Transcription of the 138 RP genes takes place through the action of five TFs, namely Rap1, Hmo1, Fhl1, Ifh1 and Sfp1. In the last years, numerous efforts have been focused on the genome-wide
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analysis of the binding of these factors. Recently, our and Pugh’s labs produced genome-wide ChIP-Seq data for Rap1, Hmo1, Ifh1 and Fhl1, providing a complete landscape of the in vivo binding of these factors (Knight et al., 2014; Reja et al., 2015). Though the results of our analysis will be extensively presented in the Results section of this thesis, here we are going to mention few important features of RP gene transcription revealed by our work and to describe the TFs that bind RP gene promoters.
The 138 yeast RP gene promoters can be classified in two main types (here after referred as categories) with respect to the presence and organization of the different TFs. The presence of Hmo1 defines the first category with respect to the second one; indeed, category I RP gene promoters are characterized by the strong binding of Hmo1 around 200 bp upstream of the ATG, as well as Fhl1, Ifh1 and Rap1 binding. Contrary, Hmo1 does not bind to category II RP genes whose transcription is driven by Fhl1, Ifh1 and Rap1. The vast majority of RP genes are included in category I and category II. Lastly, on a small number of promoters, category III, ChIP-seq analysis did not evidence the presence of anyone of the four transcription factors. However, recent ChIP experiments revealed the binding of another TF, Abf1, on category III promoters. Furthermore, RP gene promoters are characterized by the presence of one or more FNs in the previously defined NDR. Interestingly, category I promoters are characterized by the presence of two (or three) FNs while at category II promoters a single FN was detected (Knight et al., 2014) (Figure 19).
Figure 19. Schematic representation of RP gene promoter architectures (Knight et al., 2014).
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Rap1, the pioneer transcription factor that drives RP gene transcription
As already mentioned in this Introduction, Rap1 is considered one of a small group of relatively abundant and essential yeast transcription factors (including Abf1, Reb1, Tbf1 and Cbf1) referred to as GRFs or PTFs (Buchman et al., 1988; Yarragudi et al., 2004) and involved in positioning nucleosomes at promoters. Rap1 (Repressor Activator Protein 1) was discovered more than thirty years ago as “multi-functional” DNA binding protein involved in either transcription activation (RP and glycolytic genes) or repression (mating type loci), chromatin silencing, and telomere length maintenance (Shore, 1994; Shore and Nasmyth, 1987). Rap1 protein contains a BRCT (BRCA1 C Terminus) N-terminal domain of unknown function, a central Myb DNA binding domain and a C-terminal RCT domain. A fascinating and still partially unsolved question is how Rap1 works in different genomic contexts (telomeres, active promoters, repressed promoters) promoting both transcriptional activation and repression. Though, Rap1 role in nucleosome positioning has been described long time ago by the Morse group (Ganapathi et al., 2011; Yarragudi et al., 2004; Yarragudi et al., 2007) and then by others (Koerber et al., 2009; Lickwar et al., 2012), just recent works elucidate its contribution to chromatin architecture and transcription of RP genes. These works will be described in the Results section of this thesis. Furthermore, Rap1 promotes transcription also through a different mechanism. Indeed, some works demonstrated that Rap1 directly interacts with TFIID and TFIIA, promoting RNAPII recruitment (Garbett et al., 2007; Layer et al., 2010; Layer and Weil, 2013; Papai et al., 2010).
Hmo1, a link between rRNA and RP gene transcription
Hmo1 is one of the seven HMGB (High Mobility Group B) proteins in yeast. HMGB are non-histone proteins that interact with chromatin and contain one or more HMG structural motifs that bind to the minor groove of DNA (Lu et al., 1996). Hmo1 is prevalently observed as homodimer and contains
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two HMG-box domains, generally referred as boxA and boxB (Gadal et al., 2002). Though it is a non-essential protein, Hmo1-Δ null strains are characterized by a slow growth phenotype. Hmo1 binds the promoters of many different genes including a subset of RP genes (category I RP genes) and rDNA loci. Data from our lab revealed that -1 nucleosome position at RP genes overlaps perfectly with Hmo1 binding site (Knight et al., 2014), highlighting a possible competition between Hmo1 and the nucleosome and suggesting a role of Hmo1 in nucleosome exclusion (Kasahara et al., 2011).
Hmo1 binds the promoter of many non-RP genes; curiously among these there are Hmo1 itself and Utp22. Utp22 is a protein involved in rRNA processing (Peng et al., 2003) and a component of the CURI complex (see below) (Rudra et al., 2005). The binding of Hmo1 on Utp22 promoter and on rDNA locus discloses the role of Hmo1 in the regulation of rRNA transcription and processing and highlights its possible function of linker between RNAPII RP gene and RNAPI rRNA transcription. The role of Hmo1 in coupling two fundamental aspects of ribosome biogenesis is also sustained by some works that propos that Hmo1 activity is regulated by TORC1 (Berger et al., 2007; Dolinski and Heitman, 1999).
Fhl1 and Ifh1 pair is a marker of RP genes
Fhl1 (Forkhead-like 1) is a non-essential DNA binding protein that specifically binds the promoters of almost all RP genes. Indeed, ChIP-seq data revealed the presence of Fhl1 on few non-RP gene promoters (Knight et al., 2014). Inhibition of TORC1 by rapamycin or starvation does not affect Fhl1 binding on DNA, that it is thus stable and constitutive (Schawalder et al., 2004). The main role of Fhl1 is the recruitment of Ifh1 on RP gene promoters. This recruitment involves the interaction between the “forkhead-associated” (FHA) domain of Fhl1 and the “forkhead-associated binding” (FHB) domain of Ifh1 (Rudra et al., 2005).
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Ifh1 (Interacts with forkhead 1), contrary to Fhl1, is a TF essential for yeast cell viability. The essential region of Ifh1 protein is the FHB domain, nevertheless the C-terminal transcriptional activation domain and the acidic N-terminal region are important for the normal growth of the cell.
Furthermore, a work from Mallick and Whiteway identified a central region of Ifh1 protein important for interaction with Rap1 BRCT domain (Mallick and Whiteway, 2013). As Ifh1 deletion is lethal for yeast cell, the phenotype of several Ifh1 mutants has been deeply investigated. Importantly, data from our and other labs revealed the importance two phosphorylated residues in the FHB domain, Ser680 and Thre681, for interaction with Fhl1 (Albert et al., 2016; Cai et al., 2013). Ifh1 interaction with Fhl1 and, thus, Ifh1 recruitment to RP gene promoters, disappears rapidly following nutrient depletion, diauxic shift or rapamycin treatment (Schawalder et al., 2004). These observations suggest that TORC1 regulates Ifh1 activity, probably mediating its phosphorylation state via Sch9;
nevertheless, the molecular details of this regulation are still elusive. Importantly, It has been shown that Ifh1 binding on RP gene promoters correlates with RP gene activation, and forced over-expression of Ifh1 leads to up-regulation of RP gene transcription (Schawalder et al., 2004) (Figure 20). However, the mechanism by which Ifh1 regulates RP gene transcription is still elusive and will be matter of investigation of this thesis.
Figure 20. (A) Rapamycin treatment affects Ifh1 binding to RP promoters and impairs RP transcription. (B) Ifh1 over-expression leads to RP gene transcription up-regulation (Schawalder et al.,
2004).
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Lastly, Ifh1 is part of a protein complex named “CURI” (CK2, Utp22, Rrp7 and Ifh1) where it associates with three other proteins, Utp22, Rrp7 and CK2 (Casein Kinase 2) (Rudra et al., 2005). Utp22 and Rrp7 are two essential proteins involved in rRNA processing and interestingly, they interact with CK2 in an Ifh1-independent sub-complex having a role in 18S rRNA processing (Rudra et al., 2007). The association of Ifh1 with Utp22, Rrp7 and CK2 in the CURI complex highlights a possible function of Ifh1 in coupling rRNA and RP gene transcription. The role of Ifh1 in coupling these two aspects of ribosome biogenesis has been recently elucidated by our lab and will be presented in the Results section of this thesis (Albert et al., 2016).
Finally, another transcription factor, Crf1, has been characterized as a repressor of RP gene transcription. Crf1 is a partial homolog of Ifh1 that is recruited by Fhl1 to RP gene promoters after TORC1 inhibition (Martin et al., 2004). Nevertheless, the role of Crf1 in RP gene transcription regulation seems to be restricted to specific yeast strain backgrounds (Zhao et al., 2006).
Sfp1, a link between ribosome biogenesis and cell cycle progression
Sfp1 is a zinc-finger protein characterized by an unusually long spacer (40 amino acids instead of the typical 7) between its two zinc-finger domains. Though deletion of Sfp1 gene is not lethal, Sfp1-null cells show a tremendously slow growth, have a reduced size and defects in cell cycle progression (Jorgensen et al., 2002; Xu and Norris, 1998). Inhibition of TOR signaling, stress or starvation lead to Sfp1 exit from the nucleus and to decreased RP and RiBi gene transcription (Marion et al., 2004).
Importantly, Sfp1 over-expression leads to up-regulation of both RP and RiBi gene transcription (Fingerman et al., 2003; Jorgensen et al., 2002). However, Sfp1 binding to RP and RiBi gene promoters has never been reported (Fingerman et al., 2003). Furthermore, Sfp1 is also involved in transcription of few other genes as the cell cycle regulator Cln3 (Cipollina et al., 2008a; Cipollina et al., 2008b; Jorgensen et al., 2004), suggesting a possible role of Sfp1 in coupling ribosome biogenesis
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and cell cycle progression. Nevertheless, the mechanism by which Sfp1 promotes transcription activation is still not known.