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Analysis of Differential Gene Expression in a Myotonic Dystrophy Tissue Cultue Model

This study investigates differential gene expression in a tissue culture model of Myotonic Dystrophy Type 1 (DM1), a multisystemic disorder caused by CTG repeat expansions in the DMPK gene. We utilized HeLa cells transfected with a DMPK minigene containing 960 CTG repeats to induce a disease state. RNA sequencing and subsequent analysis with the Tuxedo Suite (TopHat, Cufflinks, Cuffdiff) enabled us to compare gene expression across various pentamidine dosages. Insights into gene ontology and potential therapeutic targets for alleviating DM1 symptoms are discussed.

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Analysis of Differential Gene Expression in a Myotonic Dystrophy Tissue Cultue Model

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  1. Analysis of Differential Gene Expression in a Myotonic Dystrophy Tissue Cultue Model Matthew Tanner Berglund Lab, Institute of Molecular Biology CIS 407 Dec. 2nd, 2013

  2. Myotonic Dystrophy 1 (DM1) Symptoms Multisystemic – affects skeletal and smooth muscle (myotonia, atrophy), eyes (cataracts), heart, and endocrine system. Genetic Pathology DM1 caused by CTG trinucleotide repeat expansion in 3’ UTR of DMPK gene. Unaffected: less than 34 repeats DM1: 50 to >1000 repeats Severity of disease increases and age of onset decreases with increasing repeat length.

  3. Potential for small molecules to alleviate molecular symptoms of DM1 Pentamidine Anti-fungal, anti-protozoan drug. Binds minor groove of DNA. Wilson et al., 2008. Coonrod, et al. (2013)

  4. Tissue Culture Model and Experiment 24 hours after transfection: harvest whole-cell RNA. DMPK960 Transfect into HeLa cells. Drug 6 hours afterwards. DMPKminigene containing 960 CTG repeats. Induces DM1 disease state. Illuminahighthroughput sequencing library preparation (several steps) IlluminaHi-Seq 2000 massively parallel sequencer

  5. RNA-Seq Library Preparation Pease & Sooknanan, 2012.

  6. Illumina Raw Output Four lines for each sequence: Coordinates of read Sequence + ASCII quality score for each base call (Phred-33)

  7. Alignment and Analysis with Tuxedo Suite Tophat 2.0 – align FASTQ reads that were cleaned up with Stacks’ process_shortreads to human genome. Pass $SAMPLE variable in at command line: qsub tophat.sh –N align0A –V SAMPLE=“0A”

  8. Alignment and Analysis with Tuxedo Suite Tophat 2.0 – align FASTQ reads that were cleaned up with Stacks’ process_shortreads to human genome. Cufflinks – take mapped reads (accepted_hits.bam) and generate transcript model of reads. Cuffmerge – take individual transcript models (transcripts.gtf)and merge into master transcriptome. Cuffdiff– take mapped reads from individual treatments (accepted_hits.bam)and, with aid of master transcriptome, compare each sample pair-wise. Take various Cuffdiff output files (differential gene expression, splicing, promoter usage, isoforms, etc.) and analyze with original scripts or explore with existing programs.

  9. Integrative Genome Viewer

  10. Integrative Genome Viewer

  11. Visualization of differential gene expression log10 (FPKM + 1) of genes at each dosage that are associated the p53 network. FPKM: fragments per kilobase of exon model per million mapped fragments

  12. none low med high Visualization of differential gene expression log10 (FPKM + 1) of genes at each dosage that are associated with the gene ontology “Regulation of RNA splicing” (GO:0043484)

  13. How about all the (significantly differentially expressed) genes? What are these genes? log2(foldchange) of 1210 genes with q < 0.05 between none and low pentamidine dosage

  14. A closer look none low med high

  15. Gene ontologies found in this subset

  16. Next steps? • Analyze this and other clustered subsets by: • Sequence analysis (motifs) • GC/AT content • Gene ontology enrichment

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