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John T Lis

Publications and source records attributed to John T Lis.

At least 19 recordsLinked to original sources
PubMed2025

Evolution of promoter-proximal pausing enabled a new layer of transcription control.

Promoter-proximal pausing of RNA polymerase (Pol) II is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here, we analyzed transcription in species across the tree of life. Unicellular eukaryotes display an accumulation of Pol II near transcription start sites, which we propose transitioned to the longer-lived, focused pause observed in metazoans. This transition coincided with the evolution of new subunits in the negative elongation factor (NELF) and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat-shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

RNA Polymerase II↗
PubMed2006

Dynamics of heat shock factor association with native gene loci in living cells.

Direct observation of transcription factor action in the living cell nucleus can provide important insights into gene regulatory mechanisms. Live-cell imaging techniques have enabled the visualization of a variety of intranuclear activities, from chromosome dynamics to gene expression. However, progress in studying transcription regulation of specific native genes has been limited, primarily as a result of difficulties in resolving individual gene loci and in detecting the small number of protein molecules functioning within active transcription units. Here we report that multiphoton microscopy imaging of polytene nuclei in living Drosophila salivary glands allows real-time analysis of transcription factor recruitment and exchange on specific native genes. After heat shock, we have visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. We show that heat shock factor (HSF), the transcription activator of hsp70, is localized to the nucleus before heat shock and translocates from nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence recovery after photobleaching show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. However, this is not a consequence of a change of HSF diffusibility, as shown here directly by fluorescence correlation spectroscopy. Our results provide strong evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription. This and previous studies indicate that transcription activators display diverse dynamic behaviours in their associations with targeted loci in living cells. Our method can be applied to study the dynamics of many factors involved in transcription and RNA processing, and in their regulation at native heat shock genes in vivo.

Animals↗
PubMed2006

An RNA aptamer that interferes with the DNA binding of the HSF transcription activator.

Heat shock factor (HSF) is a conserved and highly potent transcription activator. It is involved in a wide variety of important biological processes including the stress response and specific steps in normal development. Reagents that interfere with HSF function would be useful for both basic studies and practical applications. We selected an RNA aptamer that binds to HSF with high specificity. Deletion analysis defined the minimal binding motif of this aptamer to be two stems and one stem-loop joined by a three-way junction. This RNA aptamer interferes with normal interaction of HSF with its DNA element, which is a key regulatory step for HSF function. The DNA-binding domain plus a flanking linker region on the HSF (DL) is essential for the RNA binding. Additionally, this aptamer inhibits HSF-induced transcription in vitro in the complex milieu of a whole cell extract. In contrast to the previously characterized NF-kappaB aptamer, the HSF aptamer does not simply mimic DNA binding, but rather binds to HSF in a manner distinct from DNA binding to HSF.

Animals↗
PubMed2006

Promoter cleavage: a topoIIbeta and PARP-1 collaboration.

Hormones trigger dramatic changes in the structure and transcriptional activity of specific promoters that lead to exchange of repression complexes for activation complexes. now show that estrogen-dependent restructuring and transcription of the pS2 promoter require the generation of a DNA double-strand break by a novel protein complex containing two enzymes, topoisomerase IIbeta and poly(ADP-ribose) polymerase.

DNA Topoisomerases, Type II↗
PubMed2006

Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules.

Chromatin-remodeling enzymes can overcome strong histone-DNA interactions within the nucleosome to regulate access of DNA-binding factors to the genetic code. By unzipping individual DNA duplexes, each containing a uniquely positioned nucleosome flanked by long segments of DNA, we directly probed histone-DNA interactions. The resulting disruption-force signatures were characteristic of the types and locations of interactions and allowed measurement of the positions of nucleosomes with 2.6-base-pair (bp) precision. Nucleosomes remodeled by yeast SWI/SNF were moved bidirectionally along the DNA, resulting in a continuous position distribution. The characteristic distance of motion was approximately 28 bp per remodeling event, and each event occurred with a catalytic efficiency of 0.4 min(-1) per nM SWI/SNF. Remodeled nucleosomes had essentially identical disruption signatures to those of unremodeled nucleosomes, indicating that their overall structure remained canonical. These results impose substantial constraints on the mechanism of SWI/SNF remodeling.

Chromosomal Proteins, Non-Histone↗
PubMed2006

Drosophila Paf1 modulates chromatin structure at actively transcribed genes.

The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure.

Animals↗
PubMed2005

Improving slide-based assays by stirring: application of liquid-on-liquid mixing to immunofluorescence staining of polytene chromosomes.

A new method for stirring thin liquid films has been developed and demonstrated to increase the sensitivity of immunofluorescence staining of polytene chromosomes. This liquid-on-liquid mixing (LOLM) technique uses a stirrer fluid, immiscible with the thin film, to transmit shear at the liquid-liquid interface. Here, we stir mineral oil layered over an aqueous thin film of antibody solution, which stains transcription apparatuses on chromosomes previously fixed to a glass slide. The quality of staining was assessed at varying antibody concentrations and incubation or stirring times. Our data indicate that the LOLM technique overcomes the diffusion barrier associated with traditional slide-based biological assays.

Animals↗
PubMed2005

Distinct transcriptional responses of RNA polymerases I, II and III to aptamers that bind TBP.

The TATA-binding protein (TBP) is a general factor that is involved in transcription by all three types of nuclear RNA polymerase. To delineate the roles played by the DNA-binding surface of TBP in these transcription reactions, we used a set of RNA aptamers directed against TBP and examined their ability to perturb transcription in vitro by the different RNA polymerases. Distinct responses to the TBP aptamers were observed for transcription by different types of polymerase at either the initiation, reinitiation or both stages of the transcription cycle. We further probed the TBP interactions in the TFIIIB*DNA complex to elucidate the mechanism for the different sensitivity of Pol III dependent transcription before and after preinitiation complex (PIC) formation. Lastly, the aptamers were employed to measure the time required for Pol III PIC formation in vitro. This approach can be generalized to define the involvement of a particular region on the surface of a protein at particular stages in a biological process.

Cytochromes c↗
PubMed2005

Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.

Uninduced heat shock genes are poised for rapid activation, with RNA polymerase II (Pol II) transcriptionally engaged, but paused or stalled, within the promoter-proximal region. Upon heat shock, this Pol II is promptly released from the promoter region and additional Pol II and transcription factors are robustly recruited to the gene. Regulation of the heat shock response relies upon factors that modify the efficiency of elongation through the initially transcribed sequence. Here, we report that Pol II is susceptible to transcription arrest within the promoter-proximal region of Drosophila hsp70 and that transcript cleavage factor TFIIS is essential for rapid induction of hsp70 RNA. Moreover, using a tandem RNAi-ChIP assay, we discovered that TFIIS is not required to establish the stalled Pol II, but that TFIIS is critical for efficient release of Pol II from the hsp70 promoter region and the subsequent recruitment of additional Pol II upon heat induction.

Animals↗
PubMed2005

Interactions between subunits of Drosophila Mediator and activator proteins.

Mediator was first identified because of its activity in activator-stimulated transcription in vivo and in vitro. Later, biochemical fractionation led to the co-purification of the multi-subunit Mediator complex and RNA polymerase II (pol II). Results of these studies suggested a model whereby transcription-activator proteins, which bind to specific gene regulatory sequences, recruit both Mediator and pol II as a holoenzyme in a one-step mechanism. More recent studies of Drosophila Mediator and additional studies in yeast have demonstrated that different transcription activators can bind and recruit Mediator to promoters in vivo in a step that is independent of pol II recruitment. Moreover, the different activators in Drosophila bind and recruit Mediator via physical interactions with specific subsets of proteins. These features of Mediator function seem to be broadly conserved.

Animals↗
PubMed2004

Specific contributions of histone tails and their acetylation to the mechanical stability of nucleosomes.

The distinct contributions of histone tails and their acetylation to nucleosomal stability were examined by mechanical disruption of individual nucleosomes in a single chromatin fiber using an optical trap. Enzymatic removal of H2A/H2B tails primarily decreased the strength of histone-DNA interactions located approximately +/-36bp from the dyad axis of symmetry (off-dyad strong interactions), whereas removal of the H3/H4 tails played a greater role in regulating the total amount of DNA bound. Similarly, nucleosomes composed of histones acetylated to different degrees by the histone acetyltransferase p300 exhibited significant decreases in the off-dyad strong interactions and the total amount of DNA bound. Acetylation of H2A/H2B appears to play a particularly critical role in weakening the off-dyad strong interactions. Collectively, our results suggest that the destabilizing effects of tail acetylation may be due to elimination of specific key interactions in the nucleosome.

Acetylation↗
PubMed2004

NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1.

PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. Herein, we describe previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity.

Adenosine Triphosphate↗
PubMed2004

Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25.

21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3'-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.

Anti-Bacterial Agents↗
PubMed2004

Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes.

The TATA-binding protein (TBP) is a critical general transcription factor that associates with the core promoter and acts as a nexus for gene regulation through its interactions with other factors. A large number of proteins recognize the relatively small yet highly conserved C-terminal domain of TBP. One subset of these proteins (general transcription factors) interacts with the TBP.TATA complex and RNA polymerase II to create the preinitiation complex. To study TBP functions in preinitiation complex and other complexes, we generated a set of RNA aptamers with high affinity to yeast TBP. These aptamers act on TBP in different ways: all of them bind TBP competitively with DNA bearing the TATA element, and some can actively disrupt the TBP.TATA interaction in preformed, higher-order complexes containing the additional general transcription factors TFIIB and TFIIA. In crude cell extracts, the aptamers inhibit transcription in ways that reveal the dynamic nature of TBP interactions during initiation and reinitiation.

Base Sequence↗
PubMed2004

Transcription through chromatin: understanding a complex FACT.

In eukaryotic cells, genomic DNA is assembled with chromosomal proteins, mainly histones, in a highly compact structure termed chromatin. In this form, DNA is not readily accessible to the cellular machineries, which require DNA as a template. Dynamic changes in chromatin organization play a critical role in regulation of DNA-dependent processes such as transcription, DNA replication, recombination and repair. Chromatin structure is altered in transcriptionally active loci: the basic chromatin unit, the nucleosome, appears to be depleted for one histone H2A/H2B dimer. Previously, reconstitution of RNA polymerase II (PolII)-driven transcription on chromatin templates in a highly purified in vitro system led to identification of FACT (for facilitates chromatin transcription), which was required for productive transcript elongation through nucleosomes. FACT was proposed to promote PolII transcription through nucleosomes by removing either one or both H2A/H2B dimers. Here we present an overview of the earlier studies, which resulted in the initial identification and characterization of FACT, as well as the recent findings that refine the model for the mechanism of FACT function in transcription.

Animals↗
PubMed2004

Coordination of transcription, RNA processing, and surveillance by P-TEFb kinase on heat shock genes.

Positive transcription elongation factor b (P-TEFb) is a kinase that phosphorylates the carboxyl-terminal domain (CTD) of RNA Polymerase II (Pol II). Here, we show that flavopiridol, a highly specific P-TEFb kinase inhibitor, dramatically reduces the global levels of Ser2--but not Ser5--phosphorylated CTD at actively transcribed loci on Drosophila polytene chromosomes under both normal and heat shocked conditions. Brief treatment of Drosophila cells with flavopiridol leads to a reduction in the accumulation of induced hsp70 and hsp26 RNAs. Surprisingly, the density of transcribing Pol II and Pol II progression through hsp70 in vivo are nearly normal in flavopiridol-treated cells. The major defect in expression is at the level of 3' end processing. A similar but more modest 3' processing defect was also observed for hsp26. We propose that P-TEFb phosphorylation of Pol II CTD coordinates transcription elongation with 3' end processing, and failure to do so leads to rapid RNA degradation.

Animals↗