SUMMARY The transcription factor HIF1A is a key mediator of the cellular response to hypoxia. Despite the importance of HIF1A in homeostasis and various pathologies, little is known about how it regulates RNA polymerase II (RNAPII). We report here that HIF1A employs a specific variant of the Mediator complex to stimulate RNAPII elongation. The Mediator-associated kinase CDK8, but not the paralog CDK19, is required for induction of many HIF1A target genes. HIF1A induces binding of CDK8-Mediator and the Super Elongation Complex (SEC), containing AFF4 and CDK9, to alleviate RNAPII pausing. CDK8 is dispensable for HIF1A chromatin binding and histone acetylation, but it is essential for binding of SEC and RNAPII elongation. Global analysis of active RNAPII reveals that hypoxia-inducible genes are paused and active prior to their induction. Our results provide a mechanistic link between HIF1A and CDK8, two potent oncogenes, in the cellular response to hypoxia.

The p53 transcription factor is a potent suppressor of tumor growth. We report here an analysis of its direct transcriptional program using Global Run-On sequencing (GRO-seq). Shortly after MDM2 inhibition by Nutlin-3, low levels of p53 rapidly activate ∼200 genes, most of them not previously established as direct targets. This immediate response involves all canonical p53 effector pathways, including apoptosis. Comparative global analysis of RNA synthesis vs steady state levels revealed that microarray profiling fails to identify low abundance transcripts directly activated by p53. Interestingly, p53 represses a subset of its activation targets before MDM2 inhibition. GRO-seq uncovered a plethora of gene-specific regulatory features affecting key survival and apoptotic genes within the p53 network. p53 regulates hundreds of enhancer-derived RNAs. Strikingly, direct p53 targets harbor pre-activated enhancers highly transcribed in p53 null cells. Altogether, these results enable the study of many uncharacterized p53 target genes and unexpected regulatory mechanisms.DOI: http://dx.doi.org/10.7554/eLife.02200.001

Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 59 ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of~70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not transspliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a ''hybrid'' operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.[Supplemental material is available for this article. The sequence data from this study have been submitted to the NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) under accession nos. SRA008646 and SRA003622.] C. elegans uses two RNA processing features that distinguish it from other model organisms. First, the transcripts of many genes are trans-spliced to a spliced leader (SL). Trans-splicing is a process in which an SL replaces the 59 end of a transcript by spliceosomal splicing. The 22-nucleotide (nt) SL is donated by an ;100-nt SL snRNP (small nuclear ribonucleoprotein) to a pre-mRNA with an intron-like region, the outron, containing an unpaired 39 splice site located near the 59 end. The second distinguishing feature is that many genes are transcribed in polycistronic units, known as operons, where a single promoter serves several genes. The operons can be up to eight genes long, and the polycistronic pre-mRNAs are separated into individual cistrons by 39 end formation accompanied by SL trans-splicing.These two features have important implications for C. elegans research. For instance, deletions/insertions within an operon may affect not only the expression of the gene containing the mutation, but also the genes downstream from it in the operon (Cui et al. 2008). Similarly, in a strain with enhanced RNAi sensitivity, RNAi of an operon gene can also affect expression of genes downstream (Guang et al. 2010). Finally, the trans-splice site is at the 59 end of the mRNA, not the pre-mRNA, and thus, the promoter is often not directly adjacent to the gene, but rather upstream of the outron or the entire operon (Blumenthal and Spieth 1996). SL trans-splicing has been repor...