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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]

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]

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. [ABSTRACT FROM AUTHOR]

Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling. [Display omitted] • Luminal androgen receptor (LAR) TNBC specimens express AR splice variants (AR-SVs) • AR-SV promotes aggressive TNBC growth • AR-positive/AR-SV-positive specimens activate interferon JAK-STAT • N terminus AR degrader effective in AR- and AR-SV-positive TNBC xenografts Asemota et al. show that TNBC specimens, notably from African American women, express androgen receptor splice variants (AR-SVs), and AR-SV expression causes aggressive TNBC growth. AR- and AR-SV-positive TNBC specimens are enriched for JAK-STAT signaling. Growth of AR- and AR-SV-positive TNBC xenografts is inhibited by AR N terminus domain-binding degrader. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • SRC-2 depletion induces mesenchymal-to-epithelial transition. • SRC-2 expression negatively correlates with luminal and cell morphogenesis genes. • SRC-2 regulates the expression of the EMT biomarker Lyn. Abstract Steroid receptor coactivator 2 (SRC-2) is a nuclear receptor coactivator, important for the regulation of estrogen receptor alpha (ERα)-mediated transcriptional activity in breast cancer cells. However, the transcriptional role of SRC-2 in breast cancer is still ambiguous. Here we aimed to unravel a more precise transcriptional role of SRC-2 and uncover unique target genes in MCF-7 breast cancer cells, as opposed to the known oncogene SRC-3. Gene expression analyses of cells depleted of either SRC-2 or SRC-3 showed that they transcriptionally regulate mostly separate gene sets. However, individual unique gene sets were implicated in some of the same major gene ontology biological processes, such as cellular structure and development. This finding was supported by three-dimensional cell cultures, demonstrating that depletion of SRC-2 and SRC-3 changed the morphology of the cells into epithelial-like hollow acinar structures, indicating that both SRC proteins are involved in maintaining the hybrid E/M phenotype. In clinical ER-positive, HER2-negative breast cancer samples the expression of SRC-2 was negatively correlated with the expression of MCF-7-related luminal, cell cycle and cellular morphogenesis genes. Finally, elucidating SRC-2 unique transcriptional effects, we identified Lyn kinase (an EMT biomarker) to be upregulated exclusively after SRC-2 depletion. In conclusion, we show that both SRC-2 and SRC-3 are essential for the EMT in breast cancer cells, controlling different transcriptional niches. [ABSTRACT FROM AUTHOR]

LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin. [ABSTRACT FROM AUTHOR]

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates the expression of target genes through ligand binding. To express the target gene, coactivator binding to the VDR/ligand complex is essential. Although there are many coactivators in living cells, precise interactions between coactivators and VDR have not been clarified. Here, we synthesized two coactivator peptides, DRIP205-2 and SRC2-3, evaluated their affinity for the ligand-binding domain (LBD) of VDR using 1α,25-dihydroxyvitamin D 3 , partial agonist 1 , and antagonist 2 by surface plasmon resonance (SPR), and assessed their interaction modes with VDR-LBD using X-ray crystallographic analysis. This study showed that the SRC2-3 peptide is more sensitive to the ligands (agonist, partial agonist, and antagonist) and shows more intimate interactions with VDR-LBD than DRIP205-2 peptide. [ABSTRACT FROM AUTHOR]

The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a “protein inhibitor of activated c-Myb” in the absence of SUMOylation while, in its presence, PIAS behaves as a “protein activator of repressed c-Myb”. [ABSTRACT FROM AUTHOR]

Human 8-oxoguanine DNA glycosylase 1 (OGG1) is the major DNA repair enzyme that plays a key role in excision of oxidative damaged DNA bases such as 8-oxoguainine (8-oxoG). Recent studies suggest another function of OGG1, namely that it may be involved in the endotoxin- or oxidative stress-induced inflammatory response. In this study, we investigated the role of OGG1 in the inflammatory response. OGG1 expression is increased in the organs of endotoxin-induced or myelin oligodendrocyte glycoprotein (MOG)-immunized mice and immune cells, resulting in induction of the expression of pro-inflammatory mediators at the transcriptional levels. Biochemical studies showed that signal transducer and activator of transcription 1 (STAT1) plays a key role in endotoxin-induced OGG1 expression and inflammatory response. STAT1 regulates the transcriptional activity of OGG1 through recruiting and binding to the gamma-interferon activation site (GAS) motif of the OGG1 promoter region, and chromatin remodeling by acetylation and dimethylation of lysine-14 and -4 residues of histone H3. In addition, OGG1 acts as a STAT1 coactivator and has transcriptional activity in the presence of endotoxin. The data presented here identifies a novel mechanism, and may provide new therapeutic strategies for the treatment of endotoxin-mediated inflammatory diseases. [ABSTRACT FROM AUTHOR]

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. [ABSTRACT FROM AUTHOR]

The GLI1 oncogene and p53 tumor suppressor gene function in an inhibitory loop that controls stem cell and tumor cell numbers. Since GLI1 and p53 both interact with the coactivator TATA Binding Protein Associated Factor 9 (TAF9), we hypothesized that competition between these transcription factors for TAF9 in cancer cells may contribute to the inhibitory loop and directly affect GLI1 function and cellular phenotype. We showed that TAF9 interacts with the oncogenic GLI family members GLI1 and GLI2 but not GLI3 in cell-free pull-down assays and with GLI1 in rhabdomyosarcoma and osteosarcoma cell lines. Removal of the TAF9-binding acidic alpha helical transactivation domain of GLI1 produced a significant reduction in the ability of GLI1 to transform cells. We then introduced a point mutation into GLI1 (L1052I) that eliminates TAF9 binding and a point mutation into GLI3 (I1510L) that establishes binding. Wild-type and mutant GLI proteins that bind TAF9 showed enhanced transactivating and cell transforming activity compared with those that did not. Therefore, GLI-TAF9 binding appears important for oncogenic activity. We then determined whether wild-type p53 down-regulates GLI function by sequestering TAF9. We showed that p53 binds TAF9 with greater affinity than does GLI1 and that co-expression of p53 with GLI1 or GLI2 down-regulated GLI-induced transactivation, which could be abrogated using mutant forms of GLI1 or p53. This suggests that p53 sequesters TAF9 from GLI1, which may contribute to inhibition of GLI1 activity by p53 and potentially impact therapeutic success of agents targeting GLI-TAF9 interactions in cancer. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The LATERAL ORGAN BOUNDARIES DOMAIN/ASYMMETRIC LEAVES2-LIKE (LBD/ASL) gene family encodes a class of transcription factors harboring a conserved plant-specific lateral organ boundaries domain and plays a key role in lateral organ development of plants. Recent studies have revealed developmental functions of some LBD genes in Arabidopsis, rice, and maize. We have shown previously that LBD18/ASL20 promotes the emergence of lateral roots in Arabidopsis. LBD18 induces EXPANSIN14 (EXP14) expression by binding to a specific region of the EXP14 promoter. To further understand the molecular mechanism of LBD18 acting as a transcription factor, we isolated a protein interacting with LBD18 by screening an Arabidopsis cDNA library using the yeast two-hybrid system with LBD18 as bait. We found that GBF INTERACTING PROTEIN1 (GIP1) interacts with LBD18 in yeast and Arabidopsis protoplasts. Reverse-transcription-polymerase chain reaction analysis showed overlapping expression of GIP1 and LBD18 in various tissues of Arabidopsis such as roots, aerial parts, and rosette leaves. Transient gene expression assay results with Arabidopsis protoplasts indicated that GIP1 enhances transcriptional activity of LBD18 in the EXP14 promoter fused to the GUS reporter gene. These results show that GIP1 may act as a transcriptional coactivator of LBD18. [Copyright &y& Elsevier]

Highlights: [•] PELP1 expression is dysregulated in a significant number of cancer types. [•] Overexpression of PELP1 has been correlated with higher tumor grade and poor prognosis is some tumor types. [•] PELP1 interacts with numerous proteins and functions as a scaffolding protein in the cytoplasm and nucleus. [•] PELP1 signaling pathways modulate cellular proliferation, migration and invasion, and hormone resistance. [ABSTRACT FROM AUTHOR]

Abstract: Breast cancer is the leading cause of cancer death for women worldwide. Various therapeutic approaches have been proposed, among which endocrine therapy has recently become popular due to the high sensitivity of breast tissues to steroids such as estrogens and progesterone. The underlying mechanisms of steroid regulation in breast cancer cell proliferation, invasiveness, metastasis and endocrine resistance, however, remain largely unknown. Steroid receptor coactivator-1 (SRC-1) has attracted much attention because it is an important co-regulator and plays a pivotal role in modulating the transcriptional activities of steroid nuclear receptors. Accumulated research has established a strong correlation between SRC-1 and the pathological progression or disease-related features of breast cancer, which supports its potential as a target for specific therapeutic intervention in the clinical management of breast cancer. In addition, a diverse group of downstream molecules have also been shown to participate in various functional pathways related to SRC-1-associated regulation of breast cancer. These downstream molecules are also considered promising therapeutic targets, providing additional options for targeted treatments. In this review, the expression of SRC-1 in breast cancer and the close relationships between SRC-1 and the cell proliferation, invasiveness, metastasis and endocrine resistance of breast cancer will be discussed, followed by a brief summary of its putative functional mechanisms with an emphasis on the potential therapeutic role of SRC-1. [Copyright &y& Elsevier]

Highlights: [•] Multiple kinases phosphorylate steroid hormone receptors and their coregulators. [•] Site-specific phosphorylation modulates the selective actions of receptors. [•] Activation of cell signaling pathways impacts upon the biological response of receptors. [•] Regulation of receptor function by cell signaling is relevant to human disease. [ABSTRACT FROM AUTHOR]

Abstract: Transcription factors require coactivators and corepressors to modulate transcription in mammalian cells. The vitamin D receptor (VDR) utilizes coactivators and corepressors to gain tight control over the activity of a diverse set of genes that can regulate calcium transport, slow proliferation and promote immune responses. We have recently established the VDR/RXR cistrome in human colon cancer cells and have linked these binding sites to the genes that are regulated by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). In additional studies described herein, we demonstrate that the coactivators SRC1, CBP and MED1 are recruited to upregulated genes to facilitate transcription as expected. SRC1 was the most highly correlated to VDR/RXR binding (50%). However, we also found that corepressor molecules such as NCoR and SMRT were present along with SRC1, CBP or MED1 at these 1,25(OH)2D3 activated gene enhancers. Interestingly, genome-wide NCoR binding mimicked VDR binding by increasing its association with VDR binding in response to 1,25(OH)2D3 treatment. Overall, these data indicate a complex role for corepressor and coactivator complexes in the activation or active repression of 1,25(OH)2D3 responsive genes. This article is part of a Special Issue entitled ‘Vitamin D Workshop’. [Copyright &y& Elsevier]

Abstract: Background: Mediator is an evolutionarily conserved multisubunit complex that plays an essential regulatory role in eukaryotic transcription of protein-encoding genes. The human complex was first isolated as a transcriptional coactivator bound to the thyroid hormone receptor (TR) and has since been shown to play a key coregulatory role for a broad range of nuclear hormone receptors (NRs) as well as other signal-activated transcription factors. Scope of review: We provide a general overview of Mediator structure and function, summarize the mechanisms by which Mediator is targeted to NRs, and outline recent evidence revealing Mediator as a regulatory axis for other distinct coregulatory factors, chromatin modifying enzymes and cellular signal transduction pathways. Major conclusions: Besides serving as a functional interface with the RNA polymerase II basal transcription machinery, Mediator plays a more versatile role in regulating transcription including the ability to: a) facilitate gene-specific chromatin looping events; b) coordinate chromatin modification events with preinitiation complex assembly; and c) regulate critical steps that occur during transcriptional elongation. The variably associated MED1 subunit continues to emerge as a pivotal player in Mediator function, not only as the primary interaction site for NRs, but also as a crucial interaction hub for other coregulatory factors, and as an important regulatory target for signal-activated kinases. General significance: Mediator plays an integral coregulatory role at NR target genes by functionally interacting with the basal transcription apparatus and by coordinating the action of chromatin modifying enzymes and transcription elongation factors. This article is part of a Special Issue entitled Thyroid hormone signalling. [Copyright &y& Elsevier]

Abstract: The steroid receptor coactivator p/CIP, also known as SRC-3, is an oncogene commonly amplified in breast and ovarian cancers. p/CIP is known to associate with coactivator arginine methyltransferase 1 (CARM1) on select estrogen responsive genes. We have shown, using a ChIP-on-chip approach, that in response to stimulation with 17β-estradiol (E2), the p/CIP/CARM1 complex is recruited to 204 proximal promoters in MCF-7 cells. Many of the complex target genes have been previously implicated in signaling pathways related to oncogenesis. Jak2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the Jak2 promoter are reflective of a transcriptionally permissive gene, and modest changes in RNA and protein expression lead us to suggest that an additional factor(s) may be required for a more notable transcriptional and functional response. Bioinformatic examination of the 204 proximal promoter sequences of p/CIP/CARM1 targets supports the idea that transcription factor crosstalk is likely the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. This data may have implications towards understanding the oncogenic role of p/CIP in breast cancer and ultimately allow for the identification of new prognostic indicators and/or viable therapeutic targets. [Copyright &y& Elsevier]

Abstract: Notch signaling plays a pivotal role in embryonic and postnatal development. Upon binding of a Notch ligand, proteolytic cleavage events liberate the Notch-intracellular domain (NICD) that migrates into the nucleus. In order to activate target genes, NICD associates with the transcription factor RBP-J (also known as CSL), Mastermind and the acetyltransferase p300. Here, we identify the DEAD-box RNA helicase Ddx5 as a novel component of the RBP-J/NICD complex utilizing a biotinylation-tagging approach followed by mass-spectrometry. Biochemical assays confirm a direct interaction of Ddx5 with RBP-J. We show that Ddx5 localizes at RBP-J binding sites within the Notch target genes preTCRα, Hes1 and CD25 in a Notch-dependent manner. Moreover, knockdown of Ddx5 also downregulates a subset of Notch target genes in a murine pre T-cell model. Interestingly, also knockdown/overexpression of the RNA coactivator SRA, a cofactor of Ddx5, downregulates Hes1 and preTCRα. Using Chromatin-IP, we show that this effect is accompanied with a loss of p300 occupancy at Notch target genes and decreased histone acetylation. Together, our data demonstrate that Ddx5 and SRA function as coactivators of Notch signaling. [Copyright &y& Elsevier]

Abstract: We previously identified the methylsulfonylnitrobenzoates (MSNBs) that block the interaction of the thyroid hormone receptor with its obligate transcriptional coactivators and prevent thyroid hormone signaling. As part of our lead optimization work we demonstrated that sulfonylnitrophenylthiazoles (SNPTs), which replace the ester linkage of MSNBs with a thiazole, also inhibited coactivator binding to TR. Here we report that replacement of the ester with an amide (methylsulfonylnitrobenzamides, MSNBA) also provides active TR antagonists. [Copyright &y& Elsevier]

Abstract: Thyroid hormone receptor (TR)/peroxisome proliferator activated receptor coactivator (PGC-1α) interactions are required for T3-dependent transcriptional responses involved in adaptive thermogenesis and liver. Thus, it is important to define TR/PGC-1α contact modes and to understand their significance in gene expression. Previous studies have shown that TRβ1 recruits PGC-1α to target promoters via contacts between the hormone-dependent TRβ1 activation function 2 (AF-2) in the C-terminal ligand binding domain (LBD) and a major PGC-1α nuclear receptor (NR) interaction box (consensus LxxLL) at amino acids 142–146. While our studies verify the existence and importance of this interaction, we present evidence that TRβ1 also binds PGC-1α in a second ligand and LxxLL motif independent mode and show that this interaction requires the TRβ1 N-terminal domain (NTD) and the PGC-1α N-terminal activation domain (AD) at amino acids 1–130. Transfection assays suggest that optimal PGC-1α coactivation requires the TRβ1 NTD and that these contacts are needed for utilization of the PGC-1α C-terminal AD, which does not bind TR and is implicated in basal transcription machinery contacts. We propose that TR AF-1/PGC-1α contacts are needed for transition between activities of PGC-1α N-and C-terminal ADs in gene expression. Our findings provide insights into possible roles for TR and NR AF-1 in gene expression. [Copyright &y& Elsevier]

Abstract: Nucleus-encoded regulatory factors are major contributors to mitochondrial biogenesis and function. Several act within the organelle to regulate mitochondrial transcription and translation while others direct the expression of nuclear genes encoding the respiratory chain and other oxidative functions. Loss-of-function studies for many of these factors reveal a wide spectrum of phenotypes. These range from embryonic lethality and severe respiratory chain deficiency to relatively mild mitochondrial defects seen only under conditions of physiological stress. The PGC-1 family of regulated coactivators (PGC-1α, PGC-1β and PRC) plays an important integrative role through their interactions with transcription factors (NRF-1, NRF-2, ERRα, CREB, YY1 and others) that control respiratory gene expression. In addition, recent evidence suggests that PGC-1 coactivators may balance the cellular response to oxidant stress by promoting a pro-oxidant environment or by orchestrating an inflammatory response to severe metabolic stress. These pathways may serve as essential links between the energy generating functions of mitochondria and the cellular REDOX environment associated with longevity, senescence and disease. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. [Copyright &y& Elsevier]

Abstract: MTF-1 is a sequence-specific DNA binding protein that activates the transcription of metal responsive genes. The extent of activation is dependent on the nature of the metal challenge. Here we identify separate regions within the Drosophila MTF-1 (dMTF-1) protein that are required for efficient copper- versus cadmium-induced transcription. dMTF-1 contains a number of potential metal binding regions that might allow metal discrimination including a DNA binding domain containing six zinc fingers and a highly conserved cysteine-rich C-terminus. We find that four of the zinc fingers in the DNA binding domain are essential for function but the DNA binding domain does not contribute to the metal discrimination by dMTF-1. We find that the conserved C-terminus of the cysteine-rich domain provides cadmium specificity while copper specificity maps to the previously described copper-binding region (Chen et al.). In addition, both metal specific domains are autorepressive in the absence of metal and contribute to the low level of basal transcription from metal inducible promoters. [Copyright &y& Elsevier]

Abstract: During embryogenesis, the development of the male genital is dependent on androgens. Their actions are mediated by the androgen receptor (AR), which functions as a transcription factor. To identify AR coregulators that support AR action during the critical time window of androgen-dependent development in the genital tubercle of male mice, we performed yeast two-hybrid screenings with cDNA libraries of genital tubercles from male mouse embryos using human AR as bait. RWD domain containing 1 (RWDD1) was identified as an AR-interacting protein from three independent libraries of the embryonic days E15, E16 and E17. The interaction between the AR and RWDD1 was confirmed in vitro and in vivo and the ligand binding domain of the AR was shown to be sufficient to mediate the interaction. RWDD1 enhanced AR-dependent transactivation in reporter assays with promoters of different complexity and in different cell lines. These results suggest that RWDD1 functions as a coactivator of androgen-dependent transcription. [Copyright &y& Elsevier]

Abstract: Androgens are key regulators of male sexual differentiation and essential for development and maintenance of male reproductive tissues. The androgens testosterone and dihydrotestosterone mediate their effect by binding to, and activation of the androgen receptor (AR). Upon activation, the AR is able to recognize specific DNA sequences in gene promoters and enhancers from where it recruits coregulators to orchestrate chromatin remodeling and transcription regulation. The number of proteins that bind to the AR has surpassed 200 and many of them enhance (coactivator) or repress (corepressor) its transactivating capacity. For most of these coregulators, their AR binding interface and their exact mode of action still needs to be elucidated, but for some of the more classical coactivators and corepressors, we gained insight in their working mechanisms. Of particular interest are specific sequences (LxxLL and FxxLF-like motifs) in a subset of coactivators that interact with the AR via a coactivator binding groove in the ligand-binding domain. As compared to other steroid receptors, the conformation of the AR coactivator binding pocket is unique and preferentially binds FxxLF-like motifs. This predisposition is expected to contribute to the regulation of specific sets of target genes via recruitment of selected coregulators. This review provides an overview of these (inter)actions with a focus on the unique characteristics of the AR coactivator binding groove. [Copyright &y& Elsevier]

Abstract: Pure antiestrogens were clinically developed as alternative therapies for estrogen receptor (ER) positive breast cancers. Unlike the selective estrogen receptor modulators (SERMs), these antiestrogens are devoid of tissue-specific ER agonist activity. Many of these compounds are steroidal in nature, containing an estradiol (E2) structural core with long alkyl side chains at the C-7α position. Two novel 7α-substituted E2 derivatives were evaluated that retain high binding affinity for ER. Compared to known pure antiestrogens, these compounds, referred to as compound 13 (C13) and C14, contain shorter 7α alkyl side chains and differ only in their terminal substituent: a hydroxyl moiety versus a benzyloxy group, respectively. Herein we assessed the effects of these compounds on ER transcriptional activity and report that despite their similar overall structure, C13 and C14 produce distinct cell type-specific responses. Of note, C13 functions as a mixed agonist/antagonist in Hela cells, inducing only weak ER transcriptional activity while preventing coactivator recruitment and stabilizing ER expression. However, this compound effectively stimulates ER activity in MCF-7 cells, does not increase ER levels and promotes cell proliferation on par with E2. Conversely, C14 stimulates transcriptional activity in both cell types and enhances ER–coactivator interactions. The activities of both compounds were inhibited by the pure antiestrogen ICI 182,780. Taken together, these results reveal that C13 is a SERM while C14 is an ER agonist, and indicate that the terminal modification of the C-7α hexanyl side chain of these estradiol derivatives is an important determinant of the biocharacter of these ER ligands. [Copyright &y& Elsevier]

Abstract: Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade. [Copyright &y& Elsevier]

Abstract: This chapter presents an overview of the current status of studies on the structural and molecular biology of the retinoid X receptor subtypes α, β, and γ (RXRs, NR2B1–3), their nuclear and cytoplasmic functions, post-transcriptional processing, and recently reported ligands. Points of interest are the different changes in the ligand-binding pocket induced by variously shaped agonists, the communication of the ligand-bound pocket with the coactivator binding surface and the heterodimerization interface, and recently identified ligands that are natural products, those that function as environmental toxins or drugs that had been originally designed to interact with other targets, as well as those that were deliberately designed as RXR-selective transcriptional agonists, synergists, or antagonists. Of these synthetic ligands, the general trend in design appears to be away from fully aromatic rigid structures to those containing partial elements of the flexible tetraene side chain of 9-cis-retinoic acid. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945–2010). [Copyright &y& Elsevier]

Abstract: Hepatic gluconeogenesis is a major pathway that maintains normal plasma glucose levels during prolonged starvation. The aim of this review is to provide insights into the integration of transcriptional regulation of gluconeogenic enzyme genes in response to nutritional and hormonal changes. The roles of transcription factors/co-regulators in response to those factors will be discussed. Overall, glucagon and glucocorticoids are positive regulators of gluconeogenesis. Glucagon, via cAMP, promotes the interaction of cAMP-responsive binding protein with CREB-regulated transcription coactivator 2 which facilitates its binding to cAMP-responsive elements (CREs). The response to glucocorticoids is mediated by the glucocorticoid receptor that binds to glucocorticoid responsive elements (GREs) in the promoters of gluconeogenic genes. These CREs and GREs may be arranged as distinct elements or combined to form a “unit” to ensure the maximal transcriptional response to these hormones. The hepatocyte nuclear factors, forkhead O box, and the peroxisome proliferator-activated receptor-γ coactivator 1α can also synergistically increase transcription of gluconeogenic genes. Surtuin 1, an energy sensor can also modify the transcriptional activity of some of these transcription factors. In contrast, insulin secreted during fed conditions acts to repress transcription of gluconeogenic enzymes. This is achieved via activation of Akt/PKB and the consequent disruption of interactions between certain transcription factors/coactivators and their positive response elements in the promoters of those genes. Hypothalamic signaling via the insulin/leptin axis also regulates hepatic gluconeogenesis. Mice lacking the above transcription factors/coactivators show impaired gluconeogenesis, indicating their essential roles in the control of this vital metabolic process. [Copyright &y& Elsevier]

Abstract: Thyroid hormone (TH) has profound influence on metabolism that is closely linked to its effect on mitochondrial biogenesis and function. After a single injection of TH into mammals, physiological alterations (e.g. changes in oxygen consumption rates) are detectable after a lag period of ∼48h. This characteristic lag period is somewhat surprising since non-genomic responses are already detectable within minutes, and first genomic responses within some hours after administration of TH. This review provides a model to explain the characteristic lag period: TH regulates a first series of TH target genes via classical activation of gene expression by binding to thyroid hormone response elements. Some directly regulated target genes serve as intermediate factors and subsequently regulate a second series of indirect TH target genes. Intermediate factors are transcription factors (such as NRF-1, NRF-2 and PPARγ) and transcriptional coactivators (such as PGC-1α and PGC-1β). In concert with several post-translational modifications, these intermediate factors orchestrate the physiological response to thyroid hormone in vivo. [Copyright &y& Elsevier]

Abstract: E6-AP is a known coactivator of the estrogen receptor alpha (ERα), however the coactivation mechanism of E6-AP is not clear. This work was undertaken to elucidate the coactivation mechanism of E6-AP. In order to examine the role of E6-AP in ERα signaling, we knocked-down the expression of E6-AP and examined the transactivation functions of ERα. Knockdown of E6-AP showed reduced mRNA production of the ERα target genes pS2 and GREB1 suggesting that E6-AP is required for their proper transcription facilitated by ERα. In order to study the mechanism(s) by which E6-AP regulates the transcriptional functions of ERα, we performed chromatin immunoprecipitation (ChIP) assays under E6-AP knockdown conditions. Our ChIP data suggest that knockdown of E6-AP leads to decreased recruitment of the histone acetylase p300 to the ERα target gene pS2 promoter as well as reduced histone modifications at the promoter. Although there was reduced p300 recruitment to the pS2 promoter, loss of p300 did not account fully for the loss of histone acetylation. Taken together our data suggest that E6-AP regulates the transactivation functions of ERα in part by complexing with p300 and other chromatin modifying enzymes at target gene promoters to create a transcriptionally active promoter environment. [Copyright &y& Elsevier]

The conformationally flexible secosteroid, 1α,25(OH)2vitamin D3 (1α,25(OH)2D3) initiates biological responses via binding to the vitamin D receptor (VDR). The VDR contains two overlapping ligand binding sites, a genomic pocket (VDR-GP) and an alternative pocket (VDR-AP), that respectively bind a bowl-like ligand configuration (gene transcription) or a planar-like ligand shape (rapid responses). When occupied by 1α,25(OH)2D3, the VDR-GP interacts with the retinoid X receptor to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1α,25(OH)2D3. By recruiting complexes of either coactivators or corepressors, activated VDR modulates the transcription of genes encoding proteins that promulgate the traditional genomic functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. 1α,25(OH)2D3/VDR control of gene expression and rapid responses also delays chronic diseases of aging such as osteoporosis, cancer, type-1 and -2 diabetes, arteriosclerosis, vascular disease, and infection. [ABSTRACT FROM AUTHOR]

Abstract: Androgen receptor (AR) coregulators modulate ligand-induced gene expression in a tissue specific manner. The molecular events that follow coactivator binding to AR and the mechanisms that govern the sequence-specific effects of AR coregulators are poorly understood. Using consensus coactivator sequence D11-FxxLF and biophysical techniques, we show that coactivator association is followed by conformational rearrangement in AR ligand binding domain (AR-LBD) that is enthalpically and entropically favorable with activation energy of 29.8±4.2kJ/mol. Further characterization of ARA70 and SRC3-1 based consensus sequences reveal that each coactivator induces a distinct conformational state in the dihydrotestosterone:AR-LBD:coactivator complex. Complementary computational modeling revealed that coactivator induced specific alterations in the backbone flexibility of AR-LBD distant from the site of coactivator binding and that the intramolecular rearrangements in AR-LBD backbone induced by the two coactivator peptides were different. These data suggest that coactivators may impart specificity in the transcriptional machinery by changing the steady-state conformation of AR-LBD. These data provide direct evidence that even in the presence of same ligand, AR-LBD can occupy distinct conformational states depending on its interactions with specific coactivators in the tissues. We posit that this coactivator-specific conformational gating may then dictate subsequent binding partners and interaction/affinity for the DNA-response elements. [Copyright &y& Elsevier]

Abstract: The PGC-1 family of regulated coactivators, consisting of PGC-1α, PGC-1β and PRC, plays a central role in a regulatory network governing the transcriptional control of mitochondrial biogenesis and respiratory function. These coactivators target multiple transcription factors including NRF-1, NRF-2 and the orphan nuclear hormone receptor, ERRα, among others. In addition, they themselves are the targets of coactivator and co-repressor complexes that regulate gene expression through chromatin remodeling. The expression of PGC-1 family members is modulated by extracellular signals controlling metabolism, differentiation or cell growth and in some cases their activities are known to be regulated by post-translational modification by the energy sensors, AMPK and SIRT1. Recent gene knockout and silencing studies of many members of the PGC-1 network have revealed phenotypes of wide ranging severity suggestive of complex compensatory interactions or broadly integrative functions that are not exclusive to mitochondrial biogenesis. The results point to a central role for the PGC-1 family in integrating mitochondrial biogenesis and energy production with many diverse cellular functions. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection. [Copyright &y& Elsevier]

Abstract: Interactions between progesterone receptor (PR) and signal transducer and activator of transcription 3 (Stat3)-mediated signaling pathways have already been described. In the present study, we explored the capacity of Stat3 to functionally interact with progesterone receptor (PR) and modulate PR transcriptional activation in breast cancer cells. We found that the synthetic progestin medroxyprogesterone acetate (MPA) induced the association of a PR/Stat3 complex in which Stat3 acts as a coactivator of PR. We demonstrated that Stat3 activation is required for MPA modulation of the endogenous genes bcl-X and p21CIP1 which are involved in MPA-induced cell cycle regulation. Stat3 activity as a coactivator of PR was observed in both the classical and nonclassical ligand activated-PR transcriptional mechanisms, since the effects described were identified in the bcl-X promoter which contains a progesterone responsive element and in the p21CIP1 promoter which carries Sp1 binding sites where PR is recruited via the transcription factor Sp1. The data herein presented identifies a potential therapeutic intervention for PR-positive breast tumors consisting of targeting Stat3 function or PR/Stat3 interaction which will result in the inhibition of PR function. [Copyright &y& Elsevier]

Abstract: PNRC (proline-rich nuclear receptor coregulatory protein) was primarily identified as a coactivator of nuclear receptors (NRs) by our laboratory, which enhances NR-mediated transcription by RNA polymerase II. Recent study has shown that PNRC also stimulates RNA polymerase III-dependent transcription through interaction with the subunit RPC39 of RNA polymerase III. Here, we report that PNRC accumulates in the nucleolus and its depletion by small interfering RNA (siRNA) impairs pre-rRNA transcription by RNA polymerase I. We identified the sequence at position 94–101 (94PKKRRKKK101) of PNRC as its nucleolar localization sequence (NoLS). Fusion of this sequence to GFP directed GFP to the nucleolus. Characterization of the NoLS revealed that the stretches of six successive basic residues are sufficient to function as a NoLS. Through co-immunoprecipitation assay, we demonstrated that the NoLS is necessary and sufficient to mediate the association of PNRC with B23/nucleophosmin. Moreover, B23 depletion by siRNA disrupted the accumulation of PNRC in the nucleolus. Together, our study indicates that PNRC is a novel nucleolar protein that might be involved in regulation of pre-rRNA synthesis, and it localizes to the nucleolus by interaction with B23 via its NoLS. Our study also suggests that the stretches of six successive basic residues (lysine and/or arginine) could function as NoLS. [ABSTRACT FROM AUTHOR]

Abstract: Hepatocyte nuclear factor-4 alpha (HNF-4α) is a member of the nuclear receptor superfamily with important roles in hepatic metabolism. Fasting induces the cAMP/protein kinase A (PKA)-signaling pathway. The mechanisms whereby cAMP regulates HNF-4α transcriptional activity are incompletely understood. We have therefore investigated the role of cAMP/PKA in regulation of HNF-4α in COS-1 cells and the hepatoma HepG2 cell line. cAMP/PKA inhibited the transcriptional activity of HNF-4α in COS-1 cells, whereas a stimulatory effect was observed in HepG2 cells. The cAMP-induced inhibition of HNF-4α in COS-1 cells was counteracted by overexpression of the nuclear receptor coactivator PGC-1α, and cAMP/PKA-dependent induction of the PGC1A gene in HepG2 cells seems to explain the cell specific differences. This was further supported by knock-down of PGC-1α in HepG2 cells, which abolished the stimulatory effect of PKA on HNF-4α transcriptional activity. Similar to the cAMP/PKA-mediated regulation of HNF-4α, overexpression of the cAMP-response element binding protein (CREB) inhibited the transcriptional activity of HNF-4α in COS-1 cells, regardless of cAMP/PKA activation and CREB phosphorylation. Moreover, activation of CREB by cAMP/PKA further stimulated HNF-4α transactivation in HepG2 cells. cAMP induced the expression of the HNF-4α target genes PCK1 and G6Pase in these cells. In conclusion, our results suggest that the level of PGC-1α determines whether the cAMP/PKA-pathway overall stimulates or inhibits HNF-4α transcriptional activation. [Copyright &y& Elsevier]

Abstract: We recently reported that 22S-butyl-1α,24R-dihydroxyvitamin D3 3 recovers the agonistic activity for vitamin D receptor (VDR), although its 25,26,27-trinor analog 2 is a potent VDR antagonist. To investigate the structural features involved in the recovery of agonism, we crystallized the ternary complex of VDR-ligand-binding domain, ligand 3 and coactivator peptide, and conducted X-ray crystallographic analysis of the complex. Compared with the complex with 2, the complex with 3 recovered the following structural features: a pincer-type hydrogen bond between the 24-hydroxyl group and VDR, the conformation of Leu305, the positioning of His301 and His393, the stability of the complex, and intimate hydrophobic interactions between the ligand and helix 12. In addition, we evaluated the potency of both compounds for recruiting RXR and coactivator. The results indicate that the complex with 3 generates a suitable surface for coactivator recruitment. These studies suggest that the action of 2 as an antagonist is caused by the generation of a surface not suitable for coactivator recruitment due to the lack of hydrophobic interactions with helix 12 as well as insufficient hydrogen bond formation between the 24-hydroxyl group and VDR. We concluded that the action of 3 as an agonist is based on the elimination of these structural defects in the complex with 2. [ABSTRACT FROM AUTHOR]

Abstract: Thyroid hormones are essential for proper development and differentiation in vertebrates. Recently, concern over the disruption of thyroid hormone homeostasis by industrial chemicals and environmental pollutants has been spreading. To evaluate these chemicals, several bioassays have been developed to detect thyroid hormone ligand activity. Nevertheless, a simple and useful assay is required for the assessment of an enormous number of environmental chemicals. We established yeast reporter assays by expression of full-length thyroid hormone receptor (TRα or TRβ) cDNA and of the TR-dependent reporter gene in yeasts. By additional introduction of the general coactivator SRC-1 cDNA into the yeasts, a higher response to endogenous thyroid hormones, thyroxine (T4), and triiodothyronine (T3) was obtained. The EC50 values for T3 were 35 and 1.5nM for TRα and TRβ assay yeasts, respectively. We tested four chemicals, tetrabromobisphenol A, tetramethylbisphenol A, 2-isopropylphenol, and o-t-butylphenol, which are suspected to have thyroid hormone-disrupting activity. All four chemicals showed agonistic activities in both assay yeasts; however, their activities were weak in comparison with endogenous TR ligands. Antagonist activities of 2-isopropylphenol and o-t-butylphenol were also found in the TRα yeast assay. Taken together, these assay yeasts will be powerful tools for assessing TR ligand activity of industrial chemicals and environmental pollutants. [Copyright &y& Elsevier]

Abstract: Sox proteins are found in all metazoans and are active in many developmental processes. Their function as transcription factors is governed by their DNA-binding properties as much as by their interactions with other transcription factors and cofactors, and subject to modulation by posttranslational modifications. Although most Sox proteins predominantly function as transcriptional activators, there is also evidence for transcriptional repression and architectural roles. Further effects involve complex formation of Sox proteins with transcriptionally relevant factors off DNA. Sox proteins may be additionally active on the posttranscriptional level and are therefore remarkably versatile regulators of gene expression. [Copyright &y& Elsevier]

Abstract: The immediate early genes of the α-herpesviruses HSV and VZV are transcriptionally regulated by viral and cellular factors in a complex combinatorial manner. Despite this complexity and the apparent redundancy of activators, the expression of the viral IE genes is critically dependent upon the cellular transcriptional coactivator HCF-1. Although the role of HCF-1 had remained elusive, recent studies have demonstrated that the protein is a component of multiple chromatin modification complexes including the Set1/MLL1 histone H3K4 methyltransferases. Studies using model viral promoter–reporter systems as well as analyses of components recruited to the viral genome during the initiation of infection have elucidated the significance of HCF-1 chromatin modification complexes in contributing to the final state of modified histones assembled on the viral IE promoters. Strikingly, the absence of HCF-1 results in the accumulation of nucleosomes bearing repressive marks on the viral IE promoters and silencing of viral gene expression. [Copyright &y& Elsevier]

Background: Clinical trials implicate the estrogen receptor (ER) coactivator amplified in breast cancer 1 (AIB1) to be a prognostic and a treatment-predictive factor, although results are not unanimous. We have further investigated this using a controlled randomised trial of tamoxifen versus control. [ABSTRACT FROM PUBLISHER]

Abstract: DNA viruses have long served as model systems to elucidate various aspects of eukaryotic gene regulation, due to their ease of manipulation and relatively low complexity of their genomes. In some cases, these viruses have revealed mechanisms that are subsequently recognized to apply also to cellular genes. In other cases, viruses adopt mechanisms that prove to be exceptions to the more general rules. The double-stranded DNA viruses that replicate in the eukaryotic nucleus typically utilize the host cell RNA polymerase II (RNAP II) for viral gene expression. As a consequence, these viruses must reckon with the impact of chromatin on active transcription and replication. Unlike the small DNA tumor viruses, such as polyomaviruses and papillomaviruses, the relatively large genomes of herpesviruses are not assembled into nucleosomes in the virion and stay predominantly free of histones during lytic infection. In contrast, during latency, the herpesvirus genomes associate with histones and become nucleosomal, suggesting that regulation of chromatin per se may play a role in the switch between the two stages of infection, a long-standing puzzle in the biology of herpesviruses. In this review we will focus on how chromatin formation on the herpes simplex type-1 (HSV-1) genome is regulated, citing evidence supporting the hypothesis that the switch between the lytic and latent stages of HSV-1 infection might be determined by the chromatin state of the HSV-1. [Copyright &y& Elsevier]

Abstract: Progesterone receptor modulators (PRMs) play an important role in women''s health. They are widely used in oral contraception or hormone therapy, and provide an attractive treatment approach for gynecological disorders such as uterine leiomyomas, endometriosis or breast cancer. Due to the broad range of activities, various studies were conducted to assess progesterone receptor antagonists (PAs) and selective progesterone receptor modulators (SPRMs) with respect to progesterone receptor (PR) agonistic and antagonistic activities in vivo. These properties are not always adequately reflected in classical in vitro models, especially differences in the agonistic potential of SPRMs, such as asoprisnil, J1042, and J912, and mixed antagonists, such as mifepristone, are not sufficiently substantiated. The effects of PRMs upon gene expression in progesterone target tissues such as breast epithelium and uterus are poorly understood. This study compares the properties of PR ligands using mammalian two-hybrid assays and gene expression profiling. The protein–protein interaction analyses in HeLa cells provide for specific ligand-induced PR conformations, whereas Affymetrix GeneChip HG-U133Plus2.0 analyses in T47D breast cancer cells indicate the transcriptional activity on the level of target genes. The analyses comprise the pure agonist R5020, the non-steroidal PR modulator PRA-910, SPRMs (J1042, asoprisnil, J912), the mixed antagonist mifepristone, classical antagonists (onapristone, ZK 137316) and the pure antagonist lonaprisan to consider all types of ligands described before. Marginal differences were identified in coactivator interaction profiles at all, but significant differences between SPRMs and PR antagonists (PAs) were observed in recruiting the LXXLL-motif containing peptide (LX-H10), very similar to in vivo activities in endometrial transformation in the rabbit (McPhail test). Global gene expression profiles demonstrated progesterone-independent effects for all PR modulators examined and emphasised similarities of asoprisnil and J1042 compared to J912 and all types of PR antagonists. In summary, the data support the popular concept of PR modulator classification in agonists, selective progesterone receptor modulators, mixed and pure antagonists. It further refines previous classification models and accentuates unique effects for each PR modulator. [Copyright &y& Elsevier]

Abstract: It is desirable to obtain new antagonists for thyroid hormone receptors (TRs) and other nuclear receptors (NRs). We previously used X-ray structural models of TR ligand binding domains (LBDs) to design compounds, such as NH-3, that impair coactivator binding to activation function 2 (AF-2) and block thyroid hormone (triiodothyronine, T3) actions. However, TRs bind DNA and are transcriptionally active without ligand. Thus, NH-3 could modulate TR activity via effects on other coregulator interaction surfaces, such as activation function (AF-1) and corepressor binding sites. Here, we find that NH-3 blocks TR–LBD interactions with coactivators and corepressors and also inhibits activities of AF-1 and AF-2 in transfections. While NH-3 lacks detectable agonist activity at T3-activated genes in GC pituitary cells it nevertheless activates spot 14 (S14) in HTC liver cells with the latter effect accompanied by enhanced histone H4 acetylation and coactivator recruitment at the S14 promoter. Surprisingly, T3 promotes corepressor recruitment to target promoters. NH-3 effects vary; we observe transient recruitment of N–CoR to S14 in GC cells and dismissal and rebinding of N–CoR to the same promoter in HTC cells. We propose that NH-3 will generally behave as an antagonist by blocking AF-1 and AF-2 but that complex effects on coregulator recruitment may result in partial/mixed agonist effects that are independent of blockade of T3 binding in some contexts. These properties could ultimately be utilized in drug design and development of new selective TR modulators. [Copyright &y& Elsevier]

Abstract: Nuclear receptor (NR) dependent transcriptional action requires recruitment of diverse factors characterized as coregulators. PNRC (proline-rich nuclear receptor coregulatory protein) is a member of coregulators that are capable of potentiating the transcriptional activity of NRs. Here we identified three human PNRC splicing variants designated PNRC1c, PNRC1d and PNRC1f. PNRC1c and PNRC1f are generated through alternative recognition of the 3''-splice site in exon 1, leading to in-frame deletion of 79 amino acids (aa) and an altered reading frame, respectively. PNRC1d is generated through the alternate promoter usage and forms a truncated protein containing C-terminus 142 aa of full-length PNRC. These isoforms differ in their abilities to bind NRs and potentiate NR mediated transcriptions. Moreover, PNRC1d can modulate the activity of full-length PNRC in enhancing ER mediated transcription. Our results suggest that PNRC exists as functionally distinct isoforms and alternative splicing serves as a regulatory mechanism of PNRC coactivator activity. [Copyright &y& Elsevier]

Abstract: We established a novel method to evaluate endocrine disrupting chemicals (EDCs) by assembling the estrogen receptor–ligand binding domain (ERLBD) and GFP labeled coactivator on magnetic nanoparticles. EDC can promote or inhibit coactivator recruitment to the ligand–ERLBD complex. ERLBD was displayed on the surface of nano-sized bacterial magnetic particles (BacMPs) produced by the magnetic bacterium, Magnetospirillum magneticum AMB-1. Our method resulted in 38 molecules of ERLBD molecules on a BacMPs with diameter of 75nm. Furthermore, ligand-dependent recruitment assays of GFP labeled coactivator to ERLBD–BacMPs was performed by measuring the fluorescence intensity. 17β-estradiol (E2), estriol, diethylstilbestrol, zeralenone (full agonist), octylphenol (partial agonist) and ICI 182,780 (antagonist) were evaluated by this method. Full agonists tested showed increased fluorescence with increasing agonist concentration. Octylphenol had lower fluorescence intensity than E2. ICI 182,780 did not produce any fluorescence. The method developed in this study can evaluate the estrogenic potential of chemicals by discriminating whether they are an ER full agonist, partial agonist, or antagonist. Finally, this method is amenable adaptation into a high throughput format by using automated magnetic separation. [Copyright &y& Elsevier]

Abstract: Phthalate esters (PEs), a group of environmental chemicals, affect biological systems via endocrine and lipid metabolism modulations. These effects are believed to be mediated in part by peroxisome proliferator-activated receptors (PPARs). Evaluations of PE activities as ligands toward PPARs have been investigated in many studies on their primary metabolites, monoesters. However, the activities of various other metabolites, including oxidized derivatives, remain to be determined. Here, we have evaluated the PPAR ligand activities of these PE derivatives by in vitro coactivator recruiting assay. Mono(2-ethyl-5-hydroxyhexyl)phthalate, the most abundant metabolite of di-(2-ethylhexyl)phthalate (DEHP), was less active than mono(2-ethylhexyl)phthalate (MEHP) as a PPAR ligand. Other derivatives oxidized at the alkyl group and benzene ring of DEHP, MEHP, dibutyl phthalate and its monoester were also investigated and some affected PPAR activities. Unexpectedly, MEHP as well as its further oxidized metabolite did not show clear activity for PPARα, although MEHP is believed to interact with PPARα. This might imply indirect PPAR-mediated mechanisms that lead to observed biological effects such as peroxisome proliferation. [Copyright &y& Elsevier]

Abstract: Thyroid hormone (T3) has a profound influence on normal development, differentiation and metabolism. T3 induces complex gene expression patterns raises the question of how these expression patterns might be regulated. Since the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) induces very similar cellular energy metabolic pathways, we investigated the molecular mechanism of T3 regulation of PGC-1α. PGC-1α is rapidly regulated by T3, both in vivo and in cell culture. Transient transfection experiments demonstrated binding of the thyroid hormone receptor (TR) to a response element located at −4kb upstream of the transcriptional start site within the PGC-1α gene. Introducing of a single copy of the −4kb TRE in a heterologous promoter context is sufficient to maintain T3 responsiveness. Chromatin immunoprecipitation analysis revealed increased histone acetylation upon stimulation of T3. Finally, TR binds the −4kb TRE in electrophoretic mobility shift assays, identifying PGC-1α as a direct target of TR action. Since T3 directly regulates PGC-1α and PGC-1α coactivates liganded TR, we suggest an autoregulatory feed-forward loop of PGC-1α activation upon T3 treatment. [Copyright &y& Elsevier]