SEMINÁRIO ÂMBITO MESTRADO BIOESTATÍSTICA – Genome-wide Analysis of Intragenic Transcription Initiation Induced by SETD2 Depletion


  • Profª Ana Rita Grosso – Instituto de Medicina Molecular, Faculdade de Medicina – Universidade de Lisboa
  • FCUL (DEIO) – Campo Grande – Bloco C6 Piso 2 – Sala: 6.2.45 – 17:00h
  • Sexta-feira, 5 de Dezembro de 2014

(Jointly with: Silvia Carvalho, Ana Cláudia Raposo, Filipa Batalha Martins, Sreerama Chaitanya Sridhara, José Rino, Maria Carmo-Fonseca and Sérgio Fernandes de Almeida).

Histone H3 of nucleosomes positioned on active genes is trimethylated at Lys36 (H3K36me3) by the SETD2 (also termed KMT3A/SET2 or HYPB) methyltransferase. Previous studies in yeast indicated that H3K36me3 prevents spurious intragenic transcription initiation through recruitment of a histone deacetylase complex, a mechanism that is not conserved in mammals. Here, we report that downregulation of SETD2 in human cells leads to intragenic transcription initiation in at least 11% of active genes. We analyzed previously reported high-throughput RNA-sequencing data from human mesenchymal stem cells transfected with control and targeting siRNAs directed against SETD2 (Luco et al 2010). To identify genes with intragenic transcription initiation, we compared read counts for expressed exons (minimum of 5 reads/100bp required) in depleted and control cells. First, we selected all transcripts with significantly higher read counts for at least one exon in SETD2-depleted cells. Second, we discarded transcripts with significantly higher read counts for the first annotated and expressed exon (likely corresponding to upregulated full-length mRNAs), Third, we defined the new cryptic TSS as the first significantly higher exon and filtered out all transcripts that showed significantly higher read counts for <60% of the downstream exons (possibly corresponding to isolated alternative splicing events). Significantly higher exons were defined as those having exonic read counts (normalized reads per million) fold change higher than 1.2 between SETD2-depleted and control cells, and Fisher’s exact test (alternative hypothesis: read count proportions relative to the total library size in siSETD2 greater than siControl). To withdraw restrictions by previous gene annotation, we performed transcriptome assembly using Cufflinks.

Luco,R.F., Pan,Q., Tominaga,K., Blencowe,B.J., Pereira- Smith,O.M. and Misteli,T. (2010) Regulation of alternative splicing by histone modifications. Science, 327, 996–1000.