Model: Heat Shock Genes
Why do we study the Drosophila heat shock genes as one of our major model systems? Heat shock genes are good models to study transcription and its activation because:
- Robust activation: transcript levels are 100x greater when activated than in the unactivated state
- Rapid response: transcription activation occurs within tens of seconds after the initial temperature shift
- Synchronous activation: allows for kinetic analysis of the transcription cycle and the ability to mechanistically order the activity of specific factors
- Well-defined system: we have been studying this system for over 3 decades and know what to expect when developing new methods
- Generalizable: the principles and underlying mechanisms are proving to be applicable to other systems
- Natural amplification: Drosophila polytene chromosomes are extended and aligned interphase chromosomes providing both sensitivity and resolution to analyze the recruitment and dynamics of factors at specific loci
Mammalian Gene Regulation
We are examining the generality of models and exploring new hypothesis using both focused studies on mammalian heat shock genes and genome-wide analysis of mouse and human gene regulation. The focused studies of mouse heat shock genes are taking advantage of cell lines that are knock-outs for the master regulators HSF1 and HSF2. Genome-wide assays are defining the complete regulatory networks of the heat shock response and the relationship of the HSF-regulated pathways to cancer.
Complete transcriptional patterns revealed by our Global Run-On (GRO-seq) in Drosophila and well-studied mammalian cell lines are assessing the generality of promoter-proximal pausing as a mechanism of gene regulation. These and additional studies are also allowing us to investigate of the underlying mechanisms of this regulation.
Imaging:
Optical Imaging:
Polytene Chromosomes
Formation of Polytene Chromosomes: The chromosomes are duplicated and stay connected and aligned. Patterns of denser "bands" alternate with lighter-staining "interbands" to form a consistently mappable genome.
Polytene chromosomes occur in the secretory glands of Drosophila (as well as in some other tissues and other dipteran flies). Multiple rounds of DNA replication result in about 1000 strands of DNA, which remain aligned and attached to each other. These chromosomes are large enough to view by light microscopy. Despite their unusual structure, polytenes function as interphase chromosomes and are transcriptionally active and responsive to hormonal and environmental signals (such as heat shock).
For more information on, and classical pictures of, polytene chromosomes, see also the Wikipedia article or the Sedat Lab description.
Spread Polytene Immunofluorescence
Immunofluorescence staining of spread polytene chromosomes: Two different proteins are pseudocolored red and green. Areas of colocalization between the two proteins appear yellow.
Indirect immunofluorescence staining of polytene chromosomes provides a rapid way of determining the in vivo genomic distribution of chromosomal proteins.
Polytenes are fixed and spread out on a slide, then probed with antibodies raised against specific proteins. This method allows us to visualize where proteins are in relationship to the underlying DNA sequence, and to each other.
The Drosophila genome has been sequenced, and the polytene maps have been correlated with the DNA sequence. This allows us to interpret staining patterns with precision, in some cases down to an individual gene.
Immunostaining for two or three factors at once allows comparison of factor distributions relative to one another. The colocalization of two factors may be indicative of a physical interaction in vivo.
Selected Papers:
Multiphoton Microscopy
Multiphoton microscopy 3-D reconstruction of a nucleus: HSF (in green) and DNA (in red) localization in a living polytene nucleus after heat shock.
In collaboration with Watt Webb and Warren Zipfel's Lab, we are using the optical sectioning power of multiphoton microscopy to examine protein localization, dynamics, and interactions at specific chromosomal loci in living polytene cells and in real time.
Proteins of interest are tagged with a fluorescent protein such as EGFP and transgenic lines that express this protein with temporal and spatial control can be created. Because of the ability of multiphoton microscopy to image deeply into a sample, the localization of the tagged protein can be watched in living cells in real time.
Additionally, the dynamics of the tagged protein can be studied using techniques such as FRAP (Fluorescence Recovery After Photobleaching) or FCS (Fluorescence Correlation Spectroscopy), and its interactions with another protein can be watched using FRET (Fluorescence Resonance Energy Transfer).
Selected Papers:
Zobeck KL, Buckley MS, Zipfel WR, and Lis JT. (2010) Recruitment Timing and Dynamics of Transcription Factors at the Hsp70 Loci in Living Cells. Molec Cell. 40: 965-975. (PubMed)
Yao J et al. (2007) “Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation.” Mol Cell. 28(6):978-90. (PubMed)
Molecular Imaging:
Chromatin Immunoprecipitation
Chromatin Immunoprecipitation (ChIP) provides data on the distribution of protein factors on DNA with high temporal and spatial resolution.
The crosslinking of proteins to DNA freezes interactions in time and stabilizes weak interactions that might not persist through the immunoprecipitation protocol. We use two methods for crosslinking, chemical fixation via formaldehyde and photophysical fixation via UV light. Formaldehyde crosslinks proteins to other proteins as well as to DNA, whereas UV light predominantly forms DNA-protein crosslinks. The two methods can provide complementary information about the structure of a protein-DNA complex.
The crosslinked DNA is fragmented, and an antibody is used to purify the DNA fragments bound to a specific protein. Quantitative real-time PCR measures the relative amounts of the protein that are found at various DNA sequences. The genome-wide distribution of the protein can now be conveniently analyzed by ChIP-seq and analysis pipelines developed in our lab.
Selected Papers:
Guertin MJ, Martins AL, Siepel A, Lis JT. (2012) "Accurate prediction of inducible transcription factor binding intensities in vivo." PLoS Genet. 8(3):e1002610. (PubMed)
Zhang X, Bolt M, Guertin MJ, Chen W, Zhang S, Cherrington BD, Slade DJ, Dreyton CJ, Subramanian V, Bicker KL, Thompson PR, Mancini MA, Lis JT, Coonrod SA. (2012) "Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor α target gene activation." Proc Natl Acad Sci USA. 109(33):13331-6. (PubMed)
Fuda NJ, Buckley MS, Wei W, Core LJ, Waters CT, Reinberg D, Lis JT. (2012) "Fcp1 dephosphorylation of the RNA polymerase II C-terminal domain is required for efficient transcription of heat shock genes." Mol Cell Biol. 32(17):3428-37. (PubMed)
Guertin MJ, Lis JT. (2010) "Chromatin landscape dictates HSF binding to target DNA elements." PLoS Genet. 9;6(9). (PubMed)
Ni Z et al. (2008) "P-TEFb is critical for the maturation of RNA polymerase II into productive elongation in vivo." Mol Cell Biol. 28(3):1161-70. (PubMed)
Footprinting
Footprinting involves treating live cells with a chemical agent that causes DNA damage. If a protein is bound to the DNA, it can protect the strands it covers from this damage, and its binding site can be deduced.
Selected Papers:
- Giardina C & Lis JT. (1995) "Dynamic protein-DNA architecture of a yeast heat shock promoter." Mol Cell Biol. 15(5):2737-44. (PubMed)
Transcription Studies: Run-ons, Transcript Assays
A nuclear run-on assay measures the amount of Pol II that is either actively transcribing or is "transcriptionally engaged," tightly associated with DNA in preparation for transcription. We have recently expanded the power of these nuclear run-on assays with GRO-seq, an assay evaluates genome-wide the location, orientation, and density of transcriptionally-engage polymerases. A variation in this protocol, called PRO-seq, now allow Pol II to be mapped with base pair resolution.
Selected Papers:
Kwak H, Fuda NJ, Core LJ, Lis JT. (2013) "Precise maps of RNA polymerase reveal how promoters direct initiation and pausing." Science. 339(6122):950-3. (PubMed)
Core LJ, Waterfall JJ, Gilchrist DA, Fargo DC, Kwak H, Adelman K, Lis JT. (2012) "Defining the Status of RNA Polymerase at Promoters." Cell Reports.pii: S2211-1247(12)00274-4. [Epub ahead of print]. (PubMed)
Min IM , Waterfall JJ, CoreLJ, Munro RJ, SchimentiJ, and Lis JT (2011) "Regulating RNAPolymerase Pausing and Transcription Elongation in Embryonic Stem Cells." Genes & Devel., 25: 742-754. (PubMed)
Hah N, Danko CG., Core LJ, Waterfall JJ, Siepel A, Lis, JT, and Kraus WL. (2011) "Global Analysis of the Immediate Transcriptional Effects of Estrogen Signaling Reveals a Rapid, Extensive, and Transient Response." Cell 145: 622-34. (PubMed)
Core LJ, Waterfall JJ, and Lis JT (2008) "Nascent RNA Sequencing Reveals Widespread Pausing and Divergent Initiation at Human Promoters." Science 322: 1845-1848. (PubMed)
Rasmussen EB & Lis JT. (1993) "In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes." Proc. Natl Acad. Sci. USA 90, 7923-7927. (PubMed)
Shows that capping predominantly occurs when the nascent mRNA is 20-30 nucleotides long.
Disruption:
RNA Aptamers
RNA Aptamers: A single strand of RNA folds back upon itself to give a complicated secondary structure. In three dimensions, this aptamer forms a shape which can bind tightly to its protein target.
To disrupt not only a specific protein, but a specific interacting domain on that protein, we turn to RNA aptamer technology. We select RNA aptamers from large, highly complex RNA pools using cycles of selection and amplification (SELEX). Aptamers provide several advantages as inhibitors:
- Like an antibody, they can be made to order specifically for a particular protein
- Like a small organic molecule, they can rapidly target a specific protein domain within cells
- Like a conditional allele, they are able to exert their effect in whole organisms, but
- They are also targetable to specific tissues, cells, or stages of development.
Selected Papers:
Salamanca HH, Fuda N, Shi H, Lis JT. (2011) "An RNA aptamer perturbs heat shock transcription factor activity in Drosophila melanogaster." Nucleic Acids Res. May 16. [Epub ahead of print]. (PubMed)
Sevilimedu A, Shi H, and Lis JT (2008) "TFIIB aptamers inhibit transcription by perturbing PIC formation at distinct stages."Nucleic Acids Res. 36:3118-27.
Shi H et al. (2007) "RNA aptamers directed to discrete functional sites on a single protein structural domain." Proc Natl Acad Sci U S A. 104(10):3742-6.(PubMed)
- Zhao X et al. (2006) “An RNA aptamer that interferes with the DNA binding of the HSF transcription activator.” Nucleic Acids Res. 34(13):3755-61. (PubMed)
- Fan X et al. (2005) “Distinct transcriptional responses of RNA polymerases I, II and III to aptamers that bind TBP.” Nucleic Acids Res. 33(3):838-45. (PubMed)
- Fan X et al. (2004) “Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes.” Proc Natl Acad Sci U S A. 101(18):6934-9. (PubMed)
- Shi H et al. (1999) “RNA aptamers as effective protein antagonists in a multicellular organism.” Proc Natl Acad Sci U S A. 96(18):10033-8. (PubMed)
- First demonstration that aptamers can interfere with a protein's function in a multicellular organism.
RNAi
RNAi can be used with relative ease in Drosophila cell cultures to deplete levels of specific proteins to less than 20% of their normal levels. By reducing the levels of a transcription, elongation, or processing factor, we can examine its role in various transcriptional and co-transcriptional processes. Generally we study the effect on the localization of other protein factors by ChIP, or on the transcripts produced by quantitative reverse transcription PCR or other methods.
Selected Papers:
Fuda NJ, Buckley MS, Wei W, Core LJ, Waters CT, Reinberg D, Lis JT. (2012) "Fcp1 dephosphorylation of the RNA polymerase II C-terminal domain is required for efficient transcription of heat shock genes." Mol Cell Biol. 32(17):3428-37. (PubMed)
Petesch SJ and Lis JT (2012) "Activator Induced Spread of Poly(ADP-Ribose) Polymerase Promotes Nucleosome Loss at Hsp70." Mol Cell, 45:64-74. (PubMed)
Ardehali BM, Yao J, Adelman K, Fuda N, Petesch S, Webb WW and Lis JT (2009) "Spt6 enhances the elongation rate of RNA polymerase II in vivo." EMBO J 28(8):1067-77. Epub 2009 Mar 12. (PubMed)
Petesch SJ and Lis JT (2008) "Rapid, Transcription-Independent Loss of Nucleosomes over a Large Chromatin Domain at Hsp70 Loci." Cell 134: 74–84.
Adelman K et al. (2006) "Drosophila Paf1 modulates chromatin structure at actively transcribed genes." Mol Cell Biol. 26(1):250-60. (PubMed)
Fast-acting conditional mutants
A rapidly-acting conditional mutant can be an invaluable tool in assessing the mechanistic role of a particular transcription factor in vivo. We are using a variety of fast-acting conditional mutants that encode Drosophila proteins that we hypothesize are critical for both establishing the potentiated promoter and for its activation. We are assessing the roles of these proteins in regulating chromatin architecture and function of the heat shock promoter in the seconds or minutes following the conditional disruption of a particular protein.
Selected Papers:
Small molecule drugs
Drugs that rapidly inhibit particular active sites of proteins believed critical for transcription and its regulation are being used to evaluate the primary functions of these proteins in regulating chromatin architecture and function of the heat shock promoter.
Selected Papers: