Mohamed Guermah

RAD6-Mediated Transcription-Coupled H2B Ubiquitylation Directly Stimulates H3K4 Methylation in Human Cells

H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

Transcription coactivator TRAP220 is required for PPAR|[gamma]|2-stimulated adipogenesis

The TRAP (thyroid hormone receptor-associated proteins) transcription coactivator complex (also known as Mediator) was first isolated as a group of proteins that facilitate the function of the thyroid hormone receptor. This complex interacts physically with several nuclear receptors through the TRAP220 subunit, and with diverse activators through other subunits. TRAP220 has been reported to show ligand-enhanced interaction with peroxisome proliferator-activated receptor γ2 (PPARγ2), a nuclear receptor essential for adipogenesis. Here we show that Trap220-/- fibroblasts are refractory to PPARγ2-stimulated adipogenesis, but not to MyoD-stimulated myogenesis, and do not express adipogenesis markers or PPARγ2 target genes. These defects can be restored by expression of exogenous TRAP220. Further indicative of a direct role for TRAP220 in PPARγ2 function via the TRAP complex, TRAP functions directly as a transcriptional coactivator for PPARγ2 in a purified in vitro system and interacts with PPARγ2 in a ligand- and TRAP220-dependent manner. These data indicate that TRAP220 acts, via the TRAP complex, as a PPARγ2-selective coactivator and, accordingly, that it is specific for one fibroblast differentiation pathway (adipogenesis) relative to another (myogenesis).

The Human PAF1 Complex Acts in Chromatin Transcription Elongation Both Independently and Cooperatively with SII/TFIIS

Genetic and cell-based studies have implicated the PAF1 complex (PAF1C) in transcription-associated events, but there has been no evidence showing a direct role in facilitating transcription of a natural chromatin template. Here, we demonstrate an intrinsic ability of human PAF1C (hPAF1C) to facilitate activator (p53)- and histone acetyltransferase (p300)-dependent transcription elongation from a recombinant chromatin template in a biochemically defined RNA polymerase II transcription system. This represents a PAF1C function distinct from its established role in histone ubiquitylation and methylation. Importantly, we further demonstrate a strong synergy between hPAF1C and elongation factor SII/TFIIS and an underlying mechanism involving direct hPAF1C-SII interactions and cooperative binding to RNA polymerase II. Apart from a distinct PAF1C function, the present observations provide a molecular mechanism for the cooperative function of distinct transcription elongation factors in chromatin transcription.

The TRAP/Mediator coactivator complex interacts directly with estrogen receptors α and β through the TRAP220 subunit and directly enhances estrogen receptor function in vitro

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17β-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ERα and ERβ in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220−/− fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ERα–TRAP/Mediator complex from cultured cells expressing an epitope-tagged ERα. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

Involvement of TFIID and USA Components in Transcriptional Activation of the Human Immunodeficiency Virus Promoter by NF-κB and Sp1

ABSTRACT The purified Rel/NF-κB (p50/p65) complex and Sp1 markedly activate transcription from the human immunodeficiency virus type 1 (HIV-1) promoter in a highly purified HeLa reconstituted transcription system. Transcriptional activation by NF-κB and Sp1 requires both TFIID and the USA fraction. The USA-derived coactivators PC2 and PC4 fully reconstitute the USA coactivator activity, both by repressing the basal level of transcription and by potentiating activator function to yield large increases in the levels of transcription induction. Under limiting concentrations, PC2 and PC4 also show synergistic effects. The C-terminal portion (amino acids 416 to 550) of the p65 subunit of NF-κB is a potent activator when assayed as a Gal fusion in the reconstituted transcription system and interacts both with TATA-binding protein (TBP) and with several human TBP-associated factors (TAFs) that include TAFII250. The p65 activation domain mediates transcription activation in the presence of partially reconstituted TFIID species that include a minimal complex containing only TBP and TAFII250. These studies also show that, like USA components, TAFs can serve both to repress TBP-mediated transcription and, following activator interactions, to reverse the repression and effect a net increase in activity. Taken together, these data underscore the importance of both TAFs and specific USA-derived coactivators for optimal activation of the HIV-1 promoter, as well as certain parallels in their overall mechanisms of action.

Structural and Functional Organization of TRAP220, the TRAP/Mediator Subunit That Is Targeted by Nuclear Receptors

ABSTRACT The TRAP/Mediator complex serves as a coactivator for many transcriptional activators, including nuclear receptors such as the thyroid hormone receptor (TR) that targets the TRAP220 subunit. The critical but selective function of TRAP220 is evidenced by the embryonic lethal phenotype of Trap220 − / − mice and by the observation that Trap220 − / − fibroblasts (isolated before embryonic death) are impaired in specific nuclear receptor-dependent pathways. Here we have used a biochemical and genetic approach to understand the basis of specificity in TRAP220 function. We show that Trap220 − / − cells possess a TRAP/Mediator complex that is relatively intact and compromised in its ability to support TR-dependent, but not VP16-dependent, transcription in vitro. Transfection studies using TRAP220 mutants revealed that the N terminus of TRAP220 is necessary and sufficient for stable association with the TRAP/Mediator complex and, further, that TRAP220-dependent TR function in transfected cells requires both of the NR boxes that contain the LXXLL motif implicated in nuclear receptor binding. Similarly, an analysis of isolated TRAP/Mediator complexes with mutations in either or both of the two NR boxes confirmed a critical role for them in in vitro coactivator function. The implications of these observations are discussed in terms of our present understanding of coactivator function.

The ts13 mutation in the TAF(II)250 subunit (CCG1) of TFIID directly affects transcription of D-type cyclin genes in cells arrested in G1 at the nonpermissive temperature.

The general transcription initiation factor TFIID contains the TATA-binding protein (TBP) and TBP-associated factors (TAFs) implicated in the function of gene-specific activators. Previous studies have indicated that a hamster cell line (ts13) with a point mutation in the TAF(II)250/CCG1 (TAF(II)250) gene shows temperature-sensitive expression of a subset of genes and arrests in late G1 at 39.5 degrees C. Here, we report the identification of cell cycle-specific (G1-specific) genes that appear to be regulated directly through TAF(II)250 both in vivo and in vitro. Transcription rates of several cell cycle-regulatory genes were determined by run-on assays in nuclei from ts13 cells grown at permissive (33 degrees C) and nonpermissive (39.5 degrees C) temperatures. Temperature-dependent differences in transcription rates were observed for cyclin A, D1, and D3 genes. In transient-transfection assays, the human cyclin D1 promoter fused to a luciferase reporter showed a temperature-dependent reduction in activity in ts13 cells but not in parental BHK cells. In in vitro assays, upstream sequence-dependent transcription from the human cyclin D1 promoter was significantly reduced in ts13 nuclear extracts preincubated at 30 degrees C but not in similarly treated BHK nuclear extracts, and transcription in the ts13 extract was restored by addition of an affinity-purified human TFIID. Preincubation of the ts13 nuclear extracts did not affect the function of several GAL4-activation domain fusion proteins (GAL4-VP16, GAL4-p65, and GAL4-p53) on either the adenovirus major late or cyclin D1 core promoter bearing GAL4 sites, further indicating that the effect of the TAF(II)250 mutation is both core promoter and activator specific.

Synergistic functions of SII and p300 in productive activator-dependent transcription of chromatin templates.

We have reconstituted a highly purified RNA polymerase II transcription system containing chromatin templates assembled with purified histones and assembly factors, the histone acetyltransferase p300, and components of the general transcription machinery that, by themselves, suffice for activated transcription (initiation and elongation) on DNA templates. We show that this system mediates activator-dependent initiation, but not productive elongation, on chromatin templates. We further report the purification of a chromatin transcription-enabling activity (CTEA) that, in a manner dependent upon p300 and acetyl-CoA, strongly potentiates transcription elongation through several contiguous nucleosomes as must occur in vivo. The transcription elongation factor SII is a major component of CTEA and strongly synergizes with p300 (histone acetylation) at a step subsequent to preinitiation complex formation. The purification of CTEA also identified HMGB2 as a coactivator that, while inactive on its own, enhances SII and p300 functions.

Alternative Mechanisms by Which Mediator Subunit MED1/TRAP220 Regulates Peroxisome Proliferator-Activated Receptor γ-Stimulated Adipogenesis and Target Gene Expression

ABSTRACT Mediator is a general coactivator complex connecting transcription activators and RNA polymerase II. Recent work has shown that the nuclear receptor-interacting MED1/TRAP220 subunit of Mediator is required for peroxisome proliferator-activated receptor γ (PPARγ)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPARγ-Mediator interaction that requires the two LXXLL nuclear receptor recognition motifs on MED1/TRAP220 and, furthermore, we show that the intact LXXLL motifs are essential for optimal PPARγ function in a reconstituted cell-free transcription system. Surprisingly, a conserved N-terminal region of MED1/TRAP220 that lacks the LXXLL motifs but gets incorporated into Mediator fully supports PPARγ-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPARγ-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPARγ shows significantly reduced transcriptional activity in cells deficient for a subunit (MED24/TRAP100) important for the integrity of the Mediator complex, indicating a general Mediator requirement for PPARγ function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPARγ and Mediator through MED1/TRAP220 is not essential either for PPARγ-stimulated adipogenesis or for PPARγ target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPARγ transcriptional activity, our data indicate the presence of alternative mechanisms for Mediator recruitment, possibly through intermediate cofactors or other cofactors that are functionally redundant with MED1/TRAP220.

Positive and negative TAF(II) functions that suggest a dynamic TFIID structure and elicit synergy with traps in activator-induced transcription.

ABSTRACT Human transcription factor TFIID contains the TATA-binding protein (TBP) and several TBP-associated factors (TAFIIs). To elucidate the structural organization and function of TFIID, we expressed and characterized the product of a cloned cDNA encoding human TAFII135 (hTAFII135). Comparative far Western blots have shown that hTAFII135 interacts strongly with hTAFII20, moderately with hTAFII150, and weakly with hTAFII43 and hTAFII250. Consistent with these observations and with sequence relationships of hTAFII20 and hTAFII135 to histones H2B and H2A, respectively, TFIID preparations that contain higher levels of hTAFII135 also contain higher levels of hTAFII20, and the interaction between hTAFII20 and hTAFII135 is critical for human TFIID assembly in vitro. From a functional standpoint, hTAFII135 has been found to interact strongly and directly with hTFIIA and (within a complex that also contains hTBP and hTAFII250) to specifically cooperate with TFIIA to relieve TAFII250-mediated repression of TBP binding and function on core promoters. Finally, we report a functional synergism between TAFIIs and the TRAP/Mediator complex in activated transcription, manifested as hTAFII-mediated inhibition of basal transcription and a consequent TRAP requirement for both a high absolute level of activated transcription and a high and more physiological activated/basal transcription ratio. These results suggest a dynamic TFIID structure in which the switch from a basal hTAFII-enhanced repression state to an activator-mediated activated state on a promoter may be mediated in part through activator or coactivator interactions with hTAFII135.