Elisabeth Scheer

The human estrogen receptor has two independent nonacidic transcriptional activation functions

We have previously reported the presence of a hormone-inducible transcriptional activation function (TAF-2) within the region of the estrogen receptor (ER) that contains the hormone binding domain. We show here that the N-terminal A/B region of the ER contains an independent constitutive activation function (TAF-1) that exhibits cell type specificity since it activates transcription efficiently in chicken embryo fibroblasts, but only poorly in HeLa cells. By analyzing the ability of TAF-1, TAF-2, and the GAL4 and VP16 acidic activating domains (AADs) to homosynergize and heterosynergize with one another and with the factor binding to the upstream element (UE) of the adenovirus 2 major late promoter, we show that the activation properties of TAF-1 and TAF-2 are different and distinct from those of AADs, in agreement with the absence of acidic amino acid stretches in TAF-1 and TAF-2.

Distinct classes of transcriptional activating domains function by different mechanisms

We have previously shown that the two transcriptional activation functions (TAF-1 and TAF-2) of the human estrogen receptor (hER) have synergistic properties different from one another and from those of acidic activating domains (AADs). Here we compare the transcriptional interference/squelching properties of the hER TAFs with those of the AADs of yeast GAL4 and chimeric GAL-VP16 activators. Our results indicate that AADs interact with a factor(s) that, while required for activation by AADs, is not essential for activation by hER TAFs. In contrast, hER TAFs appear to interact with factors indispensable for mediating both their activation function and that of AADs. Thus, different classes of trans-activators may interact with different factors. In addition, the synergistic and transcriptional interference/squelching properties of the two TAFs of the human glucocorticoid receptor (hGR) indicate that both are composed of acidic and nonacidic activation functions.

Gene-Specific Modulation of TAF10 Function by SET9-Mediated Methylation

SET9 is a member of the SET domain-containing histone methyltransferase family that can specifically methylate histone 3 at lysine 4 position. Although nucleosomal histones are poor substrates for SET9, the active enzyme can stimulate activator-induced transcription. Here, we show that SET9 can monomethylate the TBP-associated factor TAF10 at a single lysine residue located at the loop 2 region within the putative histone-fold domain of the protein. Methylated TAF10 has an increased affinity for RNA polymerase II, pointing to a direct role of this modification in preinitiation complex formation. Reporter assays and studies on TAF10 null F9 cells expressing a methylation-deficient TAF10 mutant revealed that SET9-mediated methylation of TAF10 potentiates transcription of some but not all TAF10-dependent genes. This gene specificity correlated with SET9 recruitment. The promoter-specific effects of SET9-methylated TAF10 may have important implications regarding the biological function of SET domain-containing lysine methylases, whose primary targets have been presumed to be histones.

The architecture of human general transcription factor TFIID core complex

The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 Å resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8–TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.

Two Different Drosophila ADA2 Homologues Are Present in Distinct GCN5 Histone Acetyltransferase-Containing Complexes

ABSTRACT We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.

A dual role of linker histone H1.4 Lys 34 acetylation in transcriptional activation

The linker histone H1 is a key player in chromatin organization, yet our understanding of the regulation of H1 functions by post-translational modifications is very limited. We provide here the first functional characterization of H1 acetylation. We show that H1.4K34 acetylation (H1.4K34ac) is mediated by GCN5 and is preferentially enriched at promoters of active genes, where it stimulates transcription by increasing H1 mobility and recruiting a general transcription factor. H1.4K34ac is dynamic during spermatogenesis and marks undifferentiated cells such as induced pluripotent stem (iPS) cells and testicular germ cell tumors. We propose a model for H1.4K34ac as a novel regulator of chromatin function with a dual role in transcriptional activation.

Identification of hTAFII80δ Links Apoptotic Signaling Pathways to Transcription Factor TFIID Function

Abstract Apoptotic cell death is associated with altered levels of mRNA expression, yet the mechanisms that coordinate changes in gene expression with activation of the cell death machinery remain obscure. Here, we report the cloning and characterization of hTAF II 80δ, a specialized isoform of the general transcription factor TFIID subunit hTAF II 80. Several distinct apoptotic stimuli induce the expression and caspase-dependent cleavage of hTAF II 80δ. hTAF II 80δ, unlike hTAF II 80, forms a TFIID-like complex lacking hTAF II 31. Elevated expression of hTAF II 80δ in HeLa cells is sufficient to trigger apoptotic cell death and selectively alters cellular transcription, including the induction of the target genes gadd45 and p21 . These data define a signaling pathway that couples apoptotic signals to a reprogramming of RNA polymerase II transcription.

Different TBP-associated factors are required for mediating the stimulation of transcription in vitro by the acidic transactivator GAL-VP16 and the two nonacidic activation functions of the estrogen receptor

The estrogen receptor (ER) contains two nonacidic transcriptional activation functions, AF-1 and AF-2 (formerly TAF-1 and TAF-2). In this study we show that AF-1 and AF-2 are able to stimulate transcription in vitro in a HeLa cell system when fused to the DNA binding domain of the yeast activator GAL4. We also demonstrate that a factor(s) required for the function of the ER AFs is chromatographically separable from a factor(s) necessary for the activity of the acidic activation domain of VP16. Moreover, immunoprecipitation experiments using a monoclonal antibody directed against the TATA box binding protein (TBP) indicate, that these different factors are associated with TBP in distinct TFIID complexes.

TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice.

ABSTRACT TAF10 (formerly TAFII30), is a component of TFIID and the TATA box-binding protein (TBP)-free TAF-containing complexes (TFTC/PCAF/STAGA). To investigate the physiological function of TAF10, we disrupted its gene in mice by using a Cre recombinase/LoxP strategy. Interestingly, no TAF10−/− animals were born from intercrosses of TAF10+/− mice, indicating that TAF10 is required for embryogenesis. TAF10−/− embryos developed to the blastocyst stage, implanted, but died shortly after ca. 5.5 days postcoitus. Surprisingly, trophoblast cells from TAF10−/− blastocysts were viable, whereas inner cell mass cells failed to survive, highlighting that TAF10 is not generally required for transcription in all cells. TAF10-deficient cells express normal levels of TBP and TAFs other than TAF10 but contain only partially formed TFIID, are endocycle arrested, and have undetectable levels of transcription. Thus, our results demonstrate that TAF10 is required for TFIID stability, cell cycle progression, and transcription in the early mouse embryo.

TAF9b (formerly TAF9L) is a bona fide TAF that has unique and overlapping roles with TAF9.

ABSTRACT TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene. We show that TAF9Like is a subunit of TFIID, and thus, it will be called TAF9b. TFIID and TFTC complexes in which both TAF9 and TAF9b are present exist. In vitro and in vivo experiments indicate that the interactions between TAF9b and TAF6 or TAF9 and TAF6 histone fold pairs are similar. We observed a differential induction of TAF9 and TAF9b during apoptosis that, together with their different ability to stabilize p53, points to distinct requirements for the two proteins in gene regulation. Small interfering RNA (siRNA) knockdown of TAF9 and TAF9b revealed that both genes are essential for cell viability. Gene expression analysis of cells treated with either TAF9 or TAF9b siRNAs indicates that the two proteins regulate different sets of genes with only a small overlap. Taken together, these data demonstrate that TAF9 and TAF9b share some of their functions, but more importantly, they have distinct roles in the transcriptional regulatory process.