Spn1 Regulates the Recruitment of Spt6 and the Swi/Snf Complex during Transcriptional Activation by RNA Polymerase II

Abstract

We investigated the timing of the recruitment of Spn1 and its partner, Spt6, to the CYC1 gene. Like TATA binding protein and RNA polymerase II (RNAPII), Spn1 is constitutively recruited to the CYC1 promoter, although levels of transcription from this gene, which is regulated postrecruitment of RNAPII, are low. In contrast, Spt6 appears only after growth in conditions in which the gene is highly transcribed. Spn1 recruitment is via interaction with RNAPII, since an spn1 mutant defective for interaction with RNAPII is not targeted to the promoter, and Spn1 is necessary for Spt6 recruitment. Through a targeted genetic screen, strong and specific antagonizing interactions between SPN1 and genes encoding Swi/Snf subunits were identified. Like Spt6, Swi/Snf appears at CYC1 only after activation of the gene. However, Spt6 significantly precedes Swi/Snf occupancy at the promoter. In the absence of Spn1 recruitment, Swi/Snf is constitutively found at the promoter. These observations support a model whereby Spn1 negatively regulates RNAPII transcriptional activity by inhibiting recruitment of Swi/Snf to the CYC1 promoter, and this inhibition is abrogated by the Spn1-Spt6 interaction. These findings link Spn1 functions to the transition from an inactive to an actively transcribing RNAPII complex at a postrecruitment-regulated promoter.For a large number of well-characterized genes, the ratelimiting step in the transcription process is the formation of the preinitiation complex at the promoter. At these genes, the recruitment of TATA binding protein (TBP) and RNA polymerase II (RNAPII) to the promoter correlates strongly with transcriptional output (35,41,67). Indeed, delivery of TBP and RNAPII appears to be sufficient for gene activation in many contexts (for reviews, see references 63 and 64). However, there are a growing number of genes that have been found to be regulated at a step after the recruitment of RNAPII. Such genes include the yeast CYC1 gene, the Drosophila heat shock genes, and mammalian human immunodeficiency virus type 1 and the c-Myc proto-oncogene (3,39,41,51,77). Indeed, whole-genome studies suggest that a significant portion of the human genome may be regulated postrecruitment of RNAPII (26,35). As such, these mechanisms have the potential to impact the expression of thousands of human genes. Our understanding of these mechanisms and the rate-limiting steps involved is incomplete, but these observations suggest that functions critical for a high level of transcription are either inhibited or absent under noninducing conditions. To determine the nature of these functions, further characterization of genes regulated after the assembly of the general transcription machinery is imperative.The yeast CYC1 gene encodes iso-1-cytochrome c, a protein involved in the electron transport chain in the mitochondria (75). In the presence of a fermentable carbon source (such as dextrose), CYC1 gene expression is inhibited and transcriptional levels are extremely low (24,25). When cells are grown on a nonfermentable c...

Activation of a Poised RNAPII-Dependent Promoter Requires Both SAGA and Mediator

“…CYC1 is regulated after the recruitment of TBP and RNAPII (Lue et al 1989;Chen et al 1994;Kuras and Struhl 1999;Martens et al 2001;Zhang et al 2008). Therefore, RNAPII occupies the promoter to a similar degree under both uninduced and induced conditions ( Figure 4A).…”

“…Such genes include the yeast CYC1 gene, the Drosophila heat-shock genes, and mammalian c-myc and HIV-1 genes (Krumm et al 1992;Kuras and Struhl 1999;Andrulis et al 2000;Martens et al 2001;Stevens et al 2006;Zhang et al 2008). Genomewide studies also indicate that a large number of developmental and stress-inducible genes have RNAPII preloaded at promoter-proximal regions (Guenther et al 2007;Muse et al 2007;Zeitlinger et al 2007).…”

“…Chromatin immunoprecipitation analysis: Chromatin immunoprecipitations (ChIPs) were performed as described (Zhang et al 2008) with few modifications. Cells (50 ml) were grown to an OD 600 of 0.8-1.0 and were treated with formaldehyde (1%) for 15 min with swirling every 5 min.…”

“…These preloaded, yet transcriptionally inactive, promoters can be defined as poised for subsequent activation. Poised promoters are found across the evolutionary spectrum, including bacteria, yeast, Drosophila, and humans (Kuras and Struhl 1999;Andrulis et al 2000;Kim et al 2005;Reppas et al 2006;Zhang et al 2008;Kininis et al 2009;Venters and Pugh 2009). Indeed, whole-genome studies suggest that transcription of a significant part of the human genome may be regulated at rate-limiting steps after recruitment of the PIC (Kim et al 2005;Guenther et al 2007).…”

The multi-tasking P-TEFb complex

“…Iws1 binds to the REF1/Aly nuclear export adaptor, and depletion of Iws1, or overexpression of the Spt6 SH2 domain, induced bulk poly(A)+ RNAs to be retained in the nucleus [ , and thus Iws1 may help guide the exosome to the elongation complex, a process that was earlier shown to require Spt6 [55]. The yeast homolog of Iws1, Spn1, is an essential protein that functions to recruit Spt6 to the promoter at the CYC1 gene [56]; however, it is not known if the human Iws1 protein behaves similarly.…”