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LSSS 2015-2016


Life Sciences Seminar Series


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John Lis

Department of Molecular Biology and Genetics, Cornell University, USA

New views of transcription and its regulation in cells & across genomes

Selected Publications

GAGA factor maintains nucleosome-free regions and has a role in RNA polymerase II recruitment to promoters.Fuda NJ, Guertin MJ, Sharma S, Danko CG, Martins AL, Siepel A, Lis JT
PLoS Genet 2015 Mar; 11(3):e1005108


Previous studies have shown that GAGA Factor (GAF) is enriched on promoters with paused RNA Polymerase II (Pol II), but its genome-wide function and mechanism of action remain largely uncharacterized. We assayed the levels of transcriptionally-engaged polymerase using global run-on sequencing (GRO-seq) in control and GAF-RNAi Drosophila S2 cells and found promoter-proximal polymerase was significantly reduced on a large subset of paused promoters where GAF occupancy was reduced by knock down. These promoters show a dramatic increase in nucleosome occupancy upon GAF depletion. These results, in conjunction with previous studies showing that GAF directly interacts with nucleosome remodelers, strongly support a model where GAF directs nucleosome displacement at the promoter and thereby allows the entry Pol II to the promoter and pause sites. This action of GAF on nucleosomes is at least partially independent of paused Pol II because intergenic GAF binding sites with little or no Pol II also show GAF-dependent nucleosome displacement. In addition, the insulator factor BEAF, the BEAF-interacting protein Chriz, and the transcription factor M1BP are strikingly enriched on those GAF-associated genes where pausing is unaffected by knock down, suggesting insulators or the alternative promoter-associated factor M1BP protect a subset of GAF-bound paused genes from GAF knock-down effects. Thus, GAF binding at promoters can lead to the local displacement of nucleosomes, but this activity can be restricted or compensated for when insulator protein or M1BP complexes also reside at GAF bound promoters.

Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers.Core LJ, Martins AL, Danko CG, Waters CT, Siepel A, Lis JT
Nat Genet 2014 Dec; 46(12):1311-20


Despite the conventional distinction between them, promoters and enhancers share many features in mammals, including divergent transcription and similar modes of transcription factor binding. Here we examine the architecture of transcription initiation through comprehensive mapping of transcription start sites (TSSs) in human lymphoblastoid B cell (GM12878) and chronic myelogenous leukemic (K562) ENCODE Tier 1 cell lines. Using a nuclear run-on protocol called GRO-cap, which captures TSSs for both stable and unstable transcripts, we conduct detailed comparisons of thousands of promoters and enhancers in human cells. These analyses identify a common architecture of initiation, including tightly spaced (110 bp apart) divergent initiation, similar frequencies of core promoter sequence elements, highly positioned flanking nucleosomes and two modes of transcription factor binding. Post-initiation transcript stability provides a more fundamental distinction between promoters and enhancers than patterns of histone modification and association of transcription factors or co-activators. These results support a unified model of transcription initiation at promoters and enhancers.

Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons.Jonkers I, Kwak H, Lis JT
Elife 2014 Apr 29; 3:e02407


Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes.DOI:

Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.Tome JM, Ozer A, Pagano JM, Gheba D, Schroth GP, Lis JT
Nat Methods 2014 Jun; 11(6):683-8


RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E-binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E-binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.

Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions.Pagano JM, Kwak H, Waters CT, Sprouse RO, White BS, Ozer A, Szeto K, Shalloway D, Craighead HG, Lis JT
PLoS Genet 2014 Jan; 10(1):e1004090


The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes.

Precise maps of RNA polymerase reveal how promoters direct initiation and pausing.Kwak H, Fuda NJ, Core LJ, Lis JT
Science 2013 Feb 22; 339(6122):950-3


Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a precision nuclear run-on and sequencing (PRO-seq) assay to map the genome-wide distribution of transcriptionally engaged Pol II at base pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' polyadenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites, nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. This "complex interaction" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter.

Defining the status of RNA polymerase at promoters.Core LJ, Waterfall JJ, Gilchrist DA, Fargo DC, Kwak H, Adelman K, Lis JT
Cell Rep 2012 Oct 25; 2(4):1025-35


Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.