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• W2990228889 abstract "•The yeast transcription factor Rap1 can invade compact chromatin•Rap1 directly opens chromatin structure by preventing nucleosome stacking•Stable Rap1 binding requires collaboration with RSC to shift promoter nucleosomes Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression. Pioneer transcription factors (pTFs) bind to target sites within compact chromatin, initiating chromatin remodeling and controlling the recruitment of downstream factors. The mechanisms by which pTFs overcome the chromatin barrier are not well understood. Here, we reveal, using single-molecule fluorescence, how the yeast transcription factor Rap1 invades and remodels chromatin. Using a reconstituted chromatin system replicating yeast promoter architecture, we demonstrate that Rap1 can bind nucleosomal DNA within a chromatin fiber but with shortened dwell times compared to naked DNA. Moreover, we show that Rap1 binding opens chromatin fiber structure by inhibiting inter-nucleosome contacts. Finally, we reveal that Rap1 collaborates with the chromatin remodeler RSC to displace promoter nucleosomes, paving the way for long-lived bound states on newly exposed DNA. Together, our results provide a mechanistic view of how Rap1 gains access and opens chromatin, thereby establishing an active promoter architecture and controlling gene expression. Chromatin acts as a barrier for DNA binding proteins, including transcription factors (TFs), restricting both their target search and binding-site recognition (Adams and Workman, 1995Adams C.C. Workman J.L. Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.Mol. Cell. Biol. 1995; 15: 1405-1421Crossref PubMed Google Scholar, Mirny, 2010Mirny L.A. Nucleosome-mediated cooperativity between transcription factors.Proc. Natl. Acad. Sci. USA. 2010; 107: 22534-22539Crossref PubMed Scopus (179) Google Scholar). A subset of transcription factors named “pioneer transcription factors” (pTFs) can invade compact chromatin domains (Zaret and Mango, 2016Zaret K.S. Mango S.E. Pioneer transcription factors, chromatin dynamics, and cell fate control.Curr. Opin. Genet. Dev. 2016; 37: 76-81Crossref PubMed Scopus (186) Google Scholar). They then initiate chromatin structure opening (Cirillo et al., 2002Cirillo L.A. Lin F.R. Cuesta I. Friedman D. Jarnik M. Zaret K.S. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.Mol. Cell. 2002; 9: 279-289Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar, Fakhouri et al., 2010Fakhouri T.H. Stevenson J. Chisholm A.D. Mango S.E. Dynamic chromatin organization during foregut development mediated by the organ selector gene PHA-4/FoxA.PLoS Genet. 2010; 6 (e1001060)Crossref PubMed Scopus (41) Google Scholar), which can coincide with linker histone loss (Iwafuchi-Doi et al., 2016Iwafuchi-Doi M. Donahue G. Kakumanu A. Watts J.A. Mahony S. Pugh B.F. Lee D. Kaestner K.H. Zaret K.S. The pioneer transcription factor FoxA maintains an accessible nucleosome configuration at enhancers for tissue-specific gene activation.Mol. Cell. 2016; 62: 79-91Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) or nucleosome removal (Jin et al., 2009Jin C. Zang C. Wei G. Cui K. Peng W. Zhao K. Felsenfeld G. H3.3/H2A.Z double variant-containing nucleosomes mark ‘nucleosome-free regions’ of active promoters and other regulatory regions.Nat. Genet. 2009; 41: 941-945Crossref PubMed Scopus (552) Google Scholar, Knight et al., 2014Knight B. Kubik S. Ghosh B. Bruzzone M.J. Geertz M. Martin V. Dénervaud N. Jacquet P. Ozkan B. Rougemont J. et al.Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.Genes Dev. 2014; 28: 1695-1709Crossref PubMed Scopus (68) Google Scholar, Suto et al., 2000Suto R.K. Clarkson M.J. Tremethick D.J. Luger K. Crystal structure of a nucleosome core particle containing the variant histone H2A.Z.Nat. Struct. Biol. 2000; 7: 1121-1124Crossref PubMed Scopus (411) Google Scholar). Such remodeled chromatin is accessible to subsequent non-pioneer TFs (Cirillo et al., 2002Cirillo L.A. Lin F.R. Cuesta I. Friedman D. Jarnik M. Zaret K.S. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4.Mol. Cell. 2002; 9: 279-289Abstract Full Text Full Text PDF PubMed Scopus (795) Google Scholar), which together produce changes in transcriptional programs (Soufi et al., 2015Soufi A. Garcia M.F. Jaroszewicz A. Osman N. Pellegrini M. Zaret K.S. Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming.Cell. 2015; 161: 555-568Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, Zaret and Carroll, 2011Zaret K.S. Carroll J.S. Pioneer transcription factors: establishing competence for gene expression.Genes Dev. 2011; 25: 2227-2241Crossref PubMed Scopus (952) Google Scholar). A common feature of DNA binding domains (DBDs) of pTFs is their ability to bind partial sequence motifs displayed on nucleosomes (Soufi et al., 2015Soufi A. Garcia M.F. Jaroszewicz A. Osman N. Pellegrini M. Zaret K.S. Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming.Cell. 2015; 161: 555-568Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). The presence of nucleosomes may therefore have limited effects on both on-rates and residence times of pTFs. Beyond the nucleosome, higher-order chromatin structure further constrains DNA conformation and TF accessibility (Poirier et al., 2008Poirier M.G. Bussiek M. Langowski J. Widom J. Spontaneous access to DNA target sites in folded chromatin fibers.J. Mol. Biol. 2008; 379: 772-786Crossref PubMed Scopus (110) Google Scholar). Indeed, high-resolution structural studies on reconstituted chromatin revealed that local structural elements, such as tetranucleosome units, form the basis of chromatin fiber organization (Schalch et al., 2005Schalch T. Duda S. Sargent D.F. Richmond T.J. X-ray structure of a tetranucleosome and its implications for the chromatin fibre.Nature. 2005; 436: 138-141Crossref PubMed Scopus (563) Google Scholar). Genomic studies have confirmed the prevalence of tetranucleosome contacts in vivo (Hsieh et al., 2015Hsieh T.H. Weiner A. Lajoie B. Dekker J. Friedman N. Rando O.J. Mapping nucleosome resolution chromosome folding in yeast by micro-C.Cell. 2015; 162: 108-119Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, Risca et al., 2017Risca V.I. Denny S.K. Straight A.F. Greenleaf W.J. Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping.Nature. 2017; 541: 237-241Crossref PubMed Scopus (83) Google Scholar). Neighboring tetranucleosome units can interact and form fiber segments with two intertwined stacks of nucleosomes (Li et al., 2016Li W. Chen P. Yu J. Dong L. Liang D. Feng J. Yan J. Wang P.Y. Li Q. Zhang Z. et al.FACT remodels the tetranucleosomal unit of chromatin fibers for gene transcription.Mol. Cell. 2016; 64: 120-133Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, Schalch et al., 2005Schalch T. Duda S. Sargent D.F. Richmond T.J. X-ray structure of a tetranucleosome and its implications for the chromatin fibre.Nature. 2005; 436: 138-141Crossref PubMed Scopus (563) Google Scholar, Song et al., 2014Song F. Chen P. Sun D. Wang M. Dong L. Liang D. Xu R.M. Zhu P. Li G. Cryo-EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units.Science. 2014; 344: 376-380Crossref PubMed Scopus (347) Google Scholar). It is not well understood how pTFs search DNA sequences within such compact chromatin and how they invade and subsequently remodel chromatin structure. The intrinsic dynamics within chromatin fibers might provide a potential mechanism for pTF invasion (Cuvier and Fierz, 2017Cuvier O. Fierz B. Dynamic chromatin technologies: from individual molecules to epigenomic regulation in cells.Nat. Rev. Genet. 2017; 18: 457-472Crossref PubMed Scopus (38) Google Scholar). Recent studies using force spectroscopy (Li et al., 2016Li W. Chen P. Yu J. Dong L. Liang D. Feng J. Yan J. Wang P.Y. Li Q. Zhang Z. et al.FACT remodels the tetranucleosomal unit of chromatin fibers for gene transcription.Mol. Cell. 2016; 64: 120-133Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) or single-molecule Förster resonance energy transfer (FRET) (Kilic et al., 2018bKilic S. Felekyan S. Doroshenko O. Boichenko I. Dimura M. Vardanyan H. Bryan L.C. Arya G. Seidel C.A.M. Fierz B. Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α.Nat. Commun. 2018; 9: 235Crossref PubMed Scopus (53) Google Scholar) revealed conformational dynamics in chromatin fibers from microseconds to seconds. It is thus conceivable that pTFs exploit fiber dynamics to invade compact chromatin, where they then recruit additional cellular machinery to enact necessary conformational reorganization to alter gene expression (Figure 1A). Here, we test this hypothesis and reveal the mechanism of chromatin invasion, target binding, and chromatin remodeling of the pTF Rap1 (repressor activator protein 1). Rap1 is a general regulatory factor (GRF) of transcription in budding yeast (Knight et al., 2014Knight B. Kubik S. Ghosh B. Bruzzone M.J. Geertz M. Martin V. Dénervaud N. Jacquet P. Ozkan B. Rougemont J. et al.Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.Genes Dev. 2014; 28: 1695-1709Crossref PubMed Scopus (68) Google Scholar). It has multiple roles, including the transcriptional regulation of around 5% of yeast genes (Lieb et al., 2001Lieb J.D. Liu X. Botstein D. Brown P.O. Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association.Nat. Genet. 2001; 28: 327-334Crossref PubMed Scopus (537) Google Scholar), repression of noncoding transcripts (Challal et al., 2018Challal D. Barucco M. Kubik S. Feuerbach F. Candelli T. Geoffroy H. Benaksas C. Shore D. Libri D. General regulatory factors control the fidelity of transcription by restricting non-coding and ectopic initiation.Mol. Cell. 2018; 72: 955-969.e7Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, Wu et al., 2018Wu A.C.K. Patel H. Chia M. Moretto F. Frith D. Snijders A.P. van Werven F.J. Repression of divergent noncoding transcription by a sequence-specific transcription factor.Mol. Cell. 2018; 72: 942-954.e7Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), and the maintenance of telomeric integrity (Wellinger and Zakian, 2012Wellinger R.J. Zakian V.A. Everything you ever wanted to know about Saccharomyces cerevisiae telomeres: beginning to end.Genetics. 2012; 191: 1073-1105Crossref PubMed Scopus (204) Google Scholar). The Rap1 DNA binding domain (DBD) consists of dual Myb-type domains connected by a short unstructured linker (König et al., 1998König P. Fairall L. Rhodes D. Sequence-specific DNA recognition by the myb-like domain of the human telomere binding protein TRF1: a model for the protein-DNA complex.Nucleic Acids Res. 1998; 26: 1731-1740Crossref PubMed Scopus (71) Google Scholar; Figure 1B). The DBD binds a 13-bp consensus motif with high affinity (Figure S1A), only requiring direct access to one face of the DNA (Figure 1B). Rap1 can engage a single motif in multiple binding modes, involving one or both Myb domains (Feldmann and Galletto, 2014Feldmann E.A. Galletto R. The DNA-binding domain of yeast Rap1 interacts with double-stranded DNA in multiple binding modes.Biochemistry. 2014; 53: 7471-7483Crossref PubMed Scopus (10) Google Scholar), and previous in vitro studies showed that Rap1 can bind nucleosomes (Rossetti et al., 2001Rossetti L. Cacchione S. De Menna A. Chapman L. Rhodes D. Savino M. Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites.J. Mol. Biol. 2001; 306: 903-913Crossref PubMed Scopus (29) Google Scholar). In the cell, Rap1 target sites are located within nucleosome-depleted regions (NDR) upstream of the transcription start site (TSS) or within the −1 nucleosome at the two most peripheral exposed DNA major grooves (Koerber et al., 2009Koerber R.T. Rhee H.S. Jiang C. Pugh B.F. Interaction of transcriptional regulators with specific nucleosomes across the Saccharomyces genome.Mol. Cell. 2009; 35: 889-902Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). A host of cell-based studies showed that Rap1 binding at these loci results in chromatin opening (Yu and Morse, 1999Yu L. Morse R.H. Chromatin opening and transactivator potentiation by RAP1 in Saccharomyces cerevisiae.Mol. Cell. Biol. 1999; 19: 5279-5288Crossref PubMed Scopus (87) Google Scholar), nucleosome loss, and NDR formation (Badis et al., 2008Badis G. Chan E.T. van Bakel H. Pena-Castillo L. Tillo D. Tsui K. Carlson C.D. Gossett A.J. Hasinoff M.J. Warren C.L. et al.A library of yeast transcription factor motifs reveals a widespread function for Rsc3 in targeting nucleosome exclusion at promoters.Mol. Cell. 2008; 32: 878-887Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, Kubik et al., 2015Kubik S. Bruzzone M.J. Jacquet P. Falcone J.L. Rougemont J. Shore D. Nucleosome stability distinguishes two different promoter types at all protein-coding genes in yeast.Mol. Cell. 2015; 60: 422-434Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, van Bakel et al., 2013van Bakel H. Tsui K. Gebbia M. Mnaimneh S. Hughes T.R. Nislow C. A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription.PLoS Genet. 2013; 9: e1003479Crossref PubMed Scopus (76) Google Scholar, Yan et al., 2018Yan C. Chen H. Bai L. Systematic study of nucleosome-displacing factors in budding yeast.Mol. Cell. 2018; 71: 294-305.e4Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In fact, NDRs are typical for most active eukaryotic promoters (Jiang and Pugh, 2009Jiang C. Pugh B.F. Nucleosome positioning and gene regulation: advances through genomics.Nat. Rev. Genet. 2009; 10: 161-172Crossref PubMed Scopus (704) Google Scholar) and depend on the action of remodeling factors, including RSC (Badis et al., 2008Badis G. Chan E.T. van Bakel H. Pena-Castillo L. Tillo D. Tsui K. Carlson C.D. Gossett A.J. Hasinoff M.J. Warren C.L. et al.A library of yeast transcription factor motifs reveals a widespread function for Rsc3 in targeting nucleosome exclusion at promoters.Mol. Cell. 2008; 32: 878-887Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, Brahma and Henikoff, 2019Brahma S. Henikoff S. RSC-associated subnucleosomes define MNase-sensitive promoters in yeast.Mol. Cell. 2019; 73: 238-249.e3Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Cairns et al., 1996Cairns B.R. Lorch Y. Li Y. Zhang M. Lacomis L. Erdjument-Bromage H. Tempst P. Du J. Laurent B. Kornberg R.D. RSC, an essential, abundant chromatin-remodeling complex.Cell. 1996; 87: 1249-1260Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar, Hartley and Madhani, 2009Hartley P.D. Madhani H.D. Mechanisms that specify promoter nucleosome location and identity.Cell. 2009; 137: 445-458Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, Kubik et al., 2018Kubik S. O’Duibhir E. de Jonge W.J. Mattarocci S. Albert B. Falcone J.L. Bruzzone M.J. Holstege F.C.P. Shore D. Sequence-directed action of RSC remodeler and general regulatory factors modulates +1 nucleosome position to facilitate transcription.Mol. Cell. 2018; 71: 89-102.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Kubik et al., 2019Kubik S. Bruzzone M.J. Challal D. Dreos R. Mattarocci S. Bucher P. Libri D. Shore D. Opposing chromatin remodelers control transcription initiation frequency and start site selection.Nat. Struct. Mol. Biol. 2019; 26: 744-754Crossref PubMed Scopus (25) Google Scholar, Ng et al., 2002Ng H.H. Robert F. Young R.A. Struhl K. Genome-wide location and regulated recruitment of the RSC nucleosome-remodeling complex.Genes Dev. 2002; 16: 806-819Crossref PubMed Scopus (215) Google Scholar, Parnell et al., 2008Parnell T.J. Huff J.T. Cairns B.R. RSC regulates nucleosome positioning at Pol II genes and density at Pol III genes.EMBO J. 2008; 27: 100-110Crossref PubMed Scopus (132) Google Scholar), SWI/SNF (Rawal et al., 2018Rawal Y. Chereji R.V. Qiu H. Ananthakrishnan S. Govind C.K. Clark D.J. Hinnebusch A.G. SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast.Genes Dev. 2018; 32: 695-710Crossref PubMed Scopus (26) Google Scholar, Yen et al., 2012Yen K. Vinayachandran V. Batta K. Koerber R.T. Pugh B.F. Genome-wide nucleosome specificity and directionality of chromatin remodelers.Cell. 2012; 149: 1461-1473Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), and INO80 (Krietenstein et al., 2016Krietenstein N. Wal M. Watanabe S. Park B. Peterson C.L. Pugh B.F. Korber P. Genomic nucleosome organization reconstituted with pure proteins.Cell. 2016; 167: 709-721.e12Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). An important gene family co-regulated by Rap1 is ribosomal protein genes. Rap1 binds to the promoter/enhancer regions of >90% of these genes and initiates the recruitment of additional TFs, including Hmo1, Fhl1, and Ifh1 (Knight et al., 2014Knight B. Kubik S. Ghosh B. Bruzzone M.J. Geertz M. Martin V. Dénervaud N. Jacquet P. Ozkan B. Rougemont J. et al.Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.Genes Dev. 2014; 28: 1695-1709Crossref PubMed Scopus (68) Google Scholar). In one of the two largest categories of ribosomal protein genes (category I), two closely spaced Rap1 binding sites are situated in the NDR upstream of the TSS (Knight et al., 2014Knight B. Kubik S. Ghosh B. Bruzzone M.J. Geertz M. Martin V. Dénervaud N. Jacquet P. Ozkan B. Rougemont J. et al.Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.Genes Dev. 2014; 28: 1695-1709Crossref PubMed Scopus (68) Google Scholar). When Rap1 is depleted, its binding sites are covered by a stable nucleosome (Kubik et al., 2015Kubik S. Bruzzone M.J. Jacquet P. Falcone J.L. Rougemont J. Shore D. Nucleosome stability distinguishes two different promoter types at all protein-coding genes in yeast.Mol. Cell. 2015; 60: 422-434Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Digestion of yeast chromatin with limited amounts of micrococcal nuclease (MNase) followed by sequencing (MNase-seq) (Zentner and Henikoff, 2012Zentner G.E. Henikoff S. Surveying the epigenomic landscape, one base at a time.Genome Biol. 2012; 13: 250Crossref PubMed Scopus (31) Google Scholar) revealed that many NDRs contain MNase-sensitive particles (Henikoff et al., 2011Henikoff J.G. Belsky J.A. Krassovsky K. MacAlpine D.M. Henikoff S. Epigenome characterization at single base-pair resolution.Proc. Natl. Acad. Sci. USA. 2011; 108: 18318-18323Crossref PubMed Scopus (205) Google Scholar, Kent et al., 2011Kent N.A. Adams S. Moorhouse A. Paszkiewicz K. Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing.Nucleic Acids Res. 2011; 39: e26Crossref PubMed Scopus (78) Google Scholar, Weiner et al., 2010Weiner A. Hughes A. Yassour M. Rando O.J. Friedman N. High-resolution nucleosome mapping reveals transcription-dependent promoter packaging.Genome Res. 2010; 20: 90-100Crossref PubMed Scopus (269) Google Scholar, Xi et al., 2011Xi Y. Yao J. Chen R. Li W. He X. Nucleosome fragility reveals novel functional states of chromatin and poises genes for activation.Genome Res. 2011; 21: 718-724Crossref PubMed Scopus (85) Google Scholar), which may correspond to destabilized promoter nucleosomes (Brahma and Henikoff, 2019Brahma S. Henikoff S. RSC-associated subnucleosomes define MNase-sensitive promoters in yeast.Mol. Cell. 2019; 73: 238-249.e3Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, Chereji et al., 2017Chereji R.V. Ocampo J. Clark D.J. MNase-sensitive complexes in yeast: nucleosomes and non-histone barriers.Mol. Cell. 2017; 65: 565-577.e3Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, Kubik et al., 2015Kubik S. Bruzzone M.J. Jacquet P. Falcone J.L. Rougemont J. Shore D. Nucleosome stability distinguishes two different promoter types at all protein-coding genes in yeast.Mol. Cell. 2015; 60: 422-434Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, Kubik et al., 2017Kubik S. Bruzzone M.J. Albert B. Shore D. A reply to “MNase-sensitive complexes in yeast: nucleosomes and non-histone barriers,” by Chereji et al.Mol. Cell. 2017; 65: 578-580Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, Kubik et al., 2018Kubik S. O’Duibhir E. de Jonge W.J. Mattarocci S. Albert B. Falcone J.L. Bruzzone M.J. Holstege F.C.P. Shore D. Sequence-directed action of RSC remodeler and general regulatory factors modulates +1 nucleosome position to facilitate transcription.Mol. Cell. 2018; 71: 89-102.e5Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In category I promoters, such MNase-sensitive nucleosome-like particles appear upstream of the +1 nucleosome, co-existing with bound Rap1 (Kubik et al., 2015Kubik S. Bruzzone M.J. Jacquet P. Falcone J.L. Rougemont J. Shore D. Nucleosome stability distinguishes two different promoter types at all protein-coding genes in yeast.Mol. Cell. 2015; 60: 422-434Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Taken together, Rap1 is thus a well-characterized factor that directly impacts chromatin organization at key genes. However, the molecular mechanism by which Rap1 finds its target in compacted chromatin and how it subsequently opens chromatin and destabilizes or displaces promoter nucleosomes is not understood. To reveal dynamic Rap1 invasion mechanisms, we reconstituted nucleosomes and chromatin fibers containing Rap1 binding sites in the configuration found in category I promoters. We find that residence times, but not binding rates, of Rap1 are strongly reduced by the presence of nucleosomes and chromatin fibers. We show that Rap1 binding alone does not disrupt or decidedly alter nucleosome conformation. In contrast, single-molecule FRET measurements reveal that Rap1 locally opens chromatin fiber structure. Finally, we demonstrate that Rap1 collaborates with RSC to displace nucleosomes from its target sites. The remodeled chromatin structure then provides an opening for stable Rap1 binding, access to further transcription factors, and finally gene regulation. To investigate the mechanism of Rap1 nucleosome binding, we chose the ribosomal protein L30 (RPL30) promoter (category I) as our model system (Figure 1C). We mapped the position of the −1 nucleosome, which contains two Rap1 binding sites and is displaced in vivo upon Rap1 binding by MNase-seq under Rap1-depleted conditions (Figure 1C; Kubik et al., 2015Kubik S. Bruzzone M.J. Jacquet P. Falcone J.L. Rougemont J. Shore D. Nucleosome stability distinguishes two different promoter types at all protein-coding genes in yeast.Mol. Cell. 2015; 60: 422-434Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Within this nucleosome, the Rap1 binding site 1 (S1) is located near super helical location (SHL) 4.5, whereas site 2 (S2) resides near the DNA entry-exit site at SHL 6.5 (Figure 1D). Importantly, Rap1 exhibits different affinities for the two sites with a dissociation constant KD of ∼10 nM for S1 and ∼30 nM for S2 (as determined by electromobility shift assays [EMSAs]; Figures S1C–S1E). In vivo, both sites contribute to the expression of the RPL30 gene product (Knight et al., 2014Knight B. Kubik S. Ghosh B. Bruzzone M.J. Geertz M. Martin V. Dénervaud N. Jacquet P. Ozkan B. Rougemont J. et al.Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.Genes Dev. 2014; 28: 1695-1709Crossref PubMed Scopus (68) Google Scholar). We then implemented a single-molecule total internal reflection fluorescence microscopy approach (smTIRFM) to directly observe dynamic Rap1 binding to promoter nucleosomes via fluorescence colocalization (Figure 2A; Kilic et al., 2015Kilic S. Bachmann A.L. Bryan L.C. Fierz B. Multivalency governs HP1α association dynamics with the silent chromatin state.Nat. Commun. 2015; 6: 7313Crossref PubMed Scopus (48) Google Scholar). We first generated a 235-bp DNA template based on the 601 nucleosome positioning sequence (Lowary and Widom, 1998Lowary P.T. Widom J. New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.J. Mol. Biol. 1998; 276: 19-42Crossref PubMed Scopus (1077) Google Scholar), which contained one or both Rap1 binding sites, S1 or S2, at the same position as in the native −1 promoter nucleosome (Figures 1D and S1B; Tables S1, S2, and S3). Moreover, the DNA constructs contained a far-red fluorescent dye (Alexa Fluor 647) and a biotin moiety for immobilization. We then used this DNA directly for measurements or reconstituted nucleosomes using recombinantly expressed histones (Figures 2A and S2A–S2E). Second, we purified full-length Rap1 as a Halo-tag fusion from insect cells and fluorescently labeled the protein with the highly photostable green-orange dye JF-549 (Grimm et al., 2015Grimm J.B. English B.P. Chen J. Slaughter J.P. Zhang Z. Revyakin A. Patel R. Macklin J.J. Normanno D. Singer R.H. et al.A general method to improve fluorophores for live-cell and single-molecule microscopy.Nat. Methods. 2015; 12: 244-250Crossref PubMed Scopus (654) Google Scholar; Figures 2B and S2F–S2K). Labeled Rap1 exhibited similar DNA binding compared to published values (Knight et al., 2014Knight B. Kubik S. Ghosh B. Bruzzone M.J. Geertz M. Martin V. Dénervaud N. Jacquet P. Ozkan B. Rougemont J. et al.Two distinct promoter architectures centered on dynamic nucleosomes control ribosomal protein gene transcription.Genes Dev. 2014; 28: 1695-1709Crossref PubMed Scopus (68) Google Scholar; Figures S1C–S1E). Having all components in hand, in a first set of experiments, we immobilized S1- or S2-containing naked DNA strands in a microfluidic channel and determined their position by smTIRFM imaging in the far-red channel (Figure 2C). We then injected Rap1 at a concentration chosen such that individual, non-overlapping binding events could be detected as fluorescent spots in the green-orange channel (usually 50–100 pM). Colocalization of Rap1 with DNA positions indicated binding (Figure 2C). We then recorded movies that revealed the binding kinetics of Rap1 to S1- or S2-containing naked DNA. For each DNA molecule, extracted kinetic traces allowed us to determine the length of individual binding events (tbright) and intermittent search times (tdark). The effect of dye photobleaching on residence time measurements was reduced by stroboscopic imaging (Figure S3A). Although dynamic Rap1 binding was observed for the medium affinity site S2 (Figure 2D), individual binding events to the high-affinity site S1 were so long (>40 min) that we were not able to obtain suitable statistics (Figure S3B). For S2-containing DNA, we constructed cumulative lifetime histograms of bright times (tbright) (Figure 2E), which were fitted using a bi-exponential function, yielding two residence times τoff,1 and τoff,2 (Figure 2F; see Table S4 for all rate constants). Of all binding events, 35% exhibited a short residence time (τoff,1 = 12.4 ± 4.5 s), whereas the remaining 65% showed slow Rap1 dissociation kinetics (τoff,2 = 452 ± 115 s). Due to the dual Myb-type DBD, these different residence times may indicate different binding modes where either the whole or only a partial DNA binding motif is engaged. Under equilibrium binding conditions, Rap1 thus forms long-lived complexes with free DNA, resulting in residence times in the minutes to hours range for S1 and S2. In contrast, the presence of mononucleosomes (MNs) shortened the residence time of Rap1, as observed in kinetic traces for MNs containing either S1 or S2 (Figure 2G) and in the corresponding lifetime histograms (Figure 2H). Here, a tri-exponential function was required to describe the data (Figures S3C–S3E). Around 50% of all detected events were short lived, with a time constant of 0.2 < τoff,0 < 0.7 s. We attribute these fast events to nonspecific interactions of Rap1 with the nucleosomal DNA. Specific Rap1 binding to S1 or S2 further resulted in two longer time constants τoff,1 and τoff,2. Rap1 binding to the high-affinity site S1 was associated with longer residence times (τoff,1 = 18 ± 11 s and τoff,2 > 100 s) compared to S2 (τoff,1 = 8.4 ± 1.4 s; τoff,2 = 46 ± 3" @default.
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• W2990228889 title "Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor" @default.
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