In a genome-wide association study (GWAS) of individuals of European ancestry afflicted with systemic lupus erythematosus (SLE) the extensive utilization of imputation, step-wise multiple regression, lasso regularization and increasing study power by utilizing false discovery rate instead of a Bonferroni multiple test correction enabled us to identify 13 novel non-human leukocyte antigen (HLA) genes and confirmed the association of four genes previously reported to be associated. Novel genes associated with SLE susceptibility included two transcription factors (EHF and MED1), two components of the NF-κB pathway (RASSF2 and RNF114), one gene involved in adhesion and endothelial migration (CNTN6) and two genes involved in antigen presentation (BIN1 and SEC61G). In addition, the strongly significant association of multiple single-nucleotide polymorphisms (SNPs) in the HLA region was assigned to HLA alleles and serotypes and deconvoluted into four primary signals. The novel SLE-associated genes point to new directions for both the diagnosis and treatment of this debilitating autoimmune disease.

Rosiglitazone (rosi) is a powerful insulin sensitizer, but serious toxicities have curtailed its widespread clinical use. Rosi functions as a high-affinity ligand for peroxisome proliferator-activated receptor γ (PPARγ), the adipocyte-predominant nuclear receptor (NR). The classic model, involving binding of ligand to the NR on DNA, explains positive regulation of gene expression, but ligand-dependent repression is not well understood. We addressed this issue by studying the direct effects of rosi on gene transcription using global run-on sequencing (GRO-seq). Rosi-induced changes in gene body transcription were pronounced after 10 min and correlated with steady-state mRNA levels as well as with transcription at nearby enhancers (enhancer RNAs [eRNAs]). Up-regulated eRNAs occurred almost exclusively at PPARγ-binding sites, to which rosi treatment recruited coactivators, including MED1, p300, and CBP. In contrast, transcriptional repression by rosi involved a loss of coactivators from eRNA sites devoid of PPARγ and enriched for other transcription factors, including AP-1 factors and C/EBPs. Thus, rosi activates and represses transcription by fundamentally different mechanisms that could inform the future development of anti-diabetic drugs.

BACKGROUND

Human epidermal growth factor receptor 2 (HER2)-amplified breast cancers represent a tumor subtype with chromosome 17q rearrangements that lead to frequent gene amplifications. The aim of this study was to quantify the amplification of genes located on chromosome 17q and to analyze the relations between the pattern of gene amplifications and the patients' characteristics and survival.

METHODS

Patients with HER2-positive breast tumors (HER2 score of 3+ by immunohistochemistry or positive for HER2 amplification by fluorescence in situ hybridization (FISH)) (n = 86) and with HER2-negative breast tumors (n = 40) (negative controls) were included in this study. Using a quantitative polymerase chain reaction method and DNA extracted from frozen tumor specimens, 11 genes (MED1, STARD3, HER2, GRB7, THRA, RARA, TOP2A, IGFBP4, CCR7, KRT20, KRT19 and GAS), which are localized within Chr17q12-q21 and have a putative role in breast cancer development, were quantified. Relapse-free and overall survival rates were estimated from the date of surgery to the date of the event of interest (recurrence or death) using the Kaplan-Meier method.

RESULTS

Gene amplification was observed only in HER2-positive tumors, and the frequency of amplification decreased with the distance of the gene from HER2. HER2 presented the highest level of amplification. TOP2A was not included in the smallest region of amplification involving HER2. Amplification of RARA, KRT20 and KRT19 was significantly associated with node-positive breast cancer (P = 0.030, P = 0.002 and P = 0.033, respectively). During a median follow-up period of 55 months (range, 6 to 81 months), the subgroup of patients with hormone receptor-negative cancer and without TOP2A amplification showed the worst survival (relapse-free survival: hazard ratio (HR) = 0.29, 95% confidence interval (95% CI), 0.13 to 0.65, P = 0.001; and overall survival: HR = 0.28, 95% CI, 0.10 to 0.76, P = 0.008).

CONCLUSIONS

HER2 amplification seems to drive genomic instability along chromosome 17q, leading to different patterns of gene amplification. This study confirms the clinical importance of identifying, among patients with HER2-positive breast tumors, the subgroup of patients with hormone receptor-negative and nonamplified TOP2A cancers as they have the worst prognosis.

The base excision repair protein MED1 (also known as MBD4), an interactor with the mismatch repair protein MLH1, has a central role in the maintenance of genomic stability with dual functions in DNA damage response and repair. MED1 acts as a thymine and uracil DNA N-glycosylase on T:G and U:G mismatches that occur at cytosine-phosphate-guanine (CpG) methylation sites due to spontaneous deamination of 5-methylcytosine and cytosine, respectively. To elucidate the mechanisms that underlie sequence discrimination by MED1, we did single-turnover kinetics with the isolated, recombinant glycosylase domain of MED1. Quantification of MED1 substrate hierarchy confirmed MED1 preference for mismatches within a CpG context and showed preference for hemimethylated base mismatches. Furthermore, the k(st) values obtained with the uracil analogues 5-fluorouracil and 5-iodouracil were over 20- to 30-fold higher than those obtained with uracil, indicating substantially higher affinity for halogenated bases. A 5-iodouracil precursor is the halogenated nucleotide 5-iododeoxyuridine (5IdU), a cytotoxic and radiosensitizing agent. Cultures of mouse embryo fibroblasts (MEF) with different Med1 genotype derived from mice with targeted inactivation of the gene were evaluated for sensitivity to 5IdU. The results revealed that Med1-null MEFs are more sensitive to 5IdU than wild-type MEFs in both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and colony formation assays. Furthermore, high-performance liquid chromatography analyses revealed that Med1-null cells exhibit increased levels of 5IdU in their DNA due to increased incorporation or reduced removal. These findings establish MED1 as a bona fide repair activity for the removal of halogenated bases and indicate that MED1 may play a significant role in 5IdU cytotoxicity.

The growth hormone (GH)-insulin-like growth factor-I (IGF-I) axis regulates somatic growth during childhood and orchestrates tissue repair throughout the life span. Recently described inactivating mutations in Stat5b in humans with impaired growth have focused attention on this transcription factor as a key agent linking GH-stimulated signals to IGF-I gene expression, and several putative Stat5b sites have been identified in the IGF-I gene. Here, we define and characterize potential GH- and Stat5b-activated chromosomal enhancers that can regulate IGF-I gene transcription. Of 89 recognizable Stat5 sequences in 200 kb centering on the rat IGF-I gene, 22 resided within conserved regions and/or were identical among different species. Only 15 of these sites, organized into 7 distinct domains, were found to bind Stat5b by quantitative chromatin immunoprecipitation assays in liver chromatin of rats, but only after acute GH treatment. These sites could bind Stat5b in vitro, and individual domains could mediate GH- and Stat5b-stimulated IGF-I promoter activity in cultured cells. Further analyses revealed that four Stat5b domains possessed chromatin signatures of enhancers, including binding of co-activators p300 and Med1, and RNA polymerase II. These modifications preceded GH-stimulated recruitment of Stat5b, as did lysine 4 monomethylation of histone H3, which was enriched in 6/7 Stat5b-binding elements. In contrast, histone acetylation was induced by GH but was limited to Stat5b binding domains found within the IGF-I transcription unit. We conclude that GH stimulates recruitment of Stat5b to multiple dispersed regions within the igf1 locus, including several with properties consistent with long range transcriptional enhancers that collectively regulate GH-activated IGF-I gene transcription.

Many of the long-term physiological effects of GH require hormone-mediated changes in gene expression. The transcription factor signal transducer and activator of transcription 5b (Stat5b) plays a critical role in the actions of GH on growth and metabolism by regulating a large number of GH-dependent genes by incompletely understood mechanisms. Here we have assessed the impact of GH-initiated and Stat5b-mediated signaling on the chromatin landscape of hormone-regulated genes in the liver of pituitary-deficient young adult male rats. In the absence of GH there was minimal ongoing transcription at the Socs2, Cish, Igfals, and Spi 2.1 promoters, minimal occupancy of Stat5b at proximal promoter sites, and relatively closed chromatin, as evidenced by low levels of core histone acetylation. In contrast, transcriptionally silent Igf1 promoter 1 appeared poised to be activated, based on binding of coactivators p300 and Med1/Trap220, high levels of histone acetylation, and the presence of RNA polymerase II. GH treatment led to a 8- to 20-fold rise in transcriptional activity of all five genes within 30-60 min and was accompanied by binding of Stat5b to the proximal Socs2, Cish, Igfals, and Spi 2.1 promoters and to seven distal Igf1 Stat5b elements, by enhanced histone acetylation at all five promoters, by recruitment of RNA polymerase II to the Socs2, Cish, Igfals, and Spi 2.1 promoters, and by loss of the transcriptional repressor Bcl6 from Socs2, Cish, and Igfals Stat5b sites, but not from two Igf1 Stat5b domains. We conclude that GH actions induce rapid and dramatic changes in hepatic chromatin at target promoters and propose that the chromatin signature of Igf1 differs from other GH-and Stat5b-dependent genes.

Interactions between transcriptional promoters and their distal regulatory elements play an important role in transcriptional regulation; however, the extent to which these interactions are subject to rapid modulations in response to signals is unknown. Here, we use promoter capture Hi-C to demonstrate a rapid reorganization of promoter-anchored chromatin loops within 4 hr after inducing differentiation of 3T3-L1 preadipocytes. The establishment of new promoter-enhancer loops is tightly coupled to activation of poised (histone H3 lysine 4 mono- and dimethylated) enhancers, as evidenced by the acquisition of histone H3 lysine 27 acetylation and the binding of MED1, SMC1, and P300 proteins to these regions, as well as to activation of target genes. Intriguingly, formation of loops connecting activated enhancers and promoters is also associated with extensive recruitment of corepressors such as NCoR and HDACs, indicating that this class of coregulators may play a previously unrecognized role during enhancer activation.

Peroxisome proliferator-activated receptor-binding protein (PBP), also known as thyroid hormone receptor-associated protein 220/vitamin D receptor-interacting protein 205/mediator 1, an anchor for multisubunit mediator transcription complex, functions as a transcription coactivator for nuclear receptors. Disruption of the PBP gene results in embryonic lethality around embryonic day 11.5 by affecting placental and multiorgan development. Here, we report that targeted deletion of PBP in liver parenchymal cells (PBP(Liv-/-)) results in the abrogation of hypertrophic and hyperplastic influences in liver mediated by constitutive androstane receptor (CAR) ligands phenobarbital (PB) and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, and of acetaminophen-induced hepatotoxicity. CAR interacts with the two nuclear receptor-interacting LXXLL (L, leucine; X, any amino acid) motifs in PBP in a ligand-dependent manner. We also show that PBP interacts with the C-terminal portion of CAR, suggesting that PBP is involved in the regulation of CAR function. Although the full-length PBP only minimally increased CAR transcriptional activity, a truncated form of PBP (amino acids 487-735) functioned as a dominant negative repressor, establishing that PBP functions as a coactivator for CAR. A reduction in CAR mRNA and protein level observed in PBP(Liv-/-) mouse liver suggests that PBP may regulate hepatic CAR expression. PBP-deficient hepatocytes in liver failed to reveal PB-dependent translocation of CAR to the nucleus. Adenoviral reconstitution of PBP in PBP(Liv-/-) mouse livers restored PB-mediated nuclear translocation of CAR as well as inducibility of CYP1A2, CYP2B10, CYP3A11, and CYP7A1 expression. We conclude that transcription coactivator PBP/TRAP220/MED1 is involved in the regulation of hepatic CAR function and that PBP deficiency in liver abrogates acetaminophen hepatotoxicity.

Understanding the molecular pathways that contribute to the development of tamoxifen resistance is a critical research priority as acquired tamoxifen resistance is the principal cause of poor prognosis and death of patients with originally good prognosis hormone-responsive breast tumors. In this report, we provide evidence that Med1, an important subunit of mediator coactivator complex, is spontaneously upregulated during acquired tamoxifen-resistance development potentiating agonist activities of tamoxifen. Phosphorylated Med1 and estrogen receptor (ER) are abundant in tamoxifen-resistant breast cancer cells due to persistent activation of extracellular signal-regulated kinases. Mechanistically, phosphorylated Med1 exhibits nuclear accumulation, increased interaction with ER and higher tamoxifen-induced recruitment to ER-responsive promoters, which is abrogated by inhibition of Med1 phosphorylation. Stable knockdown of Med1 in tamoxifen-resistant cells not only reverses tamoxifen resistance in vitro but also in vivo. Finally, higher expression levels of Med1 in the tumor significantly correlated with tamoxifen resistance in ER-positive breast cancer patients on adjuvant tamoxifen monotherapy. In silico analysis of breast cancer, utilizing published profiling studies showed that Med1 is overexpressed in aggressive subsets. These findings provide what we believe is the first evidence for a critical role for Med1 in tamoxifen resistance and identify this coactivator protein as an essential effector of the tamoxifen-induced breast cancer growth.

We have previously shown that adenoviral expression of peroxisome proliferator-activated receptors (PPARs) leads to rapid establishment of transcriptionally active complexes and activation of target gene expression within 5-8 h after transduction. Here we have used the adenoviral delivery system combined with expression array analysis to identify novel putative PPARgamma target genes in murine fibroblasts and to determine the role of the A/B-domain in PPARgamma-mediated transactivation of genomic target genes. Of the 257 genes found to be induced by PPARgamma2 expression, only 25 displayed A/B-domain dependency, i.e. significantly reduced induction in the cells expressing the truncated PPARgamma lacking the A/B-domain (PPARgammaCDE). Nine of the 25 A/B-domain-dependent genes were involved in lipid storage, and in line with this, triglyceride accumulation was considerably decreased in the cells expressing PPARgammaCDE compared with cells expressing full-length PPARgamma2. Using chromatin immunoprecipitation, we demonstrate that PPARgamma binding to genomic target sites and recruitment of the mediator component TRAP220/MED1/PBP/DRIP205 is not affected by the deletion of the A/B-domain. By contrast, the PPARgamma-mediated cAMP response element-binding protein (CREB)-binding protein (CBP) and p300 recruitment to A/B-domain-dependent target genes is compromised by deletion of the A/B-domain. These results indicate that the A/B-domain of PPARgamma2 is specifically involved in the recruitment or stabilization of CBP- and p300-containing cofactor complexes to a subset of target genes.

Mediator is the central coactivator complex required for regulated transcription by RNA polymerase (Pol) II. Mediator consists of 25 subunits arranged in the head, middle, tail and kinase modules. Structural and functional studies of Mediator are limited by the availability of protocols for the preparation of recombinant modules. Here, we describe protocols for obtaining pure endogenous and recombinant complete Mediator middle module from Saccharomyces cerevisiae that consists of seven subunits: <em>Med1</em>, 4, 7, 9, 10, 21 and 31. Native mass spectrometry reveals that all subunits are present in equimolar stoichiometry. Ion-mobility mass spectrometry, limited proteolysis, light scattering and small-angle X-ray scattering all indicate a high degree of intrinsic flexibility and an elongated shape of the middle module. Protein-protein interaction assays combined with previously published data suggest that the Med7 and Med4 subunits serve as a binding platform to form the three heterodimeric subcomplexes, Med7N/21, Med7C/31 and Med4/9. The subunits, <em>Med1</em> and <em>Med1</em>0, which bridge to the Mediator tail module, bind to both Med7 and Med4.

Transcription factor CCAAT/enhancer-binding protein (C/EBP)-beta is crucial for regulating transcription of genes involved in a number of diverse cellular processes, including those involved in some cytokine-induced responses. However, the mechanisms that contribute to its diverse transcriptional activity are not yet fully understood. To gain an understanding into its mechanisms of action, we took a proteomic approach and identified cellular proteins that associate with C/EBP-beta in an interferon (IFN)-gamma-dependent manner. Transcriptional mediator (Mediator) is a multisubunit protein complex that regulates signal-induced cellular gene transcription from enhancer-bound transcription factor(s). Here, we report that the Med1 subunit of the Mediator as a C/EBP-beta-interacting protein. Using gene knock-out cells and mutational and RNA interference approaches, we show that Med1 is critical for IFN-induced expression of certain genes. Med1 associates with C/EBP-beta through a domain located between amino acids 125 and 155 of its N terminus. We also show that the MAPK, ERK1/2, and an ERK phosphorylation site within regulatory domain 2, more specifically the Thr(189) residue, of C/EBP-beta are essential for it to bind to Med1. Last, an ERK-regulated site in Med1 protein is also essential for up-regulating IFN-induced transcription although not critical for binding to C/EBP-beta.

The human hyaluronan synthase 2 (HAS2) gene encodes for an enzyme making hyaluronan, altered concentrations of which are associated with many pathological situations including wounding, several inflammatory conditions, and malignant tumors. In this study we showed that HAS2 is a primary target of the cAMP activator forskolin and the nuclear hormone all-trans-retinoic acid (RA). The first 2250 bp of the promoter contain three response elements (REs) for the transcription factor CREB1 as well as two REs for the nuclear receptor RAR. Chromatin immunoprecipitation and re-chromatin immunoprecipitation assays using selected fragments of the promoter containing the putative REs showed that forskolin and all-trans-RA modulate the formation of complexes between CREB1 and RAR with various co-regulators at the predicted sites. Interestingly, CREB1 complexes are regulated by all-trans-RA as are RAR complexes by forskolin. Reporter gene assays using nested promoter fragments supported these findings. Forskolin and all-trans-RA co-stimulation reduced the binding of CREB1, RAR, and the co-repressor nuclear receptor co-repressor 1 (NCoR1), but enhanced the association of co-activators MED1 and CREB-binding protein (CBP). RNA interference experiments suggested that MED1 and NCoR1 are central for the all-trans-RA induction of the HAS2 gene and CBP dominates its forskolin response. In general, our findings suggest a convergence of CREB1 and RAR signaling, and demonstrate the individual character of each RE in terms of co-regulator use.

PGC-1alpha is an inducible nuclear receptor coactivator with direct functions in both p300-mediated chromatin remodeling and Mediator-dependent transcription in vitro. Here, we have employed the PPARgamma- and TRalpha-activated brown adipose tissue-specific UCP-1 enhancer to investigate mechanistic aspects of PGC-1alpha function. We first demonstrate a cellular role for the PGC-1alpha-interacting MED1 subunit of Mediator in UCP-1 induction, as well as the accumulation of TRalpha, PPARgamma, PGC-1alpha, and MED1 on the UCP-1 enhancer in brown adipocytes. We then use biochemical assays to show that (i) PGC-1alpha is recruited to the TRalpha-RXRalpha-UCP-1 enhancer complex through interaction of an N-terminal LXXLL domain with TRalpha, (ii) MED1/Mediator displaces PGC-1alpha from TRalpha through LXXLL domain competition, and (iii) upon loss of PGC-1alpha-TRalpha interactions, PGC-1alpha remains associated with the enhancer complex through an interaction between PGC-1alpha and MED1 C-terminal domains. These results indicate dynamic MED1-dependent PGC-1alpha interactions related to functions in both chromatin remodeling and the transition to subsequent transcription initiation.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression found in more than 200 diverse organisms. Although it is still not fully established if RNA viruses could generate miRNAs, there are examples of miRNA like sequences from RNA viruses with regulatory functions. In the case of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with viral replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA-based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Overall, 950 hairpin structured sequences were extracted from the virus genome and based on the prediction results, 29 of them could be precursor miRNAs. Targeting analysis showed that 30 viral mature miRNA-like sequences could target 1,367 different human genes. PANTHER gene function analysis results indicated that viral derived miRNA candidates could target various human genes involved in crucial cellular processes including transcription, metabolism, defense system and several signaling pathways such as Wnt and EGFR signalings. Protein class-based grouping of targeted human genes showed that host transcription might be one of the main targets of the virus since 96 genes involved in transcriptional processes were potential targets of predicted viral miRNAs. For instance, basal transcription machinery elements including several components of human mediator complex (<em>MED1</em>, MED9, <em>MED1</em>2L, <em>MED1</em>9), basal transcription factors such as TAF4, TAF5, TAF7L and site-specific transcription factors such as STAT1 were found to be targeted. In addition, many known human miRNAs appeared to be able to target viral genes involved in viral life cycle such as S, M, N, E proteins and ORF1ab, ORF3a, ORF8, ORF7a and ORF10. Considering the fact that miRNA-based therapies have been paid attention, based on the findings of this study, comprehending mode of actions of miRNAs and their possible roles during SARS-CoV-2 infections could create new opportunities for the development and improvement of new therapeutics.

Keywords: COVID19; Host–virus interaction; MicroRNA; SARS-CoV-2.

Mammalian physiology exhibits 24-hour cyclicity due to circadian rhythms of gene expression controlled by transcription factors that constitute molecular clocks. Core clock transcription factors bind to the genome at enhancer sequences to regulate circadian gene expression, but not all binding sites are equally functional. We found that in mice, circadian gene expression in the liver is controlled by rhythmic chromatin interactions between enhancers and promoters. Rev-erbα, a core repressive transcription factor of the clock, opposes functional loop formation between Rev-erbα-regulated enhancers and circadian target gene promoters by recruitment of the NCoR-HDAC3 co-repressor complex, histone deacetylation, and eviction of the elongation factor BRD4 and the looping factor MED1. Thus, a repressive arm of the molecular clock operates by rhythmically modulating chromatin loops to control circadian gene transcription.

Androgen receptor (AR) signaling pathways are important for the survival and proliferation of prostate cancer cells. Because AR activity is facilitated by distinct coregulatory factors and complexes, it is conceivable that some of these proteins might also play a role in promoting prostate oncogenesis. The multisubunit Mediator complex is an important coactivator for a broad range of regulatory transcriptional factors including AR, yet its role in prostate cancer is unclear. Here, we used RNA interference to knock down the expression of two integral Mediator components, MED1/TRAP220 and MED1MED1/TRAP220 plays a particularly important role in androgen signaling in that it serves as a direct binding target for AR. We found that the knockdown of either subunit markedly decreases transcription from transiently transfected androgen-responsive reporter genes, as well as inhibits androgen-dependent expression of endogenous AR target genes. We show for the first time that loss of either MED1/TRAP220 or MED1MED1/TRAP220 is overexpressed in both AR-positive and -negative prostate cancer cells lines, as well as in 50% (10 of 20) of the clinically localized human prostate cancers we examined, thus suggesting that MED1/TRAP220 hyperactivity may have implications in prostate oncogenesis. In sum, our data suggest that Mediator plays an important coregulatory role in prostate cancer cell proliferation and survival, and therefore, may represent a new target for therapeutic intervention.

PR domain-containing 16 (PRDM16) induces expression of brown fat-specific genes in brown and beige adipocytes, although the underlying transcription-related mechanisms remain largely unknown. Here, in vitro studies show that PRDM16, through its zinc finger domains, directly interacts with the MED1 subunit of the Mediator complex, is recruited to the enhancer of the brown fat-specific uncoupling protein 1 (Ucp1) gene through this interaction, and enhances thyroid hormone receptor (TR)-driven transcription in a biochemically defined system in a Mediator-dependent manner, thus providing a direct link to the general transcription machinery. Complementary cell-based studies show that upon forskolin treatment, PRDM16 induces Ucp1 expression in undifferentiated murine embryonic fibroblasts, that this induction depends on MED1 and TR, and, consistent with a direct effect, that PRDM16 is recruited to the Ucp1 enhancer. Related studies have defined MED1 and PRDM16 interaction domains important for Ucp1 versus Ppargc1a induction by PRDM16. These results reveal novel mechanisms for PRDM16 function through the Mediator complex.

Peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor, when overexpressed in liver stimulates the induction of adipocyte-specific and lipogenesis-related genes and causes hepatic steatosis. We report here that Mediator 1 (MED1; also known as PBP or TRAP220), a key subunit of the Mediator complex, is required for high-fat diet-induced hepatic steatosis as well as PPARγ-stimulated adipogenic hepatic steatosis. Mediator forms the bridge between transcriptional activators and RNA polymerase II. MED1 interacts with nuclear receptors such as PPARγ and other transcriptional activators. Liver-specific MED1 knockout (MED1(ΔLiv) ) mice, when fed a high-fat (60% kcal fat) diet for up to 4 months failed to develop fatty liver. Similarly, MED1(ΔLiv) mice injected with adenovirus-PPARγ (Ad/PPARγ) by tail vein also did not develop fatty liver, whereas mice with MED1 (MED1(fl/fl) ) fed a high-fat diet or injected with Ad/PPARγ developed severe hepatic steatosis. Gene expression profiling and northern blot analyses of Ad/PPARγ-injected mouse livers showed impaired induction in MED1(ΔLiv) mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin, and lipid droplet-associated genes, including caveolin-1, CideA, S3-12, and others. These adipocyte-specific and lipogenesis-related genes are strongly induced in MED1(fl/fl) mouse liver in response to Ad/PPARγ. Re-expression of MED1 using adenovirally-driven MED1 (Ad/MED1) in MED1(ΔLiv) mouse liver restored PPARγ-stimulated hepatic adipogenic response. These studies also demonstrate that disruption of genes encoding other coactivators such as SRC-1, PRIC285, PRIP, and PIMT had no effect on hepatic adipogenesis induced by PPARγ overexpression.

CONCLUSIONS

We conclude that transcription coactivator MED1 is required for high-fat diet-induced and PPARγ-stimulated fatty liver development, which suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis. (HEPATOLOGY 2011;).

Estrogen receptor is a nuclear receptor superfamily member of transcriptional activators that regulate gene expression by recruiting diverese transcriptional coregulators. The Mediator complex is a central transcriptional coactivator complex that acts as a bridge between transcriptional activators and RNA polymerase II. MED1 (Mediator subunit 1) is the key Mediator subunit that directly interacts with estrogen receptor to mediate its functions both in vitro and in vivo. Interestingly, our previous biochemical analyses indicated that MED1 exists only in a subpopulation of the Mediator complex that is enriched with a number of distinct Mediator subunits and RNA polymerase II. Here, we report ARGLU1 as a MED1/Mediator-associated protein. We found that ARGLU1 (arginine and glutamate rich 1) not only colocalizes with MED1 in the nucleus, but also directly interacts with a far C-terminal region of MED1. Reporter assays indicate that ARGLU1 is able to cooperate with MED1 to regulate estrogen receptor-mediated gene transcription. Importantly, ARGLU1 is recruited, in a ligand-dependent manner, to endogenous estrogen receptor target gene promoters and is required for their expression. Furthermore, by ChIP-reChIP assay, we confirm that ARGLU1 and MED1 colocalize on the same estrogen receptor target gene promoter upon estrogen induction. Moreover, we found that depletion of ARGLU1 significantly impairs the growth, as well as anchorage-dependent and -independent colony formation of breast cancer cells. Taken together, these results establish ARGLU1 as a new MED1-interacting protein required for estrogen-dependent gene transcription and breast cancer cell growth.

A mutant at the yeast MED1 locus was isolated in a screen for sporulation-proficient, meiotic-lethal mutants. Synaptonemal complex formation in the med1 mutant is apparently normal and med1 strains undergo meiotic crossing over at approximately 50% of the wild-type level. The med1 mutant undergoes homolog nondisjunction at meiosis I, presumably as a consequence of the decrease in crossing over. In addition, the mutant undergoes precocious separation of sister chromatids, resulting in chromosome missegregation at both meiotic divisions. We suggest that the med1 mutation perturbs chromosome structure, leading to a reduction in recombination and a defect in sister chromatid cohesion.

The Mediator complex forms the bridge between transcriptional activators and the RNA polymerase II. Med1 (also known as PBP or TRAP220) is a key component of Mediator that interacts with nuclear hormone receptors and GATA transcription factors. Here, we show dynamic recruitment of GATA-1, TFIIB, Mediator, and RNA polymerase II to the β-globin locus in induced mouse erythroid leukemia cells and in an erythropoietin-inducible hematopoietic progenitor cell line. Using Med1 conditional knockout mice, we demonstrate a specific block in erythroid development but not in myeloid or lymphoid development, highlighted by the complete absence of β-globin gene expression. Thus, Mediator subunit Med1 plays a pivotal role in erythroid development and in β-globin gene activation.

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (Pbp(DeltaLiv)) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G(2)/M phase in Pbp(DeltaLiv) hepatocytes after partial hepatectomy. Pbp(DeltaLiv) hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl(4)-induced hepatocellular proliferation and hepatotoxicity occurred in Pbp(DeltaLiv) mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl(4) activation. Pbp(DeltaLiv) mice, chronically exposed to Wy-14,643, a PPARalpha ligand, revealed a striking proliferative response and clonal expansion of a few Pbp(fl/fl) hepatocytes that escaped Cre-mediated gene deletion in Pbp(DeltaLiv) livers, but no proliferative expansion of PBP null hepatocytes was observed. In these Pbp(DeltaLiv) mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBP(DeltaLiv) hepatocytes; all liver tumors developing in Pbp(DeltaLiv) mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.

MED1 is a base excision repair enzyme that interacts with the mismatch repair protein MLH1 and maintains genomic integrity by binding methylated DNA and repairing spontaneous deamination events. MED1 mutations have been associated with microsatellite instability and accelerated colorectal cancer (CRC) tumorigenesis. We propose that promoter methylation may serve as an alternative epigenetic mechanism for MED1 gene suppression during sporadic CRC tumorigenesis. Methylation status of the MED1 promoter was investigated in a panel of ovarian and colorectal cancer cell lines. The MED1 promoter region was sequenced following bisulfite treatment and sequence analysis identified a CpG island within the MED1 promoter which is frequently and preferentially methylated >> or =50%) in ovarian and colorectal cancer cell lines with low/reduced MED1 expression. In vitro reversal of methylation restored MED1 expression. In colorectal cancer patients, when MED1 methylation was present, both tumor and matched mucosa were affected equally (mean frequency of methylation 24%) and there was no correlation between methylation and tumor stage. Patients without history of CRC showed significantly lower frequency of methylation (mean 14%, p < 0.05). Decreased MED1 transcript levels were observed in matched normal mucosa when compared to controls (median fold difference 8.0). Additional decreased expression was seen between mucosa and matched tumor (median fold decrease 4.4). Thus, MED1 promoter methylation and gene silencing occur in sporadic CRC patients and represent an early event in CRC tumorigenesis. Detection of MED1 methylation and gene suppression in normal colon mucosa may contribute to identifying patients at higher risk of developing CRC during screening procedures.