SUMMARY 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 (i) that hRAD6 serves as the cognate E2 conjugating enzyme, (ii) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120, (iii) 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, (iv) that H2B ubiquitylation per se does not affect the level of hPAF-, SII- and p300-dependent transcription and likely functions downstream and (v) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and tri-methylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.

SUMMARY 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 (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 gamma(2) (PPAR gamma(2)), a nuclear receptor essential for adipogenesis. Here we show that Trap220(-/-) fibroblasts are refractory to PPAR gamma(2)-stimulated adipogenesis, but not to MyoD-stimulated myogenesis, and do not express adipogenesis markers or PPAR gamma(2) target genes. These defects can be restored by expression of exogenous TRAP220. Further indicative of a direct role for TRAP220 in PPAR gamma(2) function via the TRAP complex, TRAP functions directly as a transcriptional coactivator for PPAR gamma(2) in a purified in vitro system and interacts with PPAR gamma(2) in a ligand- and TRAP220-dependent manner. These data indicate that TRAP220 acts, via the TRAP complex, as a PPAR gamma(2)-selective coactivator and, accordingly, that it is specific for one fibroblast differentiation pathway (adipogenesis) relative to another (myogenesis).

Mitosis in higher eukaryotes is accompanied by a general inhibition of transcription. To begin to understand the mechanisms underlying this inhibition we have examined the behavior of the general transcription factor TFIID during mitosis. Immunocytochemistry and subcellular fractionation studies indicate that the majority of TFIID is displaced from the disassembling prophase nucleus to the mitotic cytoplasm around the time of nuclear envelope breakdown. However, a subpopulation of TFIID remains associated tightly with the condensed mitotic chromosomes. Metabolic labeling of mitotic cells revealed that several subunits of TFIID undergo mitosis-specific phosphorylation, but in spite of these changes, the TFIID complex remains intact. Functional analysis of purified TFIID from mitotic cells shows that phosphorylated forms are unable to direct activator-dependent transcription, but that this activity is restored upon dephosphorylation. These results demonstrate that TFIID regulation by phosphorylation is likely to have an important role in mitotic inhibition of RNA polymerase II transcription. In addition, they suggest a mechanism for regulating gene expression through the selective disruption of polymerase II promoter structures during mitosis.[Key Words: Mitosis; transcription; TFIID; TAFs; phosphorylation; chromosome localization] Received June 21, 1996; revised version accepted August 8, 1996.The assembly of chromatin into a regulated network of expressing and nonexpressing genes occurs in the context of the cell cycle. The pattern of gene expression that emerges is characterized both by a general stability from one cell division to the next, as well as by a capacity to establish new patterns of stable expression in a developmentally regulated way. These changes, from one stable state to another, are likely to require the temporal disruption of chromatin structure that is imposed by passage through the different stages of the cell cycle. For instance, DNA replication is invoked not only as the time of de novo chromatin assembly, but also as a time of temporary chromatin disruption, when transcription stops and newly synthesized transcription factors or other mediators of differentiative change can gain access to the DNA (for review, see Brown 1984;Villarreal 1991;Laurenson and Rine 1992;Wolffe 1994). Similarly, the dramatic changes in nuclear and chromatin structure that occur at mitosis in higher eukaryotes are accompanied by a general inhibition of transcription (Taylor 4present address:

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 extractderived proteins that interact with immobilized ER ligand binding domains in a 17␤-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)͞Mediator coactivator complex, which interacts with ER␣ and ER␤ in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220 ؊͞؊ fibroblasts reveal that these interactions depend on TRAP220, a TRAP͞Mediator 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 ER␣-TRAP͞Mediator complex from cultured cells expressing an epitopetagged ER␣. Finally, the complete TRAP͞Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP͞ Mediator, through TRAP220, in ER function. N uclear hormone receptors comprise a superfamily of transcriptional activators that bind to and, in a ligand-dependent manner, activate target genes involved in diverse physiological processes (1). Conserved nuclear receptor domains include the central DNA binding domain and a C-terminal ligand binding domain (LBD) that contains the ligand-induced AF-2 activation domain. Many receptors also contain N-terminal AF-1 activation domains that are less conserved (2). The function of nuclear receptors on target genes involves a variety of commonly used coactivators that in many cases show ligand-dependent interactions (directly or indirectly) with the AF-2 domain (3-5). One prominent group includes the p160͞SRC family and the interacting p300͞CBP and PCAF proteins, which function at least in part through intrinsic histone acetyltransferase activities that modify chromatin structure to facilitate subsequent receptor͞coactivator-mediated recruitment and͞or function of the general transcription machinery (3-5).Another coactivator of increasing importance for nuclear receptors is the thyroid hormone receptor-associated protein (TRAP)͞ Mediator complex. Although now known to mediate the activity of a number of distinct activators through specific subunit interactions (refs. 6 and 7; reviewed in refs. 8 and 9), TRAP͞Mediator was identified first through a ligand-dependent interaction with thyroid hormone receptor (TR) and shown to be essential for TR function on DNA templates in a reconstituted cell-free system (10). The TRAP220 subunit was identified as the main anchor for TR on the basis of a selective ligand-dependent interaction of isolated TRAP220 with TR (6), and analyses with TRAP220 Ϫ͞Ϫ fibroblasts confirmed a receptor-selective function for TRAP220 (11,12). The early demonstration of ligand-dependent interactions of TRAP220 with a number of other nuclear receptors furth...

Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAF II 250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerasepromoter complexes to an initiation-competent state.Activation of transcription of eukaryotic mRNA encoding genes by RNA polymerase II (pol II) involves three classes of transcription factors: general transcription factors (TFIIA, -B, -D, -E, -F, and -H), which act with pol II at core promoter elements to mediate specific initiation (1); activators, which typically function from upstream sites to transduce developmental and environmental signals to target genes; and coactivators, which operationally function to integrate the activities of general factors and activators. A set of general positive cofactors derived from the upstream stimulatory activity fraction (PC1, -2, -3, -4) and from other chromatographic fractions (PC5 and PC6) have been described in human cells (2), whereas studies in yeast have revealed an RNA pol IIassociated complex of apparently distinct cofactors (3). Similarly, some TATA-binding protein (TBP)-associated factors (TAFs), which together with TBP constitute TFIID, are also thought to possess distinct types of coactivator properties (4, 5). Precisely how any of these coactivators fulfills its role remains unclear. However, because they interact with both a variety of activators and several components of the basal machinery, current models of activated transcription view them as providing an adaptor function that facilitates formation of a functional preinitiation complex (PIC) via recruitment of the general transcription machinery (1, 2, 5, 6).PC4 was identified as a 15-kDa polypeptide that serves as a potent coactivator for a diverse group of activators in standard reconstituted in vitro transcription systems (7,8). It interacts both with a variety of activation domains and with TFIIA (7), and its cofactor function strongly correlates with its ability to bind double-stranded DNA (9).To decipher the mechanism by which PC4 stimulates transcription we have now employed a highly purified reconstituted in vitro transcription system to define the factors necessary for PC4 function. Our results reveal an intrinsic repress...

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 ␥ (PPAR␥)-stimulated adipogenesis of mouse embryonic fibroblasts (MEFs). However, the molecular mechanisms remain undefined. Here, we show an intracellular PPAR␥-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 PPAR␥ 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 PPAR␥-stimulated adipogenesis. Moreover, in undifferentiated MEFs, MED1/TRAP220 is dispensable both for PPAR␥-mediated target gene activation and for recruitment of Mediator to a PPAR response element on the aP2 target gene promoter. However, PPAR␥ 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 PPAR␥ function. These results indicate that there is a conditional requirement for MED1/TRAP220 and that a direct interaction between PPAR␥ and Mediator through MED1/TRAP220 is not essential either for PPAR␥-stimulated adipogenesis or for PPAR␥ target gene expression in cultured fibroblasts. As Mediator is apparently essential for PPAR␥ 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.Peroxisome proliferator-activated receptor ␥ (PPAR␥) is a key regulator of transcriptional pathways important for adipogenesis (34). PPAR␥ Ϫ/Ϫ mice show a total absence of both brown and white adipose tissue. Furthermore, retrovirus vector-mediated ectopic expression of PPAR␥ alone can stimulate mouse embryonic fibroblasts (MEFs) to undergo adipogenesis. In such cells, the expression of CCAAT/enhancer-binding protein ␣ (C/EBP␣), another key transcriptional regulator of adipogenesis, and adipogenesis markers such as aP2, fatty acid synthase (FAS), and adipsin are induced in a PPAR␥-dependent manner.PPAR␥ and other nuclear hormone receptors comprise a superfamily of DNA binding transcription factors. However, they also require various transcriptional coactivators to activate, in a ligand-dependent manner, transcription of the specific target genes important for cell growth, homeostasis, and differentiation (36). These transcription coactivators often exist as multiprotein complexes. They may act either through chromatin remodeling and histone modification, after recruitment by promoter-bound nuclear receptors, or at steps involving subsequent preinitiation complex formation or function (transcription initiation and elongation). Transcripti...

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.Nuclear receptors constitute a large superfamily of transcription factors that control diverse biological processes such as cell growth, differentiation, and homeostasis (reviewed in references 30 and 45). In the vast majority of cases, nuclear receptor functions are accomplished through a series of molecular events that are triggered by the binding of the cognate ligand. The liganded nuclear receptor, which binds to regulatory elements of target genes, orchestrates the assembly of multiprotein complexes containing various coactivators that directly or indirectly lead to enhanced activity of the preinitiation complex (PIC), which consists of RNA polymerase II (Pol II) and its associated basal transcriptional machinery (reviewed in references 37 and 38).The various coactivators that have been implicated in the function of nuclear receptors and other transcriptional activators fall into two broad categories. One group is comprised primarily of cofactors that facilitate the initial penetration of chromatin (10, 13, 50) either through ATP-dependent remodeling (SWI/SNF and various I-SWI-containing complexes) or through covalent protein modifications such as histone acetylation (p160 family members, CBP/p300, GCN5-containing SAGA complex) or methylation (CARM1 and PRMT) (41). The other group contains coactivators that were identified, for the most part, by biochemical assays utilizing naked DNA templates and are thus thought to function directly at the level of PIC formation or function (37). This group includes the TATA box binding protein (TBP)-associated factors in TFIID (46); positive cofactors PC1, PC2, PC3, PC4, and PC52 (37) derived from the USA fra...