChIP-seq data. Based upon the assumption that the distribution of RPol II binding patterns around the TSS of microRNA and protein coding genes are similar, we designed a statistical model to mimic RPol II binding patterns around the TSS of highly expressed, well-annotated promoter regions of protein coding genes. We used this model to systematically scan the regions upstream of all intergenic microRNAs for RPol II binding patterns similar to those of TSS from protein coding genes. We validated our findings by examining the conservation, CpG content, and activating histone marks in the identified promoter regions. We applied our model to assess changes in microRNA transcription in steroid hormone-treated breast cancer cells. The results demonstrate many microRNA genes have lost hormone-dependent regulation in tamoxifen-resistant breast cancer cells. MicroRNA promoter identification based upon RPol II binding patterns provides important temporal and spatial measurements regarding the initiation of transcription, and therefore allows comparison of transcription activities between different conditions, such as normal and disease states.
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    A Study of Alterations in DNA Epigenetic Modifications (5mC and 5hmC) and Gene Expression Influenced by Simulated Microgravity in Human Lymphoblastoid Cells.
    (PLOS, 2016) Chowdhury, Basudev; Seetharam, Arun; Wang, Zhiping; Liu, Yunlong; Lossie, Amy C.; Thimmapuram, Jyothi; Irudayaraj, Joseph; Department of Medical & Molecular Genetics, IU School of Medicine
    Cells alter their gene expression in response to exposure to various environmental changes. Epigenetic mechanisms such as DNA methylation are believed to regulate the alterations in gene expression patterns. In vitro and in vivo studies have documented changes in cellular proliferation, cytoskeletal remodeling, signal transduction, bone mineralization and immune deficiency under the influence of microgravity conditions experienced in space. However microgravity induced changes in the epigenome have not been well characterized. In this study we have used Next-generation Sequencing (NGS) to profile ground-based “simulated” microgravity induced changes on DNA methylation (5-methylcytosine or 5mC), hydroxymethylation (5-hydroxymethylcytosine or 5hmC), and simultaneous gene expression in cultured human lymphoblastoid cells. Our results indicate that simulated microgravity induced alterations in the methylome (~60% of the differentially methylated regions or DMRs are hypomethylated and ~92% of the differentially hydroxymethylated regions or DHMRs are hyperhydroxymethylated). Simulated microgravity also induced differential expression in 370 transcripts that were associated with crucial biological processes such as oxidative stress response, carbohydrate metabolism and regulation of transcription. While we were not able to obtain any global trend correlating the changes of methylation/ hydroxylation with gene expression, we have been able to profile the simulated microgravity induced changes of 5mC over some of the differentially expressed genes that includes five genes undergoing differential methylation over their promoters and twenty five genes undergoing differential methylation over their gene-bodies. To the best of our knowledge, this is the first NGS-based study to profile epigenomic patterns induced by short time exposure of simulated microgravity and we believe that our findings can be a valuable resource for future explorations.
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    Toxoplasma gondii Lysine Acetyltransferase GCN5-A Functions in the Cellular Response to Alkaline Stress and Expression of Cyst Genes
    (Public Library of Science, 2010-12-16) Naguleswaran, Arunasalam; Elias, Eliana V.; McClintick, Jeanette; Edenberg, Howard J.; Sullivan, William J., Jr.; Pharmacology and Toxicology, School of Medicine
    Parasitic protozoa such as the apicomplexan Toxoplasma gondii progress through their life cycle in response to stimuli in the environment or host organism. Very little is known about how proliferating tachyzoites reprogram their expressed genome in response to stresses that prompt development into latent bradyzoite cysts. We have previously linked histone acetylation with the expression of stage-specific genes, but the factors involved remain to be determined. We sought to determine if GCN5, which operates as a transcriptional co-activator by virtue of its histone acetyltransferase (HAT) activity, contributed to stress-induced changes in gene expression in Toxoplasma. In contrast to other lower eukaryotes, Toxoplasma has duplicated its GCN5 lysine acetyltransferase (KAT). Disruption of the gene encoding for TgGCN5-A in type I RH strain did not produce a severe phenotype under normal culture conditions, but here we show that the TgGCN5-A null mutant is deficient in responding to alkaline pH, a common stress used to induce bradyzoite differentiation in vitro. We performed a genome-wide analysis of the Toxoplasma transcriptional response to alkaline pH stress, finding that parasites deleted for TgGCN5-A fail to up-regulate 74% of the stress response genes that are induced 2-fold or more in wild-type. Using chromatin immunoprecipitation, we verify an enrichment of TgGCN5-A at the upstream regions of genes activated by alkaline pH exposure. The TgGCN5-A knockout is also incapable of up-regulating key marker genes expressed during development of the latent cyst form, and is impaired in its ability to recover from alkaline stress. Complementation of the TgGCN5-A knockout restores the expression of these stress-induced genes and reverses the stress recovery defect. These results establish TgGCN5-A as a major contributor to the alkaline stress response in RH strain Toxoplasma.