Our studies have identified the TFIID subunit TAF7 as a novel regulator of cyclin D1 and cyclin A gene transcription. We propose that activation of cyclin D1 and cyclin A gene transcription can occur via a phosphorylation-dependent mechanism in which TAF7 is phosphorylated by the TAF1 subunit of TFIID. This phosphorylation event results in release of TAF7 from the TFIID complex, stimulation of TAF1 HAT activity, acetylation of core promoter histones, and increased transcription. The phosphorylation and dissociation of TAF7 from TFIID complexes at the cyclin D1 and cyclin A promoters is most prevalent during the G1 phase of the cell cycle and potentially represents a novel downstream event of mitogenic signaling pathways that induce cyclin D1 and cyclin A gene transcription.
Acetylation of histones is well established as an important mechanism for activating gene transcription. Much effort has been placed on the characterization of histone acetyltransferases (HATs), enzymes that catalyze these posttranslational modifications. HATs can be regulated through a variety of molecular mechanisms. Autoacetylation within the activation loop of p300 and phosphorylation of CREB binding protein (CBP) stimulate their rates of histone acetylation (1
). CBP HAT activity also can be stimulated by its interaction with select transcriptional activators (2
). We have discovered that TAF1 HAT activity is regulated by an inhibitory protein interaction that is disrupted by protein phosphorylation. Intriguingly, the protein kinase responsible resides in TAF1, the same polypeptide that possesses the HAT domain subject to regulation. To our knowledge, TAF1 is the first example of a polypeptide that possesses two catalytic activities that are functionally connected. Whether this mode of regulation takes place within a single molecule in cis
or between two TAF1 proteins in trans
remains to be determined.
ts13 cells are conditional mutants that arrest in the late G1
phase of the cell cycle when shifted to the nonpermissive temperature of 39.5°C. Characterization of these mutant cells established that the G1
/S phase cell cycle arrest is due to a missense mutation in the TAF1 HAT domain that abrogates catalytic activity (7
). Expression of WT TAF1 rescued the ts13 cell cycle arrest while expression of HAT-deficient mutants was ineffective (7
). Unexpectedly, the introduction of TAF1 constructs containing disruptive kinase domain mutations also failed to complement the ts13 mutant phenotype (26
). These data indicate that both TAF1 HAT and kinase activities are required for normal cell cycle progression. Our studies have extended these findings by placing both enzymatic activities of TAF1 in the same signaling pathway and adding to our molecular understanding of their function during G1
to S phase progression in mammalian cells.
We and others have reported previously that the HAT activity of TAF1 is required for efficient transcription from a subset of protein encoding genes (25
). TAF7 is a negative regulator of TAF1 HAT activity and inhibits transcription from only a subset of genes (10
). Based on these results, we anticipated that transcription levels of genes driven by promoters dependent on TAF1 HAT activity also would be subject to inhibitory actions by TAF7. Unexpectedly, elevating TAF7 levels only inhibited transcription of the TAF1-dependent cyclin A and D1 genes and had no effect on cyclin E gene transcription, another gene that requires TAF1 HAT activity for efficient promoter activity. These data indicate that although the subset of genes dependent on TAF1 HAT activity overlaps genes regulated by TAF7, these two subpopulations are not identical. One possible explanation is that the inhibitory effects of TAF7 can be bypassed by the recruitment of another enzyme that catalyzes the histone modifications necessary for efficient gene transcription. Further studies will be necessary to identify additional TAF1-dependent genes resistant to the inhibitory effects of TAF7 and to determine the specific set of factors present at each of these promoters that accounts for their differential regulation.
Overexpression of TAF7 was sufficient to inhibit cyclin D1 and cyclin A mRNA expression levels. A question that comes to mind is “how does TAF7 overexpression mechanistically work in the cell to repress gene transcription?” One hypothesis is that excess levels of TAF7 protein would change the dynamics of TFIID assembly and create more complexes containing TAF7. This shift in TFIID composition would increase the number of DNA-bound TAF7-containing complexes at the cyclin D1 and cyclin A promoters, thereby inhibiting transcription of these genes.
When first isolated, it was generally thought that the subunit composition of a functional TFIID complex was invariant and contained a defined set of TAFs conserved from yeast to humans. Here we present data suggesting that TAF7 dissociates from the TFIID complex following phosphorylation by the TAF1 kinase, as cells transition through the G1
phase of the cell cycle ( and ). Immunoelectron microscopy performed with a TAF7 antibody mapped TAF7 at the periphery of the TFIID complex, near the TAF1 HAT domain (20
). This peripheral localization suggests that TAF7 could easily enter and exit the TFIID complex. Our findings are consistent with recent studies that have demonstrated TFIID is variable and dynamic in both its subunit composition and overall structure. Several groups have identified TAF variants that incorporate into the TFIID complex at different stages of development or in different cell types (9
). The disruption and replacement of the canonical TFIID with a novel complex composed of the TBP-related factor 3 (TRF3) and TAF3 also have been reported in myoblasts undergoing differentiation into myotubes (4
). In addition, TAF subunits originally identified in TFIID can be found in other transcription regulatory complexes (40
). Therefore, regulating the composition of canonical and noncanonical TFIID complexes represents an important mechanism for controlling gene transcription.
Loss of the TAF2 subunit of TFIID relative to the other subunits has been repeatedly observed during the purification of TFIID from yeast and human cells, suggesting that the TAF2 subunit readily dissociates from the complex (3
). Subsequent structural analysis of yeast TFIID by cryoelectron microscopy and electron tomography confirmed the existence of two subpopulations, TFIID complexes containing the TAF2 subunit and those lacking TAF2 (28
). In the absence of TAF2, significant reorganization of different domains was observed, suggesting that TFIID demonstrates significant plasticity and is capable of varying its overall structure. However, the presence of TAF2 appeared to stabilize one of four abundant states identified for TFIID. The ts13 single missense mutation in TAF1, which disrupts the ability of TAF1 to acetylate histones, is thought to shift the TAF1 protein to an inactive state for HAT activity under nonpermissive conditions. These results have led us to hypothesize that TAF7 binds to TAF1 and locks the TFIID complex into a conformation in which the structure of the TAF1 HAT domain is no longer favorable for catalytic activity. To test this hypothesis, we have established a collaboration to determine the structure of a TAF1-TAF7 dimer using X-ray crystallography.
We have discovered a novel role for TAF7 as a negative regulator of transcription and cell cycle progression. Our studies have determined that activation of cyclin D1 and cyclin A gene transcription requires a previously uncharacterized phosphorylation-dependent switch in the composition of TFIID. Phosphorylation catalyzed by the TAF1 kinase leads to dissociation of the TAF7 subunit from TFIID and activation of TAF1 HAT activity. There is a growing body of evidence that TFIID is a highly dynamic molecule, and we now demonstrate that altering its subunit composition can have profound consequences on TFIID function and gene transcription.