Identification of a transcriptional activation domain in yeast repressor activator protein 1 (Rap1) using an altered DNA-binding specificity variant

Abstract

Edited by Joel GottesfeldRepressor activator protein 1 (Rap1) performs multiple vital cellular functions in the budding yeast Saccharomyces cerevisiae. These include regulation of telomere length, transcriptional repression of both telomere-proximal genes and the silent mating type loci, and transcriptional activation of hundreds of mRNA-encoding genes, including the highly transcribed ribosomal protein-and glycolytic enzyme-encoding genes. Studies of the contributions of Rap1 to telomere length regulation and transcriptional repression have yielded significant mechanistic insights. However, the mechanism of Rap1 transcriptional activation remains poorly understood because Rap1 is encoded by a single copy essential gene and is involved in many disparate and essential cellular functions, preventing easy interpretation of attempts to directly dissect Rap1 structure-function relationships. Moreover, conflicting reports on the ability of Rap1-heterologous DNA-binding domain fusion proteins to serve as chimeric transcriptional activators challenge use of this approach to study Rap1. Described here is the development of an altered DNA-binding specificity variant of Rap1 (Rap1 AS ). We used Rap1AS to map and characterize a 41-amino acid activation domain (AD) within the Rap1 C terminus. We found that this AD is required for transcription of both chimeric reporter genes and authentic chromosomal Rap1 enhancer-containing target genes. Finally, as predicted for a bona fide AD, mutation of this newly identified AD reduced the efficiency of Rap1 binding to a known transcriptional coactivator TFIID-binding target, Taf5. In summary, we show here that Rap1 contains an AD required for Rap1-dependent gene transcription. The Rap1 AS variant will likely also be useful for studies of the functions of Rap1 in other biological pathways.

Repression of divergent noncoding transcription by a sequence-specific transcription factor

“…Fourth, the Rap1 binding site ectopically represses divergent noncoding transcription without affecting transcription in the protein-coding direction. Conversely, the activation domain of Rap1, which directs transcription in the protein-coding direction, is not required for repressing divergent transcription (Johnson and Weil, 2017;Layer et al, 2010). Finally, we found that a chromatin assembly factor (Rlf2), regulators of histone gene expression (Spt10 and Spt21), and co-transcriptional chromatin remodellers (Spt6 and Spt16) also repress divergent transcription by mechanisms distinct from Rap1.…”

“…Thus the N-terminus, but not the C-terminus, of Rap1 is dispensable for repression of divergent transcription. Important functions of the C-terminus of Rap1 are exerted by the silencing domain, the activation domain (AD), and the toxicity domain (Tox) (Freeman et al, 1995;Garbett et al, 2007;Johnson and Weil, 2017;Kurtz and Shore, 1991;Layer et al, 2010;Sussel and Shore, 1991). We assessed whether Rap1 constructs with different domain deletions could repress divergent transcription ( Figure 5A) .…”

General Regulatory Factors control the fidelity of transcription by restricting non-coding and ectopic initiation

“…Rap1 interacts directly with the SWI/SNF chromatin remodelling complex (Tomar et al, 2008) and has been shown to be required for the recruitment of Esa1, a histone H4 acetylase (Reid et al, 2000). Activation of the expression of model genes by Rap1 depends on a domain that is C-terminal to the DNA binding domain (DBD) (Garbett et al, 2007;Johnson and Weil, 2017;Papai et al, 2010;Tomar et al, 2008), which has been implicated in the interaction with TFIID and SWI/SNF (Johnson and Weil, 2017). To assess whether the role of Rap1 in controlling the fidelity of initiation is related to the recruitment or function of TFIID or SWI/SNF, we depleted Rap1 from the nucleus of cells ectopically expressing only the DBD of Rap1 (aa.…”

“…Rap1 (Repressor Activator Protein 1) has been originally described as an activator and a repressor of gene expression (for a recent review see: Azad and Tomar, 2016). Gene activation has been shown to depend on a domain in the C-terminal portion of the protein, which can activate transcription alone when fused to a DNA binding domain with altered specificity (Johnson and Weil, 2017). This region participates to the interaction of Rap1 with PIC components, TFIID and TFIIA (Garbett et al, 2007;Johnson and Weil, 2017;Papai et al, 2010), which has been proposed to be required for transcription activation.…”

“…Gene activation has been shown to depend on a domain in the C-terminal portion of the protein, which can activate transcription alone when fused to a DNA binding domain with altered specificity (Johnson and Weil, 2017). This region participates to the interaction of Rap1 with PIC components, TFIID and TFIIA (Garbett et al, 2007;Johnson and Weil, 2017;Papai et al, 2010), which has been proposed to be required for transcription activation. Repression of gene expression has been observed at mating type genes and other loci, and it has been suggested that Rap1 negatively affects gene expression by directly interacting with and inhibiting TBP binding to the DNA (Bendjennat and Weil, 2008;Giesman et al, 1991;Kurtz and Shore, 1991;Yarragudi et al, 2004).…”