Occupancy of the Drosophila hsp70 promoter by a subset of basal transcription factors diminishes upon transcriptional activation

Abstract

The presence of general transcription factors and other coactivators at the Drosophila hsp70 gene promoter in vivo has been examined by polytene chromosome immunofluorescence and chromatin immunoprecipitation at endogenous heat-shock loci or at a hsp70 promoter-containing transgene. These studies indicate that the hsp70 promoter is already occupied by TATA-binding protein (TBP) and several TBP-associated factors (TAFs), TFIIB, TFIIF (RAP30), TFIIH (XPB), TBP-free͞TAF-containg complex (GCN5 and TRRAP), and the Mediator complex subunit 13 before heat shock. After heat shock, there is a significant recruitment of the heatshock transcription factor, RNA polymerase II, XPD, GCN5, TRRAP, or Mediator complex 13 to the hsp70 promoter. Surprisingly, upon heat shock, there is a marked diminution in the occupancy of TBP, six different TAFs, TFIIB, and TFIIF, whereas there is no change in the occupancy of these factors at ecdysone-induced loci under the same conditions. Hence, these findings reveal a distinct mechanism of transcriptional induction at the hsp70 promoters, and further indicate that the apparent promoter occupancy of the general transcriptional factors does not necessarily reflect the transcriptional state of a gene.polytene chromosomes ͉ TATA-binding protein, ͉ TATA-binding protein-associated factors ͉ TFIID ͉ transcription T ranscription initiation of protein-coding genes by RNA polymerase II (Pol II) involves the polymerase along with the general transcription initiation factors (GTFs), which include TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (1). TFIID, which comprises TATA box-binding protein (TBP) and TBPassociated factors (TAFs), recognizes the core promoter in a sequence-specific manner (2, 3). TAFs are also present in other transcription-related multiprotein complexes, such as TBP-free͞ TAF-containing complex (TFTC), STAGA, and PCAF͞GCN5 (4-7). These complexes are homologous to yeast SAGA complexes and contain GCN5 histone acetyltransferase, TRRAP protein, and several TAFs (5). In addition, other transcriptional coactivators, such as Mediator complexes, promote regulatory interactions between transcriptional activators and the GTFs (refs. 8-10 and references therein).The hsp70 gene cluster has served as an important focus for the analysis of transcriptional activation upon heat shock in Drosophila melanogaster. The region upstream of the hsp70 TATA element contains multiple binding sites for the sequence-specific regulatory proteins GAGA factor (GAF) and heat-shock factor (HSF). Before heat shock, GAF has been observed to reside on the hsp70 promoter (11). The binding of GAF appears to maintain the promoter region in a nucleosome-free conformation (12-16). The existence of an open chromatin conformation of the hsp70 promoter in noninduced conditions has been thought to allow access of the GTFs to the core promoter for rapid transcriptional induction.The GTFs form a transcription complex wherein the polymerase initiates transcription and then pauses Ϸ17-37 nt downstream of the start site (17). Then, upon he...

Chromatin interaction of TATA-binding protein is dynamically regulated in human cells

“…Together, our results add to previously published observations (Darzacq et al, 2007) and further suggest that the pol II PICs are very dynamic structures and that TBP bound to pol II promoters is rapidly exchanging (van Werven et al, 2009). This challenges models of pol II mRNA factories consisting of tightly bound and rather immobile protein assemblies (Sutherland and Bickmore, 2009), but rather support a model where transcription sites are rapidly assembling and disassembling entities (Lebedeva et al, 2005). Importantly, this kinetic model allows for a rapid response to changes in mRNA requirement of cells.…”

TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription

“…With the TATA-containing CG4500 promoter, there is increased ChIP signal for both TBP and Rpb3 in the promoter region upon 20HE induction. In the control/reference TATA-containing hsp70 promoter, we also observed an increase in ChIP of TBP and Rpb3 in the promoter region (Lebedeva et al 2005). By comparison, with the DPE-containing Glut1 and CG16876 promoters, there is increased ChIP of Rpb3 in the promoter region upon 20HE induction; however, the ChIP signal for TBP does not increase under the same conditions.…”

Distinct GCN5/PCAF-containing complexes function as co-activators and are involved in transcription factor and global histone acetylation

“…The role of the GCN5-and PCAF-containing complexes as co-activators at specific loci Many metazoan transcription activators and cell-cycle regulatory proteins (that are, nuclear receptor, IRF, IFN, STAT, GAGA, HSF, Smad, Myc and E2F family of proteins) contact directly and recruit GCN5-or PCAF-containing complexes to specific promoters (McMahon et al, 1998;Masumi et al, 1999;Agalioti et al, 2000;Anafi et al, 2000;Flinn et al, 2002;Paulson et al, 2002;Yanagisawa et al, 2002;Liu et al, 2003;Kahata et al, 2004;Lebedeva et al, 2005;Figure 3b). According to the most accepted model (Carrozza et al, 2003), these activators would directly contact the TRRAP subunit of the HAT complexes; however, it is also possible that different activators can contact distinct subunits in these 2 MDa assemblies.…”