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Eukaryotic transcription factors activate gene expression with their DNA-binding domains and activation domains. DNA-binding domains bind the genome by recognizing structurally related DNA sequences; they are structured, conserved, and predictable from protein sequences. Activation domains recruit chromatin modifiers, coactivator complexes, or basal transcriptional machinery via structurally diverse protein-protein interactions. Activation domains and DNA-binding domains have been called independent, modular units, but there are many departures from modularity, including interactions between these regions and overlap in function. Compared to DNA-binding domains, activation domains are poorly understood because they are poorly conserved, intrinsically disordered, and difficult to predict from protein sequences. This review, organized around commonly asked questions, describes recent progress that the field has made in understanding the sequence features that control activation domains and predicting them from sequence.

Skin provides a physical and immune barrier to protect the body from foreign substances, microbial invasion, and desiccation. Aging reduces the barrier function of skin and its rate of repair. Aged skin exhibits decreased mitochondrial function and prolonged low-level inflammation that can be seen in other organs with aging. Peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), an important transcriptional coactivator, plays a central role in modulating mitochondrial function and antioxidant production. Mitochondrial function and inflammation have been linked to epidermal function, but the mechanisms are unclear. The aim of this review is to discuss the mechanisms by which PGC-1α might exert a positive effect on aged skin barrier function. Initially, we provide an overview of the function of skin under physiological and aging conditions, focusing on the epidermis. We then discuss mitochondrial function, oxidative stress, cellular senescence, and inflamm-aging, the chronic low-level inflammation observed in aging individuals. Finally, we discuss the effects of PGC-1α on mitochondrial function, as well as the regulation and role of PGC-1α in the aging epidermis. [ABSTRACT FROM AUTHOR]

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]

Transcriptional dysregulation of genes is a hallmark of tumors and can serve as targets for cancer drug development. However, it is extremely challenging to develop small-molecule inhibitors to target abnormally expressed transcription factors (TFs) except for the nuclear receptor family of TFs. Little is known about the interaction between TFs and transcription cofactors in gastroesophageal adenocarcinoma (GEA) or the therapeutic effects of targeting TF and transcription cofactor complexes. In this study, we found that ETS homologous factor (EHF) expression is promoted by a core transcriptional regulatory circuitry (CRC), specifically ELF3-KLF5-GATA6, and interference with its expression suppressed the malignant biological behavior of GEA cells. Importantly, we identified Ajuba LIM protein (AJUBA) as a new coactivator of EHF that cooperatively orchestrates transcriptional network activity in GEA. Furthermore, we identified KRAS signaling as a common pathway downstream of EHF and AJUBA. Applicably, dual targeting of EHF and AJUBA by lipid nanoparticles cooperatively attenuated the malignant biological behaviors of GEA in vitro and in vivo. In conclusion, EHF is upregulated by the CRC and promotes GEA malignancy by interacting with AJUBA through the KRAS pathway. Targeting of both EHF and its coactivator AJUBA through lipid nanoparticles is a novel potential therapeutic strategy. EHF is upregulated by a core transcriptional regulatory circuitry and promotes malignancy of gastroesophageal adenocarcinoma via AJUBA coactivation through the KRAS pathway, offering a novel dual-targeting therapeutic strategy. [Display omitted] [ABSTRACT FROM AUTHOR]

Three peroxisome proliferator-activated receptor subtypes, PPARα, PPAR(ß/)δ, and PPARγ, exert ligand-dependent transcriptional control in concert with retinoid X receptors (RXRs) on various gene sets harboring PPAR response elements (PPREs) in their promoter regions. Ligand-bound PPAR/RXR complexes do not directly regulate transcription; instead, they recruit multiprotein coactivator complexes to specific genomic regulatory loci to cooperatively activate gene transcription. Several coactivators are expressed in a single cell; however, a ligand-bound PPAR can be associated with only one coactivator through a consensus LXXLL motif. Therefore, altered gene transcription induced by PPAR subtypes/agonists may be attributed to the recruitment of various coactivator species. Using a time-resolved fluorescence resonance energy transfer assay, we analyzed the recruitment of four coactivator peptides (PGC1α, CBP, SRC1, and TRAP220) to human PPARα/δ/γ-ligand-binding domains (LBDs) using eight PPAR dual/pan agonists (bezafibrate, fenofibric acid, pemafibrate, pioglitazone, elafibranor, lanifibranor, saroglitazar, and seladelpar) that are/were anticipated to treat nonalcoholic fatty liver disease. These agonists all recruited four coactivators to PPARα/γ-LBD with varying potencies and efficacy. Only five agonists (bezafibrate, pemafibrate, elafibranor, lanifibranor, and seladelpar) recruited all four coactivators to PPARδ-LBD, and their concentration-dependent responses differed from those of PPARα/γ-LBD. These results indicate that altered gene expression through consensus PPREs by different PPAR subtypes/agonists may be caused, in part, by different coactivators, which may be responsible for the unique pharmacological properties of these PPAR agonists. [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]

How distal cis-regulatory elements (e.g., enhancers) communicate with promoters remains an unresolved question of fundamental importance. Although transcription factors and cofactors are known to mediate this communication, the mechanism by which diffusible molecules relay regulatory information from one position to another along the chromosome is a biophysical puzzle-one that needs to be revisited in light of recent data that cannot easily fit into previous solutions. Here we propose a new model that diverges from the textbook enhancer-promoter looping paradigm and offer a synthesis of the literature to make a case for its plausibility, focusing on the coactivator p300.

Skin provides a physical and immune barrier to protect the body from foreign substances, microbial invasion, and desiccation. Aging reduces the barrier function of skin and its rate of repair. Aged skin exhibits decreased mitochondrial function and prolonged low-level inflammation that can be seen in other organs with aging. Peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α), an important transcriptional coactivator, plays a central role in modulating mitochondrial function and antioxidant production. Mitochondrial function and inflammation have been linked to epidermal function, but the mechanisms are unclear. The aim of this review is to discuss the mechanisms by which PGC-1α might exert a positive effect on aged skin barrier function. Initially, we provide an overview of the function of skin under physiological and aging conditions, focusing on the epidermis. We then discuss mitochondrial function, oxidative stress, cellular senescence, and inflamm-aging, the chronic low-level inflammation observed in aging individuals. Finally, we discuss the effects of PGC-1α on mitochondrial function, as well as the regulation and role of PGC-1α in the aging epidermis.

Female mosquitoes transmit numerous devastating human diseases because they require vertebrate blood meal for egg development. MicroRNAs (miRNAs) play critical roles across multiple reproductive processes in female Aedes aegypti mosquitoes. However, how miRNAs are controlled to coordinate their activity with the demands of mosquito reproduction remains largely unknown. We report that the ecdysone receptor (EcR)-mediated 20-hydroxyecdysone (20E) signaling regulates miRNA expression in female mosquitoes. EcR RNA-interference silencing linked to small RNA-sequencing analysis reveals that EcR not only activates but also represses miRNA expression in the female mosquito fat body, a functional analog of the vertebrate liver. EcR directly represses the expression of clustered miR-275 and miR-305 before blood feeding when the 20E titer is low, whereas it activates their expression in response to the increased 20E titer after a blood meal. Furthermore, we find that SMRTER, an insect analog of the vertebrate nuclear receptor corepressors SMRT and N-CoR, interacts with EcR in a 20E-sensitive manner and is required for EcR-mediated repression of miRNA expression in Ae. aegypti mosquitoes. In addition, we demonstrate that miR-275 and miR-305 directly target glutamate semialdehyde dehydrogenase and AAEL009899, respectively, to facilitate egg development. This study reveals a mechanism for how miRNAs are controlled by the 20E signaling pathway to coordinate their activity with the demands of mosquito reproduction.

Nuclear receptor coregulators are the principal regulators of Estrogen Receptor (ER)-mediated transcription. ERβ, an ER subtype first identified in 1996, is associated with poor outcomes in breast cancer (BCa) subtypes, and the coexpression of the ERβ1 isoform and AIB-1 and TIF-2 coactivators in BCa-associated myofibroblasts is associated with high-grade BCa. We aimed to identify the specific coactivators that are involved in the progression of ERβ-expressing BCa. ERβ isoforms, coactivators, and prognostic markers were tested using standard immunohistochemistry. AIB-1, TIF-2, NF-kB, p-c-Jun, and/or cyclin D1 were differentially correlated with ERβ isoform expression in the BCa subtypes and subgroups. The coexpression of the ERβ5 and/or ERβ1 isoforms and the coactivators were found to be correlated with a high expression of P53, Ki-67, and Her2/neu and large-sized and/or high-grade tumors in BCa. Our study supports the notion that ERβ isoforms and coactivators seemingly coregulate the proliferation and progression of BCa and may provide insight into the potential therapeutic uses of the coactivators in BCa.

Three peroxisome proliferator-activated receptor subtypes, PPARα, PPAR(ß/)δ, and PPARγ, exert ligand-dependent transcriptional control in concert with retinoid X receptors (RXRs) on various gene sets harboring PPAR response elements (PPREs) in their promoter regions. Ligand-bound PPAR/RXR complexes do not directly regulate transcription; instead, they recruit multiprotein coactivator complexes to specific genomic regulatory loci to cooperatively activate gene transcription. Several coactivators are expressed in a single cell; however, a ligand-bound PPAR can be associated with only one coactivator through a consensus LXXLL motif. Therefore, altered gene transcription induced by PPAR subtypes/agonists may be attributed to the recruitment of various coactivator species. Using a time-resolved fluorescence resonance energy transfer assay, we analyzed the recruitment of four coactivator peptides (PGC1α, CBP, SRC1, and TRAP220) to human PPARα/δ/γ-ligand-binding domains (LBDs) using eight PPAR dual/pan agonists (bezafibrate, fenofibric acid, pemafibrate, pioglitazone, elafibranor, lanifibranor, saroglitazar, and seladelpar) that are/were anticipated to treat nonalcoholic fatty liver disease. These agonists all recruited four coactivators to PPARα/γ-LBD with varying potencies and efficacy. Only five agonists (bezafibrate, pemafibrate, elafibranor, lanifibranor, and seladelpar) recruited all four coactivators to PPARδ-LBD, and their concentration-dependent responses differed from those of PPARα/γ-LBD. These results indicate that altered gene expression through consensus PPREs by different PPAR subtypes/agonists may be caused, in part, by different coactivators, which may be responsible for the unique pharmacological properties of these PPAR agonists.

Binding of general transcription factors TFIID and TFIIA to basal promoters is rate-limiting for transcriptional initiation of eukaryotic protein-coding genes. Consequently, activator proteins interacting with subunits of TFIID and/or TFIIA can drastically increase the rate of initiation events. Yeast transcriptional activator Ino2 interacts with several Taf subunits of TFIID, among them the multifunctional Taf1 protein. In contrast to mammalian Taf1, yeast Taf1 lacks bromodomains which are instead encoded by separate proteins Bdf1 and Bdf2. In this work, we show that Bdf1 not only binds to acetylated histone H4 but can also be recruited by Ino2 and unrelated activators such as Gal4, Rap1, Leu3 and Flo8. An activator-binding domain was mapped in the N-terminus of Bdf1. Subunits Toa1 and Toa2 of yeast TFIIA directly contact sequences of basal promoters and TFIID subunit TBP but may also mediate the influence of activators. Indeed, Ino2 efficiently binds to two separate structural domains of Toa1, specifically with its N-terminal four-helix bundle structure required for dimerization with Toa2 and its C-terminal β-barrel domain contacting TBP and sequences of the TATA element. These findings complete the functional analysis of yeast general transcription factors Bdf1 and Toa1 and identify them as targets of activator proteins. [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]

Recent advances in cryo-electron microscopy have led to multiple structures of Mediator in complex with the RNA polymerase II (Pol II) transcription initiation machinery. As a result we now hold in hands near-complete structures of both yeast and human Mediator complexes and have a better understanding of their interactions with the Pol II pre-initiation complex (PIC). Herein, we provide a summary of recent achievements and discuss their implications for future studies of Mediator and its role in gene regulation. [ABSTRACT FROM AUTHOR]

Uncovering the cis -regulatory code that governs when and how much each gene is transcribed in a given genome and cellular state remains a central goal of biology. Here, we discuss major layers of regulation that influence how transcriptional outputs are encoded by DNA sequence and cellular context. We first discuss how transcription factors bind specific DNA sequences in a dosage-dependent and cooperative manner and then proceed to the cofactors that facilitate transcription factor function and mediate the activity of modular cis -regulatory elements such as enhancers, silencers, and promoters. We then consider the complex and poorly understood interplay of these diverse elements within regulatory landscapes and its relationships with chromatin states and nuclear organization. We propose that a mechanistically informed, quantitative model of transcriptional regulation that integrates these multiple regulatory layers will be the key to ultimately cracking the cis -regulatory code. The cis -regulatory code dictates how DNA sequence controls quantitative transcription levels of each gene depending on cellular context. Kim and Wysocka review progress and challenges in understanding the layers of this code, from transcription factor binding to cofactor recruitment and ultimately cis -regulatory element specificity and function within complex regulatory landscapes. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor and has been exploited as a well-established druggable target for the treatment of diabetes mellitus (DM). Traditionally, small-molecule compounds have been developed to attack at the ligand site and Ser273 phosphorylation site of PPARγ. In this study, we derived helical peptide segments from the LXXLL motif region of coactivator proteins as antidiabetic PPARγ antagonists, which were expected to competitively disrupt the native interaction between PPARγ and its cognate coactivators by rebinding at PPARγ coactivator site. Structural analysis, dynamics simulation and energetics dissection revealed that these peptides cannot be well folded into active helical structure when splitting from the protein context of their parent coactivators and exhibit a large flexibility and intrinsic disorder in the free state, which would, therefore, incur a considerable entropy penalty upon rebinding to PPARγ. Hydrocarbon stapling strategy was employed to constrain these free coactivator peptides into ordered helical conformation, thus largely minimizing unfavorable entropy penalty but having only a moderate effect on favorable enthalpy contribution. The computational findings were further substantiated by fluorescence-based assays; the binding affinity of three potent SRC1, NCoA6 and p300 coactivator peptides to PPARγ was observed to be improved by 7.2-fold, 4.2-fold and 5.7-fold upon the stapling, which were also measured to have an efficient competitive potency with their unstapled counterparts for PPARγ coactivator site, with CC50 = 0.096, 0.12 and 0.18 μM, respectively. [ABSTRACT FROM AUTHOR]

The peroxisome proliferator-activated receptor c coactivator 1 α (PGC-1α) is central to the regulation of cellular and mitochondrial energy homeostasis in mammals, but its role in other vertebrates remains unclear. Indeed, previous work suggests extensive structural and functional divergence of PGC-1α in teleosts but this remains to be directly tested. Here, we describe the initial characterization of heterozygous PGC-1a mutant zebrafish lines created by CRISPR-Cas9 disruptions of an evolutionarily conserved regulatory region of the PGC-1α proximal promoter. Using qPCR, we confirmed the disruption of PGC-1a gene expression in striated muscle, leading to a simultaneous fourfold increase in mixed skeletal muscle PGC-1a mRNA levels and an opposite fourfold downregulation in cardiac muscle. In mixed skeletal muscle, most downstream effector genes were largely unaffected yet two mitochondrial lipid transporters, carnitine palmitoyltransferase-1 and -2, were strongly induced. Conversely, PGC-1α depression in cardiac muscle reduced the expression of several transcriptional regulators (estrogen-related receptor a, nuclear respiratory factor 1, and PGC-1β) without altering metabolic gene expression. Using high-resolution respirometry, we determined that white muscle exhibited increased lipid oxidative capacity with little difference in markers of mitochondrial abundance. Finally, using whole animal intermittent respirometry, we show that mutant fish exhibit a twofold higher basal metabolism than their wild-type counterparts. Altogether, this new model confirms a central but complex role for PGC-1α in mediating energy utilization in zebrafish, and we propose its use as a valuable tool to explore the intricate regulatory pathways of energy homeostasis in a popular biomedical model. [ABSTRACT FROM AUTHOR]

Abstract The SAGA complex is an evolutionarily conserved transcriptional coactivator that regulates gene expression through its histone acetyltransferase and deubiquitylase activities, recognition of specific histone modifications, and interactions with transcription factors. Multiple lines of evidence indicate the existence of distinct variants of SAGA among organisms as well as within a species, permitting diverse functions to dynamically regulate cellular pathways. Our co-expression analysis of genes encoding human SAGA components showed enrichment in reproductive organs, brain tissues and the skeletal muscle, which corresponds to their established roles in developmental programs, emerging roles in neurodegenerative diseases, and understudied functions in specific cell types. SAGA subunits modulate growth, development and response to various stresses from yeast to plants and metazoans. In metazoans, SAGA further participates in the regulation of differentiation and maturation of both innate and adaptive immune cells, and is associated with initiation and progression of diseases including a broad range of cancers. The evolutionary conservation of SAGA highlights its indispensable role in eukaryotic life, thus deciphering the mechanisms of action of SAGA is key to understanding fundamental biological processes throughout evolution. To illuminate the diversity and conservation of this essential complex, here we discuss variations in composition, essentiality and co-expression of component genes, and its prominent functions across Fungi, Plantae and Animalia kingdoms.

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.

As the downstream effectors of Hippo pathway, YAP/TAZ are identified to participate in organ growth, regeneration and tumorigenesis. However, owing to lack of a DNA-binding domain, YAP/TAZ usually act as coactivators and cooperate with other transcription factors or partners to mediate their transcriptional outputs. In this article, we first present an overview of the core components and the upstream regulators of Hippo-YAP/TAZ signaling in mammals, and then systematically summarize the identified transcription factors or partners that are responsible for the downstream transcriptional output of YAP/TAZ in various cancers. [ABSTRACT FROM AUTHOR]

The androgen receptor (AR) plays an essential role in the development of prostate cancer, and androgen-deprivation therapy is used as a first-line treatment for prostate cancer. However, under androgen-deprivation therapy, castration-resistant prostate cancer inevitably arises, suggesting that the interacting transcriptional coregulators of AR are promising targets for developing novel therapeutics. In this study, we used novel proteomic techniques to evaluate the AR interactome, including biochemically labile binding proteins, which might go undetected by conventional purification methods. Using rapid immunoprecipitation mass spectrometry of endogenous proteins, we identified enhanced at puberty 1 (EAP1) as a novel AR coregulator, whereas its interaction with AR could not be detected under standard biochemical conditions. EAP1 enhanced the transcriptional activity of AR via the E3 ubiquitin ligase activity, and its ubiquitination substrate proteins included AR and HDAC1. Furthermore, in prostate cancer specimens, EAP1 expression was significantly correlated with AR expression as well as a poor prognosis of prostate cancer. Together, these results suggest that EAP1 is a novel AR coregulator that promotes AR activity and potentially plays a role in prostate cancer progression. [ABSTRACT FROM AUTHOR]

The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating-type genes. We report here a tup1(S649F) mutant that displays mating irregularities and an a-predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 occupancy changes in mutant vs wild type in both a and a cells. Increased Tup1(S649F) occupancy tended to occur upstream of upregulated genes, whereas locations with decreased occupancy usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including a2. We also found that large changes in mating-type-specific gene expression between a and a or between mutant and wild type were not easily explained by the range of Tup1 occupancy levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 occupancy upstream of the a-specific gene MFA2 and the a-specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with the identification of additional mating-related genes upregulated in the tup1(S649F) a strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated. [ABSTRACT FROM AUTHOR]

Gene transcription is strictly controlled by transcriptional complexes, which are assemblies of transcription factors, transcriptional regulators, and co-regulators. Mammalian genomes encode two C-terminal-binding proteins (CtBPs), CtBP1 and CtBP2, which are both well-known transcriptional corepressors of oncogenic processes. Their overexpression in tumors is associated with malignant behavior, such as uncontrolled cell proliferation, migration, and invasion, as well as with an increase in the epithelial-mesenchymal transition. CtBPs coordinate with other transcriptional regulators, such as histone deacetylases (HDACs) and histone acetyltransferases (p300 and CBP [CREBP-binding protein]) that contain the PXDLS motif, and with transcription factors to assemble transcriptional complexes that dock onto the promoters of genes to initiate gene transcription. Emerging evidence suggests that CtBPs function as both corepressors and coactivators in different biological processes ranging from apoptosis to inflammation and osteogenesis. Therapeutic targeting of CtBPs or the interactions required to form transcriptional complexes has also shown promising effects in preventing disease progression. This review summarizes the most recent progress in the study of CtBP functions and therapeutic inhibitors in different biological processes. This knowledge may enable a better understanding of the complexity of the roles of CtBPs, while providing new insights into therapeutic strategies that target CtBPs. [ABSTRACT FROM AUTHOR]

Summary: PBRM1, a subunit of the PBAF coactivator complex that transcription factors use to activate target genes, is genetically inactivated in almost all clear cell renal cell cancers (RCCs). Using unbiased proteomic analyses, we find that PAX8, a master transcription factor driver of proximal tubule epithelial fates, recruits PBRM1/PBAF. Reverse analyses of the PAX8 interactome confirm recruitment specifically of PBRM1/PBAF and not functionally similar BAF. More conspicuous in the PAX8 hub in RCC cells, however, are corepressors, which functionally oppose coactivators. Accordingly, key PAX8 target genes are repressed in RCC versus normal kidneys, with the loss of histone lysine-27 acetylation, but intact lysine-4 trimethylation, activation marks. Re-introduction of PBRM1, or depletion of opposing corepressors using siRNA or drugs, redress coregulator imbalance and release RCC cells to terminal epithelial fates. These mechanisms thus explain RCC resemblance to the proximal tubule lineage but with suppression of the late-epithelial program that normally terminates lineage-precursor proliferation.

Human Hippo signaling pathway has been recognized as a new and promising therapeutic target of gastrointestinal cancers, which is regulated by the intermolecular recognition between the TEA domain (TEAD) transcription factor and its prime coactivators. The coactivator proteins adopt two hotspot sites, namely α-helix and Ω-loop, to interact with TEAD. Here, we demonstrate that both the α-helix and Ω-loop peptides cannot maintain in structured state when splitting from the full-length coactivator proteins; they exhibit a large intrinsic disorder in free state that prevents the coactivator peptide recognition by TEAD. Rational design is used to optimize the interfacial residues of coactivator α-helix peptides, which can effectively improve the favorable direct readout effect upon the peptide binding to TEAD. Chemical modification is employed to constrain the free α-helix peptide into native ordered conformation. The method introduces an all-hydrocarbon bridge across i and i + 4 residues to stabilize the helical structure of a free coactivator peptide, which can considerably reduce the unfavorable indirect readout effect upon the peptide binding to TEAD. The all-hydrocarbon bridge is designed to point out of the TEAD–peptide complex interface, which would not disrupt the direct intermolecular interaction between the TEAD and peptide. Therefore, the stapling only improves peptide affinity, but does not alter peptide specificity, to TEAD. Affinity assay confirms that the binding potency of coactivator α-helix peptides is improved substantially by >5-fold upon the rational design and chemical modification. Structural analysis reveals that the optimized/stapled peptides can form diverse nonbonded interactions such as hydrogen bonds and hydrophobic contacts with TEAD, thus conferring stability and specificity to the TEAD–peptide complex systems. [ABSTRACT FROM AUTHOR]

Female mosquitoes transmit numerous devastating human diseases because they require vertebrate blood meal for egg development. MicroRNAs (miRNAs) play critical roles across multiple reproductive processes in female Aedes aegypti mosquitoes. However, how miRNAs are controlled to coordinate their activity with the demands of mosquito reproduction remains largely unknown. We report that the ecdysone receptor (EcR)-mediated 20-hydroxyecdysone (20E) signaling regulates miRNA expression in female mosquitoes. EcR RNA-interference silencing linked to small RNA-sequencing analysis reveals that EcR not only activates but also represses miRNA expression in the female mosquito fat body, a functional analog of the vertebrate liver. EcR directly represses the expression of clustered miR-275 and miR-305 before blood feeding when the 20E titer is low, whereas it activates their expression in response to the increased 20E titer after a blood meal. Furthermore, we find that SMRTER, an insect analog of the vertebrate nuclear receptor corepressors SMRT and N-CoR, interacts with EcR in a 20E-sensitive manner and is required for EcR-mediated repression of miRNA expression in Ae. aegypti mosquitoes. In addition, we demonstrate that miR-275 and miR-305 directly target glutamate semialdehyde dehydrogenase and AAEL009899, respectively, to facilitate egg development. This study reveals a mechanism for how miRNAs are controlled by the 20E signaling pathway to coordinate their activity with the demands of mosquito reproduction. [ABSTRACT FROM AUTHOR]

The SAGA complex is an evolutionarily conserved transcriptional coactivator that regulates gene expression through its histone acetyltransferase and deubiquitylase activities, recognition of specific histone modifications, and interactions with transcription factors. Multiple lines of evidence indicate the existence of distinct variants of SAGA among organisms as well as within a species, permitting diverse functions to dynamically regulate cellular pathways. Our co-expression analysis of genes encoding human SAGA components showed enrichment in reproductive organs, brain tissues and the skeletal muscle, which corresponds to their established roles in developmental programs, emerging roles in neurodegenerative diseases, and understudied functions in specific cell types. SAGA subunits modulate growth, development and response to various stresses from yeast to plants and metazoans. In metazoans, SAGA further participates in the regulation of differentiation and maturation of both innate and adaptive immune cells, and is associated with initiation and progression of diseases including a broad range of cancers. The evolutionary conservation of SAGA highlights its indispensable role in eukaryotic life, thus deciphering the mechanisms of action of SAGA is key to understanding fundamental biological processes throughout evolution. To illuminate the diversity and conservation of this essential complex, here we discuss variations in composition, essentiality and co-expression of component genes, and its prominent functions across Fungi, Plantae and Animalia kingdoms. [ABSTRACT FROM AUTHOR]

The transcriptional enhanced associate domain (TEAD) transcription factor is a core regulator of Hippo signaling pathway and has been recognized as a promising therapeutic target against gastrointestinal tumors. The intermolecular interaction of TEAD with its transcription coactivator vestigial-like protein (VGLL) is involved in the tumorigenesis of esophageal cancer, which possesses an additional double-stranded β-sheet (DSβS) motif in its TEAD-recognition domain as compared to the classic coactivators YAP and TAZ. Here, the DSβS motif is examined systematically based on the complex crystal structure of TEAD with VGL. It is found that the DSβS is one of the most important recognition sites at TEAD–VGLL complex interface, which can confer stability and specificity to the complex recognition and elicit the transcription activation event. Protein segments with different lengths are derived from the DSβS motif of VGLL to define a number of linear peptides; their binding modes and affinities to TEAD are computationally analyzed to reveal that these linear peptides cannot maintain in native double-strand conformation in free solution state, thus unfavorable for their rebinding to TEAD–VGLL interface. A head-to-tail cyclization strategy is described to constrain the conformation of linear peptides, which can considerably reduce the peptide flexibility and help the peptide folding to an approximately native conformation in solution. We also demonstrate that the binding affinity and biological activity of cyclic peptides are improved effectively relative to their linear counterparts at molecular and cellular levels. [ABSTRACT FROM AUTHOR]

MYC is an oncogenic DNA-binding transcription activator of many genes and is often upregulated in human cancers. MYC has an N-terminal transcription activation domain (TAD) that is also required for cell transformation. Various MYC TAD-interacting coactivators have been identified, including the transcription/transformation-associated protein (TRRAP), a subunit of different histone acetyltransferase (HAT) complexes such as the human "SPT3-TAF9-GCN5 Acetyltransferase" (STAGA) complex involved in MYC transactivation of the TERT gene. However, it remains unclear whether TRRAP and/or other subunits are directly contacted by MYC within these macromolecular complexes. Here, we characterize the interactions of MYC TAD with the STAGA complex. By protein crosslinking we identify both TRRAP and the GCN5 acetyltransferase as MYC TAD-interacting subunits within native STAGA. We show that purified GCN5 binds to an N-terminal sub-domain of MYC TAD (residues 21-108) and that the interaction of GCN5 and STAGA with this sub-domain is dependent on two related sequence motifs: M2 within the conserved MYC homology box I (MBI), and M3 located between residues 100-106. Interestingly, specific substitutions within the M2/3 motifs that only moderately reduce the intracellular MYC-STAGA interaction and do not influence dimerization of MYC with its DNA-binding partner MAX, strongly inhibit MYC acetylation by GCN5 and reduce MYC binding and transactivation of the GCN5-dependent TERT promoter in vivo. Hence, we propose that MYC associates with STAGA through extended interactions of the TAD with both TRRAP and GCN5 and that the TAD-GCN5 interaction is important for MYC acetylation and MYC binding to certain chromatin loci.

Ubiquitin-protein ligase E3A (UBE3A) has dual functions as a E3 ubiquitin-protein ligase and coactivator of nuclear hormone receptors. Mutations or deletions of the maternally inherited UBE3A gene cause Angelman syndrome. Here, we performed transcriptome profiling in the hippocampus of Ube3am+/p+ and Ube3am–/p+ mice, and determined that the expression of the retinoic acid (RA) signalling pathway was downregulated in Ube3a-deficient mice compared to WT mice. Furthermore, we demonstrated that UBE3A directly interacts with RARα and may function as a coactivator of the nuclear receptor RARα to participate in the regulation of gene expression. Loss of UBE3A expression caused the downregulation of the expression of RA-related genes, including Erbb4, Dpysl3, Calb1, Pten, and Arhgap5 in Ube3am–/p+ mice brain tissues. This work revealed a new role for UBE3A in regulating retinoic acid (RA) signalling downstream genes and hopefully to shed light on the potential drug target of AS.

Ubiquitin-protein ligase E3A (UBE3A) has dual functions as a E3 ubiquitin-protein ligase and coactivator of nuclear hormone receptors. Mutations or deletions of the maternally inherited UBE3A gene cause Angelman syndrome. Here, we performed transcriptome profiling in the hippocampus of Ube3a m +/p+ and Ube3a m–/p + mice, and determined that the expression of the retinoic acid (RA) signalling pathway was downregulated in Ube3a-deficient mice compared to WT mice. Furthermore, we demonstrated that UBE3A directly interacts with RARα and may function as a coactivator of the nuclear receptor RARα to participate in the regulation of gene expression. Loss of UBE3A expression caused the downregulation of the expression of RA-related genes, including Erbb4, Dpysl3, Calb1, Pten , and Arhgap5 in Ube3a m–/p + mice brain tissues. This work revealed a new role for UBE3A in regulating retinoic acid (RA) signalling downstream genes and hopefully to shed light on the potential drug target of AS. [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]

DAF-12 is nematode-specific nuclear receptor that has been proposed to govern development of the infectious stage of parasitic species, including Strongyloides stercoralis. Here, we identified a parasite-specific coactivator, called DAF-12 interacting protein-1 (DIP-1), that is required for DAF-12 ligand-dependent transcriptional activity. DIP-1 is found only in Strongyloides spp. and selectively interacts with DAF-12 through an atypical receptor binding motif. Using CRISPR/Cas9-directed mutagenesis, we demonstrate that DAF-12 is required for the requisite developmental arrest and the ligand-dependent reactivation of infectious S. stercoralis infective third-stage larvae, and that these effects require the DIP-1 coactivator. These studies reveal the existence of a distinct nuclear receptor/coactivator signaling pathway that governs parasite development. [ABSTRACT FROM AUTHOR]

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work. [ABSTRACT FROM AUTHOR]

Posttranslational modification of histones regulates transcription but the exact role that acetylation of specific lysine residues plays in biological processes in vivo is still not clearly understood. To assess the contribution of different histone modifications to transcriptional activation in vivo, we determined the acetylation patterns on the ecdysone induced Eip74EF and Eip75B genes in Drosophila melanogaster larvae by chromatin immunoprecipitation. We found that acetylation of histone H3 lysine 23 is localized to promoters and correlates with endogenous ecdysone induced gene activation. In contrast, acetylation of lysines 8, 12 and 16 of histone H4 and lysine 9 of histone H3 showed minor differences in their distribution on the regulatory and transcribed regions tested, and had limited or no correlation with ecdysone induced transcriptional activity. We found that dCBP, which is encoded by the nejire gene, acetylates H3 lysine 23 in vivo, and silencing of nejire leads to reduced expression of the Eip74EF and Eip75B genes. Our results suggest that acetylation of specific lysine residues of histones contribute specifically to the dynamic regulation of transcription. Furthermore, along with previous studies identify CBP dependent H3 lysine 23 acetylation as an evolutionarily conserved chromatin modification involved in steroid induced gene activation.

The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator’s synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.

Deletions within genes coding for subunits of the transcription coactivator SAGA caused strong genome-wide defects in transcription and SAGA-mediated chromatin modifications. In contrast, rapid SAGA depletion produced only modest transcription defects at 13% of protein-coding genes – genes that are generally more sensitive to rapid TFIID depletion. However, transcription of these ‘coactivator-redundant’ genes is strongly affected by rapid depletion of both factors, showing the overlapping functions of TFIID and SAGA at this gene set. We suggest that this overlapping function is linked to TBP-DNA recruitment. The remaining 87% of expressed genes that we term ‘TFIID-dependent’ are highly sensitive to rapid TFIID depletion and insensitive to rapid SAGA depletion. Genome-wide mapping of SAGA and TFIID found binding of both factors at many genes independent of gene class. Promoter analysis suggests that the distinction between the gene classes is due to multiple components rather than any single regulatory factor or promoter sequence motif.

Background: PXR is a xenobiotic-responsive nuclear receptor that controls the expression of drug-metabolizing enzymes. Drug-induced activation of PXR sometimes causes drug–drug interactions due to the induced metabolism of co-administered drugs. Our group recently reported a possible drug–drug interaction mechanism via an interaction between the nuclear receptors CAR and PPARα. As CAR and PXR are structurally and functionally related receptors, we investigated possible crosstalk between PXR and PPARα. Methods: Human hepatocyte-like HepaRG cells were treated with various PXR ligands, and mRNA levels were determined by quantitative reverse transcription PCR. Reporter assays using the HMGCS2 promoter containing a PPARα-binding motif and mammalian two-hybrid assays were performed in HepG2 or COS-1 cells. Results: Treatment with PXR activators reduced the mRNA levels of PPARα target genes in HepaRG cells. In reporter assays, PXR suppressed PPARα-dependent gene expression in HepG2 cells. In COS-1 cells, co-expression of PGC1α, a common coactivator of PPARα and PXR, enhanced PPARα-dependent gene transcription, which was clearly suppressed by PXR. Consistently, in mammalian two-hybrid assays, the interaction between PGC1α and PPARα was attenuated by ligand-activated PXR. Conclusion: The present results suggest that ligand-activated PXR suppresses PPARα-dependent gene expression by inhibiting PGC1α recruitment.

The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator's synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression. [ABSTRACT FROM AUTHOR]

A key functional event in eukaryotic gene activation is the formation of dynamic protein-protein interaction networks between transcriptional activators and transcriptional coactivators. Seemingly incongruent with the tight regulation of transcription, many biochemical and biophysical studies suggest that activators use nonspecific hydrophobic and/or electrostatic interactions to bind to coactivators, with few if any specific contacts. Here a mechanistic dissection of a set of representative dynamic activator•coactivator complexes, comprised of the ETV/PEA3 family of activators and the coactivator Med25, reveals a different molecular recognition model. The data demonstrate that small sequence variations within an activator family significantly redistribute the conformational ensemble of the complex while not affecting overall affinity, and distal residues within the activator--not often considered as contributing to binding--play a key role in mediating conformational redistribution. The ETV/PEA3•Med25 ensembles are directed by specific contacts between the disordered activator and the Med25 interface, which is facilitated by structural shifts of the coactivator binding surface. Taken together, these data highlight the critical role coactivator plasticity plays in recognition of disordered activators and indicate that molecular recognition models of disordered proteins must consider the ability of the binding partners to mediate specificity. [ABSTRACT FROM AUTHOR]

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription. [ABSTRACT FROM AUTHOR]

The PPARγ coactivator 1α (PGC-1α) is a transcriptional regulator of mitochondrial biogenesis and oxidative metabolism. Recent studies have highlighted a fundamental role of PGC-1α in promoting breast cancer progression and metastasis, but the physiological role of this coactivator in the development of mammary glands is still unknown. First, we show that PGC-1α is highly expressed during puberty and involution, but nearly disappeared in pregnancy and lactation. Then, taking advantage of a newly generated transgenic mouse model with a stable and specific overexpression of PGC-1α in mammary glands, we demonstrate that the re-expression of this coactivator during the lactation stage leads to a precocious regression of the mammary glands. Thus, we propose that PGC-1α action is non-essential during pregnancy and lactation, whereas it is indispensable during involution. The rapid preadipocyte–adipocyte transition, together with an increased rate of apoptosis promotes a premature mammary glands involution that cause lactation defects and pup growth retardation. Overall, we provide new insights in the comprehension of female reproductive cycles and lactation deficiency, thus opening new roads for mothers that cannot breastfeed. [ABSTRACT FROM AUTHOR]

Transcription coactivators are proteins or protein complexes that mediate transcription factor (TF) function. However, they lack DNA-binding capacity, prompting the question of how they engage target loci. Three non-exclusive hypotheses have been posited: coactivators are recruited by complexing with TFs, by binding histones through epigenetic reader domains, or by partitioning into condensates through their extensive intrinsically disordered regions. Using p300 as a prototypical coactivator, we systematically mutated its annotated domains and show by single-molecule tracking in live U2OS cells that coactivator-chromatin binding depends entirely on combinatorial binding of multiple TF-interaction domains. Furthermore, we demonstrate that acetyltransferase activity opposes p300-chromatin association and that the N-terminal TF-interaction domains regulate that activity. Single TF-interaction domains are insufficient for chromatin binding and regulation of catalytic activity, implying a principle that we speculate could broadly apply to eukaryotic gene regulation: a TF must act in coordination with other TFs to recruit coactivator activity. [Display omitted] • The structured Core domain of p300 is dispensable for chromatin engagement • p300-chromatin binding requires multiple TF-interaction domains • p300 KAT activity opposes chromatin binding • p300 KAT activity may be regulated by its N-terminal TF-interaction domains Ferrie et al. demonstrate through live-cell single-molecule imaging that the coactivator p300, a central player in eukaryotic gene regulation, carries out its function of integrating multiple transcription factor (TF) inputs at the level of chromatin binding. Their results support a TF- rather than histone-mediated model of coactivator recruitment. [ABSTRACT FROM AUTHOR]