Mot1 activates and represses transcription by direct, ATPase-dependent mechanisms

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4␣ (HNF4␣) and transcription factor IID (TFIID). A systematic characterization of the HNF4␣-TFIID link revealed that the HNF4␣ DNA-binding domain binds directly to the TATA boxbinding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4␣-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4␣ mutation that blocks binding of TBP to HNF4␣ interferes with HNF4␣ transactivation activity in cells. These findings identify an unexpected role for the HNF4␣ DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4␣ and TFIID in RNA polymerase II transcription.Promoter-specific transcription by RNA polymerase II depends on the general initiation factors TFIIA, 2 -B, -D, -E, -F, and -H, which are the minimal set of factors needed for assembly of the preinitiation complex and subsequent initiation of transcription (1-4). The activity of RNA polymerase II and the general transcription factors is controlled by a host of DNAbinding transcription factors, the Mediator of RNA polymerase II transcription, and other coactivators and corepressors (5-7).Preinitiation complex assembly begins with DNA sequencespecific binding of TFIID to the core promoter to provide a nucleoprotein recognition site for subsequent binding of polymerase II and the remaining general transcription factors. The TATA box-binding protein (TBP) subunit of TFIID recognizes the TATA box element and can substitute for TFIID in transcription in vitro. Additional TBP-associated factor (TAF) subunits of TFIID recognize the initiator (Inr) and other core promoter elements and contribute to functional interactions between the general transcription machinery and DNA-binding transcription factors and coregulators (1,8,9). TBP has also been shown to be an essential component of SL1 and TFIIIB, which are multisubunit general transcription factors for RNA polymerases I and III, respectively (10 -16).Because of its central role in transcriptional regulation, there has been considerable interest in defining the repertoire of TBP-interacting proteins. Biochemical studies that led to definition of TFIID, SL1, TFIIIB, and other TBP-associated proteins have typically used conventional chromatography and/or immunoaffinity purification methods to purify TBP-containing complexes (17), followed by protein isolation by reverse-phase chromatography or SDS-PAGE and identification by Edman sequencing or mass spectrometry (e....

Section: Resultsmentioning

confidence: 90%

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Takahashi

Martin-Brown

Washburn

et al. 2009

“…NC2 is a good candidate for a factor that impacts Mot1 function in vivo. Mot1 and NC2 regulate many of the same genes and co-occupy many of the same promoters (52)(53)(54)(55)(56). It has been proposed that the NC2-containing TBP complex is a physiologically relevant Mot1 target (57), although a significant difference in Mot1 biochemical behavior in the presence of NC2 has not been detected thus far (10,14).…”

Section: Discussionmentioning

confidence: 99%

“…Because DNA sequence can influence Mot1 conformation, the combined results suggest that Mot1-TBP-DNA complexes can have differential stability dictated by the promoter sequence. Although the function of Mot1 in gene activation is not fully understood, the presence of Mot1 in complexes at active promoters (53,55,59) suggests that its dissociation activity may be regulated. We suggest that the influence of DNA sequence on Mot1 conformation may be relevant for control of Mot1 activity in vivo, possibly helping it to distinguish active versus repressed promoters.…”

Section: Dna Sequence Effects On Mot1mentioning

confidence: 99%

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme

Viswanathan

True

Auble

2016

Self Cite

The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short singlestranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.Snf2/Swi2 enzymes play critical roles in regulation of essentially all DNA metabolic processes by utilizing the energy of ATP hydrolysis to alter protein-DNA interactions (1-4). Mot1, 2 a Snf2/Swi2 protein, dissociates the TATA-binding protein (TBP) from promoters and thereby regulates TBP distribution in the cell (5-9). By exploiting the Mot1 experimental system, biochemical analyses of the TBP-DNA dissociation reaction have provided general insight into mechanisms by which Snf2/Swi2 ATPases catalyze rearrangements of stable protein-DNA complexes (4). The N terminus of Mot1 interacts with TBP through its flexible HEAT repeats and in this way recruits Mot1 to TBP-DNA (7, 10 -14). The C-terminal region of Mot1, which comprises the ATPase domain, interacts with DNA upstream of the TATA box (13,14). Given the sequence and structural similarity among Snf2/Swi2 ATPase domains (15, 16), a central goal in the field has been to decipher the mechanisms by which these enzymes couple ATP hydrolysis to dissociation or rearrangement of protein-DNA complexes.The Snf2/Swi2 ATPases comprise a group within the SF2 helicase superfamily (17). Thus, their mechanisms are proposed to be similar to those of helicases except that they do not catalyze DNA strand separation (15,18). Nonetheless, the Snf2/ Swi2 family has well over a thousand members, and the great majority of them have not been characterized biochemically (19). Biochemical studies of helicases have revealed diverse mechanistic properties (3), suggesting that Snf2/Swi2 enz...

“…Microarray Analysis-Poly(A) ϩ RNA was isolated from wild-type SAN1 cells (YPH499) (26) and congenic san1⌬ cells exactly as described previously (38), with the exception that cells were harvested at A 600 ϳ1.0 in YPD media at 30°C. Hybridizations to glass slide microarrays and data analyses were performed by the NIEHS Microarray Facility exactly as described previously (38); hybridizations were performed in triplicate, and the genes listed in Table II represent the total set of genes whose message levels were affected in a statistically significant manner when the data were analyzed at the 95% confidence level.…”

Section: Methodsmentioning

confidence: 99%

Sir Antagonist 1 (San1) Is a Ubiquitin Ligase

Dasgupta

Ramsey

Smith

et al. 2004

Self Cite

Mutations in Sir Antagonist 1 (SAN1) suppress defects in SIR4 and SPT16 in Saccharomyces cerevisiae. San1 contains a RING domain, suggesting that it functions by targeting mutant sir4 and spt16 proteins for degradation by a ubiquitin-mediated pathway. Consistent with this idea, mutant sir4 and spt16 proteins are unstable in SAN1 cells but are stabilized in san1⌬ cells. We demonstrate that San1 possesses ubiquitin-protein isopeptide ligase activity in vitro, and the ubiquitin-protein isopeptide ligase activity of San1 is required for its function in vivo. Wild-type Sir4 has a half-life of about 21 min, and san1⌬ increased Sir4 half-life to >90 min. In contrast, san1⌬ did not affect the stability of wild-type Spt16, Sir3, Sir2, or the Spt16-associated proteins Pob3 and Nhp6. Loss of SAN1 also did not affect the stability of Ste6-166, a highly unstable protein in yeast. These results support the idea that San1 controls the turnover of a specific class of unstable nuclear proteins. Sir4 nucleates the assembly of silent chromatin at telomeres and the silent mating-type loci (HM) in S. cerevisiae. Sir4 can also affect silencing in the rDNA indirectly by sequestering limiting Sir2. Increasing the stability of wild-type Sir4 by deleting SAN1 had only subtle effects on silencing, suggesting that silent chromatin in yeast is robustly buffered against changes in Sir4 stability. Consistent with the idea that San1 participates as an accessory factor to regulate silent chromatin, including the silent mating-type loci, microarray analysis defined a small but statistically significant role for San1 in transcription of several mating pheromone-responsive genes.