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Molecular Mechanisms of Ribosomal Protein Gene Coregulation

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Molecular Mechanisms of Ribosomal Protein Gene Coregulation Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Molecular mechanisms of ribosomal protein gene coregulation Rohit Reja, Vinesh Vinayachandran, Sujana Ghosh, and B. Franklin Pugh Center for Eukaryotic Gene Regulation, Pennsylvania State University, University Park, Pennsylvania 16802, USA The 137 ribosomal protein genes (RPGs) of Saccharomyces provide a model for gene coregulation. We examined the positional and functional organization of their regulators (Rap1 [repressor activator protein 1], Fhl1, Ifh1, Sfp1, and Hmo1), the transcription machinery (TFIIB, TFIID, and RNA polymerase II), and chromatin at near-base-pair res- olution using ChIP-exo, as RPGs are coordinately reprogrammed. Where Hmo1 is enriched, Fhl1, Ifh1, Sfp1, and Hmo1 cross-linked broadly to promoter DNA in an RPG-specific manner and demarcated by general minor groove widening. Importantly, Hmo1 extended 20–50 base pairs (bp) downstream from Fhl1. Upon RPG repression, Fhl1 remained in place. Hmo1 dissociated, which was coupled to an upstream shift of the +1 nucleosome, as reflected by the Hmo1 extension and core promoter region. Fhl1 and Hmo1 may create two regulatable and positionally distinct barriers, against which chromatin remodelers position the +1 nucleosome into either an activating or a repressive state. Consistent with in vitro studies, we found that specific TFIID subunits, in addition to cross-linking at the core promoter, made precise cross-links at Rap1 sites, which we interpret to reflect native Rap1–TFIID interactions. Our findings suggest how sequence-specific DNA binding regulates nucleosome positioning and transcription complex assembly >300 bp away and how coregulation coevolved with coding sequences. [Keywords: gene regulation mechanisms; ChIP-seq; chromatin; transcription factor] Supplemental material is available for this article. Received July 16, 2015; revised version accepted August 24, 2015. Saccharomyces contains 78 distinct ribosomal proteins regulatory mechanisms via either drift or selection for (RPs) encoded by 137 genes (RPGs): 19 single genes and reasons unrelated to coding sequence (Tanay et al. 59 paralogous gene pairs. Gene pairs arose from whole-ge- 2005; Wapinski et al. 2010). It remains unclear to what nome duplication ∼100 million years ago (Wolfe and extent coregulated or even paralogous genes have sim- Shields 1997). RPGs are the most highly and coordinately ilar mechanisms of regulation and transcription factor expressed genes in the cell (Li et al. 1999; Warner 1999) organization. and thus are ideal for understanding mechanisms of Several transcription factors are associated with RPG coregulation. Their expression is coordinately activated regulation (Jorgensen et al. 2004; Marion et al. 2004; Mar- by nutrient and growth sensing and is rapidly repressed tin et al. 2004; Schawalder et al. 2004; Wade et al. 2004; upon stress or starvation (Gasch et al. 2000). Rudra et al. 2005; Hall et al. 2006; Zhao et al. 2006; Joo The tight coregulation of RPGs raises the simple expec- et al. 2011; Knight et al. 2014). Of these, Rap1 (repressor tation that the structural organization of transcription activator protein 1) is the clearest sequence-specific factors and the mechanism of regulation is essentially DNA-binding protein (Shore 1994), binding ∼90% of the same for all RPGs. However, this has not been demon- RPG promoters (Warner 1999; Lieb et al. 2001). Rap1- strated for RPGs or any other set of coregulated genes be- binding sites at RPGs predominantly occur in pairs, 5– yond the presence of a common set of regulatory factors. 15 base pairs (bp) apart, and with preference for one orien- Differences in RPG mRNA and protein turnover, protein tation (Kurtz and Shore 1991; Bosio et al. 2011; Knight stoichiometry, gene copy number, and potential non- et al. 2014). redundancy of some paralogs may create gene-specific Rap1 alters chromatin structure (Yu and Morse 1999) constraints on regulation that are encoded within individ- and recruits Fhl1, Ifh1, Sfp1 (hereafter FIS), and Hmo1 spe- ual RPG promoter sequences (Zeevi et al. 2011). As such, cifically to RPG promoters (Wade et al. 2004; Rudra et al. they may be distinctly regulated. Even paralogous genes 2005; Hall et al. 2006; Zhao et al. 2006). Fhl1 binding also that produce identical proteins may have diverged their depends on Hmo1 but only where Hmo1 is normally bound (Hall et al. 2006; Kasahara et al. 2011); there may Corresponding author: [email protected] Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.268896. © 2015 Reja et al. This article, published in Genes & Development,is 115. Freely available online through the Genes & Development Open ac- available under a Creative Commons License (Attribution 4.0 Internation- cess option. al), as described at http://creativecommons.org/licenses/by/4.0/. 1942 GENES & DEVELOPMENT 29:1942–1954 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org Downloaded from genesdev.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Genomic RPG regulation mechanisms also be Hmo1-independent Fhl1 recruitment mechanisms ies have defined the approximate promoter locations of (Zeevi et al. 2011; Knight et al. 2014), which needs to be RPG-specific factors but have resolution limits of ∼50– reconciled. Fhl1 interacts with Ifh1 to activate RPG tran- 100 bp. Here we used the high-resolution ChIP-exo assay scription, which is stimulated by the TOR and PKA sig- (Rhee and Pugh 2011) to map the precise organization of naling pathways, Sirtuins, and Sfp1 (Jorgensen et al. RPG-specific factors, PIC components, and chromatin at 2004; Marion et al. 2004; Martin et al. 2004; Schawalder et all RPGs. In the context of well-established signaling al. 2004; Wade et al. 2004; Rudra et al. 2005; Hall et al. mechanisms that turn RPG expression on and off and 2006; Zhao et al. 2006; Berger et al. 2007; Xiao et al. the known factors that bind RPGs, we investigated the 2011; Downey et al. 2013). The binding of Sfp1 to RPG molecular mechanisms of RPG coregulation. We con- promoters has been questionable (Jorgensen et al. 2004; firmed many prior observations associated with RPG pro- Marion et al. 2004; Kasahara et al. 2007). moter organization, except for the role of nucleosomes. A Stress and nutrient starvation repress RPGs by dissoci- major challenge for us was the spatial deconvolution of in- ating all but Rap1 and Fhl1 (Wade et al. 2004; Rudra dividual subunits of complexes that have multiple points et al. 2005). To this end, Crf1 binds Fhl1 to displace Ifh1 of cross-linking to DNA and can cross-link indirectly via (Cherel and Thuriaux 1995; Mencia et al. 2002; Martin other subunits. Our findings suggest that, in addition to et al. 2004). Ifh1 is further inhibited when acetylated by the classical activator–PIC structure (Rap1/TFIID), a sec- SAGA/Gcn5 (Downey et al. 2013), and Sfp1 translocates ond Rap1 molecule uses RPG-specific factors as regulat- to the cytoplasm (Jorgensen et al. 2004; Marion et al. able rulers that precisely set the +1 nucleosome position 2004). Rap1 then acts as a repressor instead of an activator, via chromatin remodelers into active and repressive posi- but its mechanism remains unclear. tions at the transcriptional start site (TSS). RPG activation by Rap1 involves recruitment of the TFIIA and TFIID components of the transcription preini- tiation complex (PIC) (Mencia et al. 2002; Garbett et al. Results 2007; Papai et al. 2010). In vitro, Rap1 makes specific func- RPG-specific factors have a well-defined tional contacts with the TBP-associated factor 4 (Taf4), spatial organization Taf5, and Taf12 subunits of TFIID (Layer et al. 2010) and perhaps Taf1 and Taf2 (Ohtsuki et al. 2010; Papai et al. The 5′ ends of ChIP-exo sequencing tags correspond to 2010), but whether these contacts occur in vivo at native strand-specific exonuclease stop sites on cross-linked RPGs has not been established. Hmo1 is important for DNA molecules, as exemplified for the RPS11B and TFIID binding to RPGs (Kasahara et al. 2008). Given the RPL35A genes (Fig. 1A). Each factor location was repre- 200- to 400-bp distance between Rap1-binding sites and sented by a complex set of peaks on both strands that tend- the core promoter, the intervening DNA might be looped ed to be offset from each other by a short distance in the 3′ out, as has been visualized by electron microscopy (Papai direction (reflecting the exonuclease “headroom”). Fhl1, et al. 2010). This loop may be filled with RPG-specific reg- Ifh1, Sfp1, and Hmo1 each displayed multiple deduced ulators and/or nucleosomes, with the latter reportedly cross-linking points spread over ∼200 bp of DNA despite regulating PIC assembly (Zeevi et al. 2011; Knight et al. the potential resolution of the assay being a few base 2014). pair. The entire regulated PIC, from Rap1 to Sua7 (TFIIB) With the same set of transcription factors being associ- in the core transcription machinery, encompassed up to ated with RPGs, it comes as a surprise that they lack a 400 bp of DNA. consistent repertoire of well-defined cis-regulatory ele- The strongest positional reinforcement of RPG-specific ments beyond Rap1 sites. Nonetheless, these other ele- factor patterning across all RPGs occurred when aligned ments may contribute to RPG regulation (Zeevi et al. by their Rap1 sites rather than their TSSs (Fig. 1B, left 2011). They include poly(dA:dT) tracts, IFHL motifs vs. right graphs). Upstream of Rap1, nucleosomes were po- (Wade et al. 2004; Bosio et al. 2011), and Fhl1 motifs (Badis sitionally well organized with respect to Rap1 sites. In et al. 2008). In addition, Sfp1 has been linked to specific contrast to RPG-specific factors, the core PIC (represented sites such as AAAAWTTTT (IUPAC) (Zhu et al. 2009; by Sua7/TFIIB) was more positionally linked to the TSS Zeevi et al.
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