Sfp1 regulates transcriptional networks driving cell growth and division through multiple promoter binding modes

Abstract

16Understanding how transcriptional programs help to coordinate cell growth and division is an 17 important unresolved problem. Here we report that the nutrient-and stress-regulated transcription 18 factor Sfp1 is rate-limiting for expression of several large classes of genes involved in yeast cell growth, 19 including ribosomal protein, ribosome biogenesis, and snoRNA genes. Remarkably, the spectrum of 20 Sfp1 transcription effects is concordant with a combination of chromatin immunoprecipitation and 21 chromatin endogenous cleavage binding analyses, which together provide evidence for two distinct 22 modes of Sfp1 promoter binding, one requiring a co-factor and the other a specific DNA-recognition 23 motif. In addition to growth-related genes, Sfp1 binds to and regulates the promoters of cell cycle 24 "START" regulon genes, including the key G1/S cyclins CLN1 and CLN2. Our findings suggest that Sfp1 25 acts as a master regulator of cell growth and cell size by coordinating the expression of genes implicated 26 in mass accumulation and cell division. 2728 48 large-cell phenotype (Jorgensen et al., 2002). Taken together, these findings suggest that Sfp1 might 49 play a key role in coordinating cell growth and cell division. Interestingly, the transcriptional and cell-50 size phenotypes of SFP1 are notably similar to those of the c-Myc proto-oncogene (Jorgensen et al.,One paradox that has limited our understanding of Sfp1's mechanism of action is that the protein has 53 been detected by Chromatin Immuno-Precipitation (ChIP) at only a small fraction of the promoters that 54 it appears to regulate. For example, although ChIP detects Sfp1 at many RP gene promoters (Reja, 55 Vinayachandran et al., 2015), it is undetectable at virtually all of the >200 RiBi gene promoters where 56 4 over-expression studies suggest that it might be a direct activator (Jorgensen et al., 2002, Jorgensen et 57 al., 2004. 58Here we vastly expand our knowledge of Sfp1 binding by Chromatin Endogenous Cleavage (ChEC)-seq 59 analysis (Schmid, Durussel et al., 2004, Zentner, Kasinathan et al., 2015. Remarkably, we find that 60 ChEC and ChIP provide a highly complementary picture of Sfp1 binding, with distinct sets of sites 61 identified by one technique or the other. Our combined analysis provides evidence that Sfp1 directly 62 orchestrates TATA-binding protein (TBP) and RNAPII recruitment at a broad array of genes that drive 63 cell growth, including most RiBi, RP and snoRNA genes. In addition, we find that Sfp1 binds to the 64 promoters of many G1/S ("START") regulon genes that are targeted by the TF Swi4. Interestingly, Sfp1 65 binding sites identified by ChEC are enriched for the motif gAAAATTTTc, whereas binding identified by 66 ChIP is often strongly dependent on another TF: Ifh1 at RP genes or Swi4 at G1/S regulon genes. These 67 findings provide an unprecedented example of how the combination of ChIP and ChEC can reveal a 68 more complete picture of TF-chromatin interactions. Taken together, our results support a role for 69 Sfp1 as ...

Mechanisms Coordinating Ribosomal Protein Gene Transcription in Response to Stress

“…Next, we assessed the consequence of depletion of stress sensitive factors Ifh1 and Sfp1. Consistent with our previous results (Albert et al, 2019b), Ifh1 depletion triggered strong downregulation of Rap1-dependent genes and a significant though smaller decrease in transcription of Cat III genes (Figure 6B). Importantly, Sfp1 depletion caused an opposite transcriptional response to that of Ifh1 depletion, with Cat III RPGs being the most downregulated, and Cat I and II genes the least affected ( Figure 6C).…”

“…On the other hand, it could also introduce a bias in ChIP assays, due to the strong bias in formaldehyde crosslinking for G/C base pairs (Rossi et al, 2018), which might lead to low detection of proteins such as Abf1, which is enriched at Cat III promoters (Fermi et al, 2016). We also noted that Cat III promoters are enriched in a motif (gAAAATTTTc) bound by Sfp1, both in vitro and in vivo (Figure 5C; (Albert et al, 2019b;Zhu et al, 2009)), which is itself largely depleted for G/C base pairs.…”

“…We recently reported (Albert et al, 2019b) that Sfp1 binding is undetectable by ChIP at RiBi promoters enriched for the Sfp1 binding motif but can be revealed only by an alternative assay that does not require formaldehyde cross linking, Chromatin Endogenous Cleavage (ChEC; (Schmid et al, 2004)). We thus used published ChEC-seq data (Albert et al, 2019b) to examine binding of Sfp1 at RPGs promoters.…”

“…We recently reported (Albert et al, 2019b) that Sfp1 binding is undetectable by ChIP at RiBi promoters enriched for the Sfp1 binding motif but can be revealed only by an alternative assay that does not require formaldehyde cross linking, Chromatin Endogenous Cleavage (ChEC; (Schmid et al, 2004)). We thus used published ChEC-seq data (Albert et al, 2019b) to examine binding of Sfp1 at RPGs promoters. Strikingly, Sfp1 ChEC-seq signal reveals a high level of binding at Cat III promoters, comparable to the signals observed at RiBi genes, and more globally shows an opposite binding pattern at promoters of the three RPG categories compared when comparing it to the Sfp1 signal generated by ChIP (Figure 5D, 5E).…”

Genetic screens identify multiple negative regulators that additively restrict recruitment of the SWI/SNF chromatin remodeler to theHOpromoter inSaccharomyces cerevisiae

“…Sfp1 is an activator of ribosomal protein (RP) and ribosome biogenesis (Ribi) genes and is one of several proteins activated through the TOR pathway that determine cell size and regulate the cellular response to nutrients and stress (Jorgensen et al 2002;Fingerman et al 2003;Jorgensen et al 2004;Marion et al 2004). A recent study demonstrated that Sfp1 also acts as a glucose-regulated repressor at G1/S genes and associates with these promoters via an interaction with Swi4 (Albert et al 2019). We and others were unable to detect binding of Sfp1 at the HO promoter in log phase cells (Benjamin Albert and David Shore, personal communication).…”