Histone H3 Lys4 (H3K4) methylation is a prevalent mark associated with transcription activation. A common feature of several H3K4 methyltransferase complexes is the presence of three structural components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Here we report the first biochemical reconstitution of a functional four-component mixed-lineage leukemia protein-1 (MLL1) core complex. This reconstitution, combined with in vivo assays, allows direct analysis of the contribution of each component to MLL1 enzymatic activity and their roles in transcriptional regulation. Moreover, taking clues from a crystal structure analysis, we demonstrate that WDR5 mediates interactions of the MLL1 catalytic unit both with the common structural platform and with the histone substrate. Mechanistic insights gained from this study can be generalized to the whole family of SET1-like histone methyltransferases in mammals.
The thyroid hormone (T3) receptors (TRs) are hormone-dependent transcription factors that regulate expression of a variety of specific target genes. To help elucidate the mechanisms that underlie this transcriptional regulation and other potential TR activities, we used the yeast interaction trap to isolate clones encoding proteins that specifically interact with the ligand binding domain of the rat TR beta. Several such proteins, called Trips (TR-interacting proteins), were isolated from independent selections carried out either in the presence or absence of T3. Surprisingly, all of the Trips were dependent on hormone for interaction with the TR, with some interacting only when T3 is present and others only when it is absent. Nearly all of the Trips also show similar ligand-dependent interaction with the retinoid X receptor (RXR), but none interact with the glucocorticoid receptor under any conditions. The sequences of three of the Trips predict specific functional roles: one is an apparent human homolog of a yeast transcriptional coactivator, one is a new member of a class of nonhistone chromosomal proteins, and one contains a conserved domain associated with ubiquitination of specific target proteins. Consistent with the pleiotropic effects of TR and RXR, several other Trips show significant amino acid sequence similarity with proteins involved in various regulatory pathways. The inherent transcriptional activity of the Trips was tested in yeast, and a chimeric protein consisting of a fusion of Trip4 to the bacterial LexA repressor protein is a relatively strong transcriptional activator. Similar LexA fusions to Trip9 and Trip10 had no transcriptional activity on their own but, when coexpressed with both TR and RXR, conferred T3-dependent activation to a reporter gene controlled by LexA binding sites. We suggest that this indirect T3 response provides a novel mechanism for hormonal activation of gene expression, and that studies of the Trips will provide important insights into the specific mechanisms of action of TRs and other receptors.
The thyroid-hormone receptors are hormone-dependent transcription factors that control expression of many target genes. This regulation is presumably a consequence of hormone-dependent contacts between the receptors and the basal transcription machinery. We used the yeast two-hybrid system to identify a candidate human transcriptional mediator that interacts with both the thyroid-hormone receptor and the retinoid-X receptor in a ligand-dependent fashion. This protein, Trip1 (for thyroid-hormone-receptor interacting protein), shares striking sequence conservation with the yeast transcriptional mediator Sug1 (refs 6, 7). Here we show that Trip1 can functionally substitute for Sug1 in yeast, and that both proteins interact in vitro with the thyroid-hormone receptor, and with the transcriptional activation domains of yeast GAL4 and of herpes virus VP16.
Many transcription coactivators interact with nuclear receptors in a ligand-and C-terminal transactivation function (AF2)-dependent manner. We isolated a nuclear factor (designated ASC-2) with such properties by using the ligand-binding domain of retinoid X receptor as a bait in a yeast two-hybrid screening. ASC-2 also interacted with other nuclear receptors, including retinoic acid receptor, thyroid hormone receptor, estrogen receptor ␣, and glucocorticoid receptor, basal factors TFIIA and TBP, and transcription integrators CBP/p300 and SRC-1. In transient cotransfections, ASC-2, either alone or in conjunction with CBP/p300 and SRC-1, stimulated ligand-dependent transactivation by wild type nuclear receptors but not mutant receptors lacking the AF2 domain. Consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo, microinjection of anti-ASC-2 antibody abrogated the liganddependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector. Surprisingly, ASC-2 was identical to a gene previously identified during a search for genes amplified and overexpressed in breast and other human cancers. From these results, we concluded that ASC-2 is a bona fide transcription coactivator molecule of nuclear receptors, and its altered expression may contribute to the development of cancers.The nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in the promoters of target genes (for a review, see Ref.1). The superfamily includes receptors for a variety of small hydrophobic ligands such as steroids, T3, 1 and retinoids as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (reviewed in Ref.2). Functional analysis of nuclear receptors has shown that there are two major activation domains. The activation function-2 (AF-2) at the extreme C-terminal region of the ligandbinding domain (LBD) exhibits ligand-dependent transactivation, whereas the N-terminal activation function-1 contains a ligand-independent transactivation domain. The AF-2 region is conserved among nuclear receptors, and deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities. X-ray crystallographic studies of the LBD of nuclear receptors revealed that the ligand binding induces a major conformational change in the AF-2 region (3-7), suggesting that this region may play a critical role in mediating transactivation by a ligand-dependent interaction with coactivators. As expected, many coactivators fail to interact with AF-2 mutants of nuclear receptors (8 -10). Transcriptional activation of most nuclear receptors involves at least two separate processes as follows: derepression and activation. Repression is mediated in part by interaction of unliganded receptors with corepressors such as N-CoR (11) and SMRT (12). H...
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