Major contributions of the lab.  

My lab develops and uses a variety of strategies to probe the structure of promoters and genes and the regulation of their activities in living cells.  We have studied the factors and mechanisms that drive transcription and its regulation for decades. While our primary model system has been the highly-inducible heat shock (HS) genes in Drosophila, we have over the past several years become deeply committed to developing and using genome-wide assays in Drosophila, mammals, and other model organisms to test the generality of our focused studies of heat shock genes and to explore new genome-wide paradigms of transcriptional regulation of genes..

Our lab has had a history of developing methodologies to provide new views of  transcription regulation. In my first several years at Cornell, my lab developed UV-crosslinking methods to map protein-DNA interactions in vivo that served as the precursor for modern chromatin immunoprecipitation (ChIP) methods [1]. More recently, my lab has developed several genome-wide assays, such as GRO-seq [2], PRO-seq [3], GRO-cap [4], and PRO-cap [3] for precisely mapping nascent RNAs and start sites, often with base pair precision.  Our goals are to understand the molecular mechanics of transcription and its regulation.  Currently, we are focused on the interplay of promoter and enhancer elements and the factors that interact with these elements to dictate RNA Polymerase II (Pol II) transcription and its regulation.

  1. Gilmour DS, & Lis JT. Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc Natl Acad Sci U S A. 81:4275-9, 1984.
  2. Core LJ, Waterfall JJ, & Lis JT. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters. Science. 322(5909):1845-8. 2008
  3. Kwak H, Fuda NJ, Core LJ, & Lis JT. Precise maps of RNA polymerase reveal howpromoters direct initiation and pausing. Science 339(6122):950-3, 2013.
  4. Core LJ, Martins AL, Danko CG, Waters CT, Siepel A, & Lis JT. Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers. Nat Genet 46:1311-20, 2014


My lab’s most significant contributions are grouped into five parts.  My role as lab leader has been to provide the major questions and direction to a very talented collection of graduate students and postdoctoral fellows who brought their own insights, drive and experimental skills to these discoveries.  In the early days, I participated in technology development directly in the lab.  Note – I apologize to colleagues whose papers I could not cover under these 5 topics.

First, the UV-based ChIP assay we developed and applied to mapping RNA Pol II on genes in the mid-1980s led to the in-depth characterization of ‘Promoter-Paused Pol II’ as a very common rate-limiting and regulated step in the transcription of metazoan genes. Our initial focused studies on heat shock promoters demonstrated that RNA Pol II was already associated with heat shock gene promoter in cells before they were induced [5]; moreover, this Pol II was transcriptionally engaged but paused [6]. This work challenged the paradigm that transcription was generally regulated at Pol II recruitment and initiation.  We expanded these analyses genome-wide with our lab’s development of a global nuclear run-on assay (GRO-seq) [3].  This mapping of the density and orientation of transcriptionally-competent Pol II genome-wide with unprecedented sensitivity demonstrated the pervasiveness of this regulated pausing mechanisms, a conclusion that has been supported in many papers from our lab and our collaborators, and other groups.  We have also characterized the features of promoters that generate the pause using a base-pair resolution run-on assay that we developed called PRO-seq [4].  Our very recent work also demonstrated that the paused Pol II is relatively stable on Drosophila Hsp70 [7] and on over 3100 genes in mouse ES cells, where we measured the half-lifes of paused Pol II at pause sites in vivo [8].  These measurements and modeling strongly support that the major mechanism of paused Pol II regulation in higher eukaryotes is through the regulation of the release of the pause to productive elongation, rather than through an anti-termination mechanism  Moreover, our recent direct measurements of pause release and termination rates in vivo on Hsp70, both before and after heat shock, give further support to this mechanism indicating that the paused Pol II  is regulated at the step of escape into productive elongation and not by anti-termination [7].

  1. Gilmour DS, & Lis JT. RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells. Mol Cell Biol. 6:3984-9, 1986.
  2. Rougvie AE, & Lis JT. The RNA polymerase II molecule at the 5′ end of the uninduced hsp70 gene of D. melanogaster is transcriptionally engaged. Cell 54:795-804, 1988.
  3. Buckley MS, Kwak H, Zipfel WR, Lis JT. Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation. Genes Dev. 28:14-9, 2014.
  4. Jonkers I, Kwak H,& Lis JT. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons. Elife 3:e02407, 2014.


Second, my lab discovered key properties of the master regulator of the heat shock response, HSF. We showed that the master regulatory protein of the heat shock response HSF was trimeric [9], and we derived the rules governing HSF’s interaction heat shock regulatory DNA elements [10], the features of chromatin that allow HSF to access its regulatory element in vivo [11,12].  The basic protein structure and type of interaction with its elements was surprising because DNA sequences generally have elements that are arranged as inverted or direct repeats and these arrangements seem inconsistent with expectations of trimer binding. The nature of the flexible arrangement of DNA sequence elements also established a new paradigm for factor binding to DNA [9,10].  The more recent analysis of the HSF binding in vivo [11,12] identified features of chromatin that only allows access of HSF to < 20% of its potential binding sites.  This inducible binding of HSF allowed us to make use of the landscape of chromatin features (from the ENCODE consortium) that existed when HSF is triggered to rapidly bind its targets.  Prior studies of transcription factor occupancy also showed occupancy of a subset of potential sites, but the interpretation of  the role of the chromatin landscape was confounded by examining chromatin states when binding had already occurred. The rules for HSF, initially reported in 2010, are proving general for other factors that show inducible binding like hormone receptors.

  1. Perisic O, Xiao H, & Lis JT. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit. Cell. 59:797-806, 1989.
  2. Xiao H, & Lis JT. Germline transformation used to define key features of heat-shock response elements. Science 239:1139-42, 1988
  3. Guertin MJ, & Lis JT. Chromatin landscape dictates HSF binding to target DNA elements. PLoS Genet. 6:e1001114, 2010
  4. Guertin MJ, Martins AL, Siepel A, & Lis JT. Accurate prediction of inducible transcription factor binding intensities in vivo. PLoS Genet.;8:e1002610, 2012.


Third, in collaboration with Watt Webb’s and Warren Zipfel’s Lab, we applied the first multiphoton laser-scanning microscopy to optically dissect Drosophila polytene nuclei and measure transcription factor dynamics at specific native loci in living tissue [13].  Using these and other optical sectioning methods, we demonstrated that the recruitment of factors was rapid and sequential, thereby a transcriptional factory that efficiently recycles RNA polymerase II (Pol II) is built during gene activation [14, 15].  These studies in polytene nuclei and FISH experiments in diploid cells demonstrate that transcription factories are assembled on heat shock genes rather than genes being transported to preexisting factories, indicating that this popular recruitment to factories model is, at minimum, not general for activated genes. The methods describing these technologies were reported in detail [16] and many dozens of our fly lines have been provided to investigators.

  1. Yao J, Munson KM, Webb WW, & Lis JT. Dynamics of heat shock factor association with native gene loci in living cells. Nature 442 :1050-3, 2006.
  2. Yao J, Ardehali MB, Fecko CJ, Webb WW, & Lis JT. Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation. Mol Cell.28:978-90, 2007.
  3. Zobeck KL, Buckley MS, Zipfel WR, & Lis JT. Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells. Mol Cell.40:965-75, 2010.
  4. Yao J, Zobeck KL, Lis JT, & Webb WW. Imaging transcription dynamics at endogenous genes in living Drosophila tissues. Methods 45:233-41, 2008.


Fourth, we identified the factors, DNA elements, and a major feature of the mechanism that drives gene activated chromosome puffing and chromatin decondensation.  Transcription requires the rearrangement of the chromatin structure to expose the DNA template to RNA Pol II.  The formation of puffs on giant polytene chromosomes is one striking manifestation of the dramatic rearrangement in chromatin that occurs at both developmentally-activated and heat shock-activate gene loci.  Our labs entry to understanding this classical cytological observation began in the 1980s with our demonstration that heat shock transgenes induce new puffs in polytene chromosomes [17], and the gene elements responsible could be assayed by rigorous transgenic analyses [18].  In the late 2000s using Drosophila cell cultures, we identified poly(ADP-ribose) polymerase (PARP) and its HSF-activated production of poly(ADP-ribose) chains as a critical part of the chromatin-altering mechanism that is activated by HSF. We found that PARP activity removes nucleosomes from the locus prior to transcription [19].  More recently, we began to understand this decondensation in terms of Tip60 activating a prebound PARP that then creates poly(ADP-ribose) at the locus which leads to immediate nucleosome loss [20].  This mechanism is independent of a variety of nucleosome remodelers and is another in the arsenal of machines that can alter nucleosome content of a locus.  Because PARP is associated with many promoters in higher eukaryotes, this mechanism may be very general.

  1. Lis JT, Simon JA, & Sutton CA. New heat shock puffs and beta-galactosidase activity resulting from transformation of Drosophila with an hsp70-lacZ hybridgene. Cell 35:403-10, 1983.
  2. Simon JA, Sutton CA, Lobell RB, Glaser RL, & Lis JT. Determinants of heat shock-induced chromosome puffing. Cell 40:805-17, 1985.
  3. Petesch SJ, & Lis JT. Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134:74-84, 2008.
  4. Petesch SJ, & Lis JT. Activator-induced spread of poly(ADP-ribose) polymerase promotes nucleosome loss at Hsp70. Mol Cell 45:64-74, 2012.


Fifth, the lab developed strategies for selecting RNA aptamers that bind specifically target proteins, and for using these RNA aptamers to inhibit a target protein’s interactions in living cells.  Macromolecular interactions are at the heart of biological regulation. Interactions between transcription factors and with DNA and RNA are numerous and critical for gene regulation.  To test the primary mechanistic function of these interactions, it is critical to have reagents that can rapidly inhibit or disrupt these interactions in cells.  Drugs that rapidly disrupt macromolecular interactions are few and often lack specificity needed. We have been dedicated for years to developing RNA aptamers as inhibitors and disrupters of these interactions, as genes encoding these RNAs can be introduced into cells and animals and rapidly induced [21]. We were the first to demonstrate aptamer selection to distinct surfaces of a target [22], which is important for teasing apart different functions of a factor, and first to demonstrate aptamer inhibition of a protein’s function in an animal [21]. We have recently, with the help of our engineering collaborators, streamlined and multiplexed RNA aptamer selections [23]. Also, recently we have devised a method, HiTS-RAP, with collaborators at Illumina, that uses an Illumina GAIIx sequencer to couple both aptamer sequencing and quantitative aptamer-RNA binding to protein targets. This allows millions of RNA aptamers to be sequenced as DNA and then locally transcribed into RNA tethered to each DNA cluster on an Illumina slide.  Flowing in different concentrations of fluorescent target proteins to the slide permits the very rapid identification and characterization of the binding properties of RNA aptamers obtained from a SELEX experiment.  Together with our rapid selection methods, HiTS-RAP is dramatically increasing the production of RNA aptamers as binders and potential inhibitors of transcription factors.

  1. Shi H, Hoffman BE, & Lis JT. RNA aptamers as effective protein antagonists in a multicellular organism. Proc Natl Acad Sci U S A. 96:10033-8, 1999.
  2. Shi H, Fan X, Sevilimedu A, & Lis JT. RNA aptamers directed to discrete functional sites on a single protein structural domain. Proc Natl Acad Sci U S A.104:3742-6, 2007.
  3. Ozer A, Pagano JM, & Lis JT. New Technologies Provide Quantum Changes in the Scale, Speed, and Success of SELEX Methods and Aptamer Characterization. Mol Ther Nucleic Acids 3:e183, 2014.
  4. Tome JM, Ozer A, Pagano JM, Gheba D, Schroth GP, & Lis JT. Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling. Nat Methods11:683-8, 2014.