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Short amphipathic peptides are capable of binding to transcriptional coactivators, often targeting the same binding surfaces as native transcriptional activation domains. However, they do so with modest affinity and generally poor selectivity, limiting their utility as synthetic modulators. Here we show that incorporation of a medium‐chain, branched fatty acid to the N‐terminus of one such heptameric lipopeptidomimetic (LPPM‐8) increases the affinity for the coactivator Med25 >20‐fold (Ki >100 μM to 4 μM), rendering it an effective inhibitor of Med25 protein–protein interactions (PPIs). The lipid structure, the peptide sequence, and the C‐terminal functionalization of the lipopeptidomimetic each influence the structural propensity of LPPM‐8 and its effectiveness as an inhibitor. LPPM‐8 engages Med25 through interaction with the H2 face of its activator interaction domain and in doing so stabilizes full‐length protein in the cellular proteome. Further, genes regulated by Med25‐activator PPIs are inhibited in a cell model of triple‐negative breast cancer. Thus, LPPM‐8 is a useful tool for studying Med25 and mediator complex biology and the results indicate that lipopeptidomimetics may be a robust source of inhibitors for activator‐coactivator complexes. [ABSTRACT FROM AUTHOR]

Background Information: Cancer cells acquire malignant characteristics and therapy resistance by employing the hypoxia‐inducible factor 1 (HIF‐1)‐dependent adaptive response to hypoxic microenvironment in solid tumors. Since the underlying molecular mechanisms remain unclear, difficulties are associated with establishing effective therapeutic strategies. Results: We herein identified DEAD‐box helicase 5 (DDX5) as a novel activator of HIF‐1 and found that it enhanced the heterodimer formation of HIF‐1α and HIF‐1β and facilitated the recruitment of the resulting HIF‐1 to its recognition sequence, hypoxia‐response element (HRE), leading to the expression of a subset of cancer‐related genes under hypoxia. Conclusions: This study reveals that the regulation of HIF‐1 recruitment to HRE is an important regulatory step in the control of HIF‐1 activity. Significance: The present study provides novel insights for the development of strategies to inhibit the HIF‐1‐dependent expression of cancer‐related genes. [ABSTRACT FROM AUTHOR]

Environmental, physiological, and pathological stimuli induce the misfolding of proteins, which results in the formation of aggregates and amyloid fibrils. To cope with proteotoxic stress, cells are equipped with adaptive mechanisms that are accompanied by changes in gene expression. The evolutionarily conserved mechanism called the heat shock response is characterized by the induction of a set of heat shock proteins (HSPs), and is mainly regulated by heat shock transcription factor 1 (HSF1) in mammals. We herein introduce the mechanisms by which HSF1 tightly controls the transcription of HSP genes via the regulation of pre‐initiation complex recruitment in their promoters under proteotoxic stress. These mechanisms involve the stress‐induced regulation of HSF1‐transcription complex formation with a number of coactivators, changes in chromatin states, and the formation of phase‐separated condensates through post‐translational modifications. [ABSTRACT FROM AUTHOR]

A significant proportion of estrogen receptor‐positive (ER+) breast cancer (BC) initially responds to endocrine therapy but eventually evolves into therapy‐resistant BC. Transcription factor AP‐2 gamma (TFAP2C) is a known regulator of ER activity, and high expression of TFAP2C is associated with a decreased response to endocrine therapies. PELP1 is a nuclear receptor coregulator, commonly overexpressed in BC, and its levels are correlated with poorer survival. In this study, we identified PELP1 as a novel interacting protein of TFAP2C. RNA‐seq analysis of PELP1 knockdown BC cells followed by transcription factor motif prediction pointed to TFAP2C being enriched in PELP1‐regulated genes. Gene set enrichment analysis (GSEA) revealed that the TFAP2C‐PELP1 axis induced a subset of common genes. Reporter gene assays confirmed PELP1 functions as a coactivator of TFAP2C. Mechanistic studies showed that PELP1‐mediated changes in histone methylation contributed to increased expression of the TFAP2C target gene RET. Furthermore, the TFAP2C‐PELP1 axis promoted the activation of the RET signaling pathway, which contributed to downstream activation of AKT and ERK pathways in ER+ BC cells. Concomitantly, knockdown of PELP1 attenuated these effects mediated by TFAP2C. Overexpression of TFAP2C contributed to increased cell proliferation and therapy resistance in ER+ BC models, while knockdown of PELP1 mitigated these effects. Utilizing ZR75‐TFAP2C xenografts with or without PELP1 knockdown, we provided genetic evidence that endogenous PELP1 is essential for TFAP2C‐driven BC progression in vivo. Collectively, our studies demonstrated that PELP1 plays a critical role in TFAP2C transcriptional and tumorigenic functions in BC and blocking the PELP1‐TFAP2C axis could have utility for treating therapy resistance. [ABSTRACT FROM AUTHOR]

The conserved transcription coactivator SAGA is comprised of several modules that are involved in activator binding, TBP binding, histone acetylation ( HAT) and deubiquitination ( DUB). Crosslinking and mass spectrometry, together with genetic and biochemical analyses, were used to determine the molecular architecture of the SAGA- TBP complex. We find that the SAGA Taf and Taf-like subunits form a TFIID-like core complex at the center of SAGA that makes extensive interactions with all other SAGA modules. SAGA- TBP binding involves a network of interactions between subunits Spt3, Spt8, Spt20, and Spt7. The HAT and DUB modules are in close proximity, and the DUB module modestly stimulates HAT function. The large activator-binding subunit Tra1 primarily connects to the TFIID-like core via its FAT domain. These combined results were used to derive a model for the arrangement of the SAGA subunits and its interactions with TBP. Our results provide new insight into SAGA function in gene regulation, its structural similarity with TFIID, and functional interactions between the SAGA modules. [ABSTRACT FROM AUTHOR]

Organ injury in sepsis is initially characterized by dysfunction without cell death and structural damage, and thus with the ability to recover organ function. Adaptive metabolic responses to sepsis can prevent bioenergetic failure and death. These studies were aimed at investigating the influence of sepsis on mitochondrial homeostasis, focusing on removal of dysfunctional mitochondria and restitution of a healthy mitochondrial population. These data demonstrate decreased hepatic oxidative phosphorylation by 31 ± 11% following murine cecal ligation and puncture (CLP) at 8 h and 34 ± 9% following LPS treatment in vitro at 12 h (P<0.05). In addition, there was a loss of mitochondrial membrane potential. Mitochondrial density and number initially decreased (relative area per micrograph of 64 ±10% at baseline vs. 39 ± 13% at 8 h following LPS; P<0.05) and was associated with an increase in autophagy and mitophagy. CLP-induced markers of mitochondrial biogenesis and mitochondrial number and density recovered over time. Furthermore, these data suggest that mitochondrial biogenesis was dependent on an autophagy and mitochondrial DNA/Toll-like receptor 9 (TLR9) signaling pathway. These results suggest that hepatocyte survival and maintenance of function in sepsis is dependent on a mitochondrial homeostasis pathway marked by mitophagy and biogenesis [ABSTRACT FROM AUTHOR]

c-Jun, the major component of the AP-1 transcription factor complex, has important functions in cellular proliferation and oncogenic transformation. The RING domain-containing protein RACO-1 functions as a c-Jun coactivator that molecularly links growth factor signalling to AP-1 transactivation. Here we demonstrate that RACO-1 is present as a nuclear dimer and that c-Jun specifically interacts with dimeric RACO-1. Moreover, RACO-1 is identified as a substrate of the arginine methyltransferase PRMT1, which methylates RACO-1 on two arginine residues. Arginine methylation of RACO-1 promotes a conformational change that stabilises RACO-1 by facilitating K63-linked ubiquitin chain formation, and enables RACO-1 dimerisation and c-Jun interaction. Abrogation of PRMT1 function impairs AP-1 activity and results in decreased expression of a large percentage of c-Jun target genes. These results demonstrate that arginine methylation of RACO-1 is required for efficient transcriptional activation by c-Jun/AP-1 and thus identify PRMT1 as an important regulator of c-Jun/AP-1 function. [ABSTRACT FROM AUTHOR]

The link between breast cancer and estrogen receptor (ER) is well established. The ER is a hormone-inducible transcription factor that, upon binding to its ligand, regulates the expression of a variety of genes mainly involved in cell proliferation and differentiation. Coactivators are proteins recruited by the hormone-activated receptor, which allow or enhance the ER transactivation functions by acting as chromatin remodeling enzymes or adaptors between ER and the transcriptional machinery. Our laboratory has previously identified the WW-domain binding protein-2 (WBP-2) as a bona fide coactivator of ER. However, the molecular mechanism underlying WBP-2 coactivation function was not clear yet. In this study, we explore and identify the mechanism by which WBP-2 acts as coactivator of ER. Our data show that WBP-2 is involved in the regulation of ER target genes, and its expression is required for the proper expression of some ER target genes. To clarify the molecular mechanism by which WBP-2 regulates ER function, we performed chromatin immunoprecipitation assays. We demonstrate here that WBP-2 binds to the ER target gene promoter pS2 promoter and is required for the binding of the phosphorylated form of RNA polymerase II (associated with active transcription/elongation) to the same promoter. Furthermore, we also show that WBP-2 is essential for the recruitment of the histone acetyl transferase p300, an important chromatin modifier enzyme and for histone acetylation at the same target region. Collectively, our data indicate that WBP-2 enhances ER transactivation function at certain genes by facilitating the recruitment and/or the stabilization of a histone modifier enzyme that favors a relaxed chromatin structure, permissive of transcription. © 2012 IUBMB Life, 65(1):76-84, 2013 [ABSTRACT FROM AUTHOR]

Abstract: The development of adipose tissue is a process which involves the concerted cooperation of numerous transcription factors together with their coactivators and corepressors. The peroxisome proliferator-activated receptor γ (PPARγ) is considered to be one of the master regulators of adipocyte differentiation. The presence of two functionally distinct types of adipose tissue, white and brown (WAT and BAT), requires an even more complex regulation of adipose tissue development. In this review we will focus on the role of PPARγ coregulators in adipogenesis and especially on the role of PPARγ coregulators in white and brown adipose tissue. Specificity in coregulator function in WAT and BAT may form an additional level of regulation of adipose tissue development. [ABSTRACT FROM AUTHOR]

The Mastermindlike (MAML) family, comprising human MAML1, MAML2, and MAML3, are transcriptional regulators in Notch signaling. MAML proteins contain two consensus sites for SUMOylation at Lysine217 and Lysine299 that are conserved in humans, mice, and Xenopus. In this report, we show that MAML1 is SUMOylated at both sites. The E2-conjugating enzyme UBC9 is essential for MAML1 SUMOylation, and the E3 ligase PIAS1 stimulates this activity. Mutation of the lysines abolishes SUMOylation of MAML1 and strongly increases MAML1-activated transcription in cell culture assays. The protease SENP1 reverses SUMOylation of MAML1 and potentiates the transcription factor activity of MAML1. Furthermore, SUMOylation enhances MAML1 interaction with HDAC7, which decreases MAML1 transcriptional activity. Taken together, our data indicate that SUMOylation of MAML1 is a mechanism for repressing MAML1 activity by influencing its interaction with HDAC7. [ABSTRACT FROM AUTHOR]

The transcriptional modulator Cited2 is induced by various biological stimuli including hypoxia, cytokines, growth factors, lipopolysaccharide (LPS) and flow shear. In this study, we report that Cited2 is required for mouse fetal liver development. Cited2−/− fetal liver displays hypoplasia with higher incidence of cell apoptosis, and exhibits disrupted cell-cell contact, disorganized sinusoidal architecture, as well as impaired lipid metabolism and hepatic gluconeogenesis. Furthermore, we demonstrated the physical and functional interaction of Cited2 with liver-enriched transcription factor HNF4α. Chromatin immunoprecipitation (ChIP) assays further confirmed the recruitment of Cited2 onto the HNF4α-responsive promoters and the reduced HNF4α binding to its target gene promoters in the absence of Cited2. Taken together, this study suggests that fetal liver defects in mice lacking Cited2 result, at least in part, from its defective coactivation function for HNF4α. [ABSTRACT FROM AUTHOR]

The transcription factor cAMP response element-binding protein (CREB) plays important roles in gene expression induced by cAMP signaling and is believed to be activated when its Ser133 is phosphorylated. However, the discovery of Ser133-independent activation by the activation of transducer of regulated CREB activity coactivators (TORC) and repression by salt inducible kinase cascades suggests that Ser133-independent regulation of CREB is also important. The activation and repression are mediated by the basic leucine zipper domain of CREB. In this review, we focus on the basic leucine zipper domain in the regulation of transcriptional activity of CREB and describe the functions of TORC and salt inducible kinase. [ABSTRACT FROM AUTHOR]

Steroid receptor coactivators (SRCs) exert profound effects on animal development and physiology. These coactivators are nuclear proteins and transcription co-regulators that function to facilitate the transcription initiation mediated by nuclear receptors, as well as by other well-known transcription factors. However, how these co-regulators are functionally regulated is poorly understood. During genome-wide screening for SRC-interacting proteins, we identified a novel ankyrin repeat containing protein, SIP (SRC-Interacting Protein), which interacts with SRC coactivators in the cytoplasm. We demonstrated that extracellular stimuli such as the addition of estrogen, induced phosphorylation of SIP in its PEST (Proline, Glutamate, Serine, and Threonine rich) domain by casein kinase II. The phosphorylation of SIP resulted in dissociation of SRC proteins from SIP in the cytoplasm and led to subsequent nuclear translocation of SRC proteins and gene coactivation. Both gain-of-function and loss-of-function experiments indicate that SIP functions to sequester SRC coactivators in the cytoplasm and buffer the availability of these coactivators, thus providing a mechanism for the regulation of the transcription regulators. [ABSTRACT FROM AUTHOR]

The ubiquitin–proteasome system plays an important role in a variety of cellular functions by means of its proteolytic activity. Interestingly, recent studies have indicated that the proteasome components are also integral parts of transcription complexes. In genome-wide screening for steroid receptor coactivator (SRC)-interacting proteins using yeast two-hybrid system, we found that the 20S proteasome β subunit LMP2 (Low Molecular mass Polypeptide 2) interacts directly with the SRC coactivators. We showed that LMP2 is required for estrogen receptor (ER)-mediated gene transcription and for estrogen-stimulated cell cycle progression. We found that LMP2-associated proteasome is recruited to the entire sequence of ER target genes, implicating a role for the proteasome in both transcription initiation and elongation. We demonstrated that the recruitment of LMP2 by SRC coactivators is necessary for cyclic association of ER-regulated transcription complexes on ER targets. These results revealed a mechanism by which the proteasome machinery is recruited in ER-mediated gene transcription. Our experiments also provided evidence implicating SRC coactivators in gene transcription elongation. [ABSTRACT FROM AUTHOR]

In yeast, the multisubunit SAGA (Spt-Ada-Gcn5-acetyltransferase) complex acts as a coactivator to recruit the TATA-binding protein (TBP) to the TATA box, a critical step in eukaryotic gene regulation. However, it is unclear which SAGA subunits are responsible for SAGA's direct interactions with TBP and precisely how SAGA recruits TBP to the promoter. We have used chemical crosslinking to identify Spt8 and Ada1 as potential SAGA subunits that interact with TBP, and we find that both Spt8 and SAGA bind directly to TBP monomer in competition with TBP dimer. We further find that Spt8 and SAGA compete with DNA to bind TBP rather than forming a triple complex. Our results suggest a handoff model for SAGA recruitment of TBP: instead of binding together with TBP at the TATA box, activator-recruited SAGA transfers TBP to the TATA box. This simple model can explain SAGA's observed ability to both activate and repress transcription. [ABSTRACT FROM AUTHOR]

Hepatocyte nuclear factor-4 (HNF-4α), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4α ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4α dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4α activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4α transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4α function. [ABSTRACT FROM AUTHOR]

Nuclear retinoic acid (RA) receptors (RARs) activate gene expression through dynamic interactions with coregulators in coordination with the ligand and phosphorylation processes. Here we show that during RA-dependent activation of the RARα isotype, the p160 coactivator pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3 is phosphorylated by p38MAPK. SRC-3 phosphorylation has been correlated to an initial facilitation of RARα-target genes activation, via the control of the dynamics of the interactions of the coactivator with RARα. Then, phosphorylation inhibits transcription via promoting the degradation of SRC-3. In line with this, inhibition of p38MAPK markedly enhances RARα-mediated transcription and RA-dependent induction of cell differentiation. SRC-3 phosphorylation and degradation occur only within the context of RARα complexes, suggesting that the RAR isotype defines a phosphorylation code through dictating the accessibility of the coactivator to p38MAPK. We propose a model in which RARα transcriptional activity is regulated by SRC-3 through coordinated events that are fine-tuned by RA and p38MAPK. [ABSTRACT FROM AUTHOR]

The interferon-β promoter has been studied extensively as a model system for combinatorial transcriptional regulation. In virus-infected cells the transcription factors ATF-2, c-Jun, interferon regulatory factor (IRF)-3, IRF-7 and NF-κB, and the coactivators p300/CBP play critical roles in the activation of this and other promoters. It remains unclear, however, why most other combinations of AP-1, IRF and Rel proteins fail to activate the interferon-β gene. Here we have explored how different IRFs may cooperate with other factors to activate transcription. First we showed in undifferentiated embryonic carcinoma cells that ectopic expression of either IRF-3 or IRF-7, but not IRF-1, was sufficient to allow virus-dependent activation of the interferon-β promoter. Moreover, the activity of IRF-3 and IRF-7 was strongly affected by promoter context, with IRF-7 preferentially being recruited to the natural interferon-β promoter. We fully reconstituted activation of this promoter in insect cells. Maximal synergy required IRF-3 and IRF-7 but not IRF-1, and was strongly dependent on the presence of p300/ CBP, even when these coactivators only modestly affected the activity of each factor by itself. These results suggest that specificity in activation of the interferon-β gene depends on a unique promoter context and on the role played by coactivators as architectural factors. [ABSTRACT FROM AUTHOR]

RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export. RHA has been reported to bind directly to the transcriptional coactivator, CREB-binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex.Using yeast twohybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor κB (NF-κB). The interaction between p65 and RHA was confirmed by glutathione-Stransferase pull-down assay in vitro, and by coimmunoprecipitation assay in vivo. In transient transfection assays, RHA enhanced NF-κB dependent reporter gene expression induced by p65, tumor necrosis factor-α, or NFκB inducing kinase. Themutant form ofRHAlackingATPbinding activity inhibited NF-κB dependent reporter gene expression induced by these activators. Moreover, depletion of RHA using short interfering RNA reduced the NF-κB dependent transactivation. These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF-κB. [ABSTRACT FROM AUTHOR]

Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. Mutation of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. We show that the conserved coactivator MBF1 is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. mbf1-null mutants live shorter than mbf1+ controls in the presence of hydrogen peroxide (H2O2). An AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1. We conclude that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress. [ABSTRACT FROM AUTHOR]

Nuclear receptors require coactivator binding in order to activate transcription of their cognate target genes. Ligands regulate nuclear receptor (NR)-mediated recruitment of coactivators by binding to the ligand-binding domain of the receptor and inducing a conformational change allowing for recognition of a specific motif contained within the coactivator protein. This motif is known as the NR box or LXXLL (where L is leucine and X is any amino acid) domain. Here, we review the discovery of the domain as well as its characterization. [ABSTRACT FROM AUTHOR]

Transcriptional coactivators such as p300 act as crucial elements in the eukaryotic gene regulation network. These proteins bind to various transcription factors which recruit them to specific gene regions whose chromatin structure subsequently is remodeled. Previously, we have shown that C/EBPβ recruits p300 by interacting with the E1A-binding site of the coactivator. We now show that C/EBPβ not only binds to p300 but also triggers massive phosphorylation of p300. This novel activity of C/EBPβ is dependent on the E1A-binding region of p300 as well as on several subdomains of C/EBPβ, all of which are involved in the p300-C/EBPβ interaction. We have identified several sites of C/EBPβ-inducible phosphorylation within the C-terminal domain of p300. Mutation of these sites substantially impairs the activity of p300 as a coactivator of C/EBPβ. Interestingly, phosphorylation of p300 is also triggered by other C/EBP family members as well as by various other transcription factors that interact with the E1A-binding domain of p300, suggesting that this novel phosphorylation mechanism may be of general relevance. [ABSTRACT FROM AUTHOR]

Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [ABSTRACT FROM AUTHOR]

We have used confocal microscopy to elucidate the effects of antiandrogens on nuclear localization of the androgen receptor (AR) with its transcriptional coactivator GRIP1. We show that the agonist-activated AR recruits GRIP1 to colocalize with the receptor in the nucleoplasm. By contrast, AR complexed to the antiandrogens hydroxyflutamide and bicalutamide fails to influence nuclear distribution of GRIP1. Likewise, the non-steroidal antiandrogens prevent the agonist-induced AR–GRIP1 colocalization from occurring. Androgen antagonists affect nuclear redistribution of AR–GRIP1 in a fashion that parallels their effects on the transcriptional activity of AR, in that the pure antagonists block GRIP1-dependent activation of AR function, whereas the mixed antagonist/agonist cyproterone acetate promotes both AR-driven redistribution of GRIP1 and activation of AR by GRIP1. [Copyright &y& Elsevier]

Transcriptional regulation by estrogen receptor α (ERα) involves protein-protein interactions among the receptor, its associated coactivators and the RNA polymerase II transcriptional machinery. We have used an in vitro chromatin assembly and transcription system to examine the biochemistry of interactions among ERα, the SRC proteins and p300/CBP. Using polypeptides designed to block specific receptor-cofactor or cofactor-cofactor interactions, we show that interactions among ERα, its coactivators and the RNA pol II machinery are all required for ERamediated transcription. Furthermore, we show that ERα-SRC-p300/CBP interactions are necessary and sufficient for the targeted acetylation of nucleosomal histones on estrogen-responsive promoters in the absence of transcription. The protein-protein interactions required for histone acetylation constitute a subset of the interactions required for transcriptional activation. Finally, we show that the major role of SRC-p300/CBP interactions is to enhance ERα-mediated transcription initiation, and they have little or no role in stimulating subsequent rounds of transcription. Together, our results indicate a specific role for the SRC and p300/CBP coactivators, as well as targeted histone acetylation, in ERα-mediated transcription. [ABSTRACT FROM AUTHOR]

The BOB.1/OBF.1 coactivator is critically involved in mediating octamer-dependent transcriptional activity in B lymphocytes. Mice tacking this coactivator show various defects in B-cell development, most notably they completely tack germinal centers. Consistent with this phenotype, BOB.1/OBF.1 levels are massively upregulated in germinal center B cells as compared with resting B cells. We have addressed the mechanism of upregulation and found that only a minor part of this regulation can be attributed to increased levels of BOB.1/OBF.1-specific mRNA. Apparently, BOB.1/ OBF.1 is also regulated at the protein level. In support of this suggestion we have been able to identify two related BOB.1/OBF.1 interacting proteins, SIAH1 and SIAH2, in a yeast two-hybrid screen. SIAH1 and SIAH2 are known regulators of protein stability. Cotransfection experiments revealed that coexpression of SIAH results in a destabilization of BOB.1/ OBF.1 protein without affecting mRNA levels. Further- more, proteasome inhibitors block the degradation of BOB..1/OBF.1 protein. Finally, B-cell receptor cross-linking also resulted in the degradation of BOB.1/ OBF.1 and consequently reduced transcriptional activation of BOB.1/OBF.1-dependent reporters. [ABSTRACT FROM AUTHOR]

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor a (hERa) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hERa A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hERa in MCF7 cells and in partially purified complexes associated with hERa from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hERa and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hERa. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ERa AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins. [ABSTRACT FROM AUTHOR]

Transcription of the herpes simplex virus 1 (HSV-1) immediate early (IE) genes is determined by multi-protein enhancer complexes. The core enhancer assembly requires the interactions of the POU-homeo-domain protein Oct-1, the viral transactivator αTIF and the cellular factor C1 (HCF). In this context, the C1 factor interacts with each protein to assemble the stable enhancer complex. In addition, the IE enhancer cores contain adjacent binding sites for other cellular transcription factors such as Sp1 and GA-binding protein (GABP). In this study, a direct interaction of the C1 factor with GABP is demonstrated, defining the C1 factor as the critical coordinator of the enhancer complex assembly. In addition, mutations that reduce the GABP transactivation potential also impair the C1-GABP interaction, indicating that the C1 factor functions as a novel coactivator of GABP-mediated transcription. The interaction and coordinated assembly of the enhancer proteins by the C1 factor may be critical for the regulation of the HSV lytic­latent cycle. [ABSTRACT FROM AUTHOR]

The control of target gene expression by nuclear receptors requires the recruitment of multiple cofactors. However, the exact mechanisms by which nuclear receptor-cofactor interactions result in tissue-specific gene regulation are unclear. Here we characterize a novel tissue-specific coactivator for the androgen receptor {AR), which is identical to a previously reported protein FHL2/DRAL with unknown function. In the adult, FHL2 is expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus. FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo. In an agonist- and AF-2-dependent manner FHL2 selectively increases the transcriptional activity of the AR, but not that of any other nuclear receptor. In addition, the transcription of the prostate-specific AR target gene probasin is coactivated by FHL2. Taken together, our data demonstrate that FHL2 is the first LIM-only coactivator of the AR with a unique tissue-specific expression pattern. [ABSTRACT FROM AUTHOR]

Ligand activation of the nuclear receptor PPARγ induces adipogenesis and increases insulin sensitivity, while activation of other PPAR isoforms (-α and -δ) induces little or no fat cell differentiation. Expression and activation of chimeras formed between PPARγ and PPARδ in fibroblasts has allowed us to localize a major domain of PPARγ responsible for adipogenesis to the N-terminal 138 amino acids, a region with AF-1 transcriptional activity. Using this region of PPARγ as bait, we have used a yeast two-hybrid screen to clone a novel protein, termed PGC-2, containing a partial SCAN domain. PGC-2 binds to and increases the transcriptional activity of PPARγ but does not interact with other PPARs or most other nuclear receptors. Ectopic expression of PGC-2 in preadipocytes containing endogenous PPARγ causes a dramatic increase in fat cell differentiation at both the morphological and molecular levels. These results suggest that interactions between PGC-2, a receptor isoform-selective cofactor and PPARγ contribute to the adipogenic action of this receptor. [ABSTRACT FROM AUTHOR]

Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. Here we show that in primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is regulated synergistically by the B-cell antigen receptor, CD40L and interleukin signaling pathways. Consistent with the requirement for multiple T cell-dependent signals to induce OCA-B, we find that OCA-B protein is highly expressed in germinal center B cells. Accordingly, germinal center formation is blocked completely in the absence of OCA-B expression in B cells, whereas the helper functions of OCA-B-deficient T cells are indistinguishable from controls. The requirement for OCA-B expression in B cells is germinal center specific since the development of primary B cell follicles, the marginal zone and plasma cells are all intact. Thus, OCA-B is the first example of a transcriptional coactivator that is both synergistically induced by and required for integration of signals that mediate cell fate decisions. [ABSTRACT FROM AUTHOR]

Resistance to thyroid hormone (RTH) has hitherto been associated with thyroid hormone β receptor (TRβ) mutations which cluster in two regions (αα310­353 and 429–461) of the hormone-binding domain and closely approximate the ligand-binding cavity. Here, we describe a third cluster of RTH mutations extending from 234–282 which constitute a third boundary of the ligand pocket. One mutant, T277A, exhibits impaired transactivation which is disproportionate to its mildly reduced ligand affinity (Ka). T3-dependent recruitment of coactivators (SRC-1, ACTR) by mutant receptor­RXR heterodimers was reduced in comparison with wild-type. Cotransfection of SRC-1 restored transactivation by T277A. In the TRβ crystal structure this helix 3 residue is surface-exposed and is in close proximity to residues L454 and E457 in helix 12 which are known to be critical for coactivator interaction, suggesting that they all constitute part of a receptor-coactivator interface. The transcriptional function of other mutants (A234T, R243W/Q, A268D, Δ276I, A279V, R282S) in this cluster correlated with their reduced Ka and they inhibited wild-type TRβ action in a dominant negative manner. DNA binding, heterodimerization and corepressor recruitment were preserved in all mutants, signifying the importance of these attributes for dominant negative activity and correlating with the absence of natural mutations in regions bordering the third cluster which mediate these functions. [ABSTRACT FROM AUTHOR]