The ubiquitin pathway has been shown to regulate iNKT cell immunity, but the deubiquitinase involved in this process has not been identified. Herein we found that ubiquitin-specific peptidase 22 (USP22) is highly expressed in iNKT cells during their early developmental stage 1. USP22 deficiency blocked the transition from stage 1 to 2 during iNKT cell development in a cell-intrinsic manner. USP22 suppression also diminishes iNKT17 and iNKT1 differentiation but favors iNKT2 polarization without altering conventional T cell activation and differentiation. USP22 interacts with the Mediator complex subunit 1 (MED1), a transcription coactivator involved in iNKT cell development. Interestingly, while interacting with MED1, USP22 does not function as a deubiquitinase to suppress MED1 ubiquitination for its stabilization. Instead, USP22 enhances MED1 functions for IL-2Rβ and T-bet gene expression through deubiquitinating histone H2A but not H2B monoubiquitination. Therefore, our study revealed USP22-mediated histone H2A deubiquitination fine-tunes MED1 transcriptional activation as a previously unappreciated molecular mechanism to control iNKT development and functions.

The underlying mechanism of transcriptional co-repressor ETO2 during early erythropoiesis and hemoglobin switching is unclear. We find that absence of ETO2 in mice interferes with down-regulation of PU.1 and GATA2 in the fetal liver, impeding a key step required for commitment to erythroid maturation. In human β-globin transgenic Eto2 null mice and in human CD34+ erythroid progenitor cells with reduced ETO2, loss of ETO2 results in ineffective silencing of embryonic/fetal globin gene expression, impeding hemoglobin switching during erythroid differentiation. ETO2 occupancy genome-wide occurs virtually exclusively at LDB1-complex binding sites in enhancers and ETO2 loss leads to increased enhancer activity and expression of target genes. ETO2 recruits the NuRD nucleosome remodeling and deacetylation complex to regulate histone acetylation and nucleosome occupancy in the β-globin locus control region and γ-globin gene. Loss of ETO2 elevates LDB1, MED1 and Pol II in the locus and facilitates fetal γ-globin/LCR looping and γ-globin transcription. Absence of the ETO2 hydrophobic heptad repeat region impairs ETO2-NuRD interaction and function in antagonizing γ-globin/LCR looping. Our results reveal a pivotal role for ETO2 in erythropoiesis and globin gene switching through its repressive role in the LDB1 complex, affecting the transcription factor and epigenetic environment and ultimately restructuring chromatin organization.

E2A, a basic helix-loop-helix (bHLH) transcription factor, plays a crucial role in determining tissue-specific cell fate, including differentiation of B cell lineages. In 5% of childhood acute lymphoblastic leukemia (ALL), the t(1,19) chromosomal translocation specifically targets the E2A gene and produces an oncogenic E2A-PBX1 fusion protein. While previous studies have demonstrated oncogenic functions of E2A-PBX1 in cell and animal models, the E2A-PBX1-enforced cistrome, the E2A-PBX1 interactome, and related mechanisms underlying leukemogenesis remain unclear. Here, by unbiased genomic profiling approaches, we identify the direct target sites of E2A-PBX1 in t(1,19)-positive pre-B ALL cells and show that, compared to normal E2A, E2A-PBX1 preferentially binds to a subset of gene loci co-bound by RUNX1 and gene-activating machineries (p300, MED1, and H3K27 acetylation). Using biochemical analyses, we further document a direct interaction of E2A-PBX1, through a region spanning the PBX1 homeodomain, with RUNX1. Our results also show that E2A-PBX1 binding to gene enhancers is dependent on the RUNX1 interaction, but not the DNA-binding activity harbored within the PBX1 homeodomain of E2A-PBX1. Transcriptome analyses and cell transformation assays further establish a significant RUNX1 requirement for E2A-PBX1-mediated target gene activation and leukemogenesis. Notably, the RUNX1 locus itself is also directly activated by E2A-PBX1, indicating a multilayered interplay between E2A-PBX1 and RUNX1. Collectively, our study provides the first unbiased profiling of the E2A-PBX1 cistrome in pre-B ALL cells and reveals a previously unappreciated pathway in which E2A-PBX1 acts in concert with RUNX1 to enforce transcriptome alterations for the development of pre-B ALL.

The genetic diversity of Yersinia pestis strains from the Mongolian natural plague foci has been investigated. A total of 32 strains isolated from western, eastern, and central aimaks, as well as from the territory of the Gobi region, have been studied. Twenty-four strains belong to the main Y. pestis subspecies, while eight belong to other subspecies. There is only one strain of biovar medievalis (genovariant 2.MED1) among the strains of the main subspecies, while the rest of the subspecies belong to the biovar antiqua. Biovar antiqua strains are split into three groups. Strains from the eastern part of the country were classified as genovariant 2.ANT3, and those from the western and central regions were classified as genovariant 3.ANT2, which was endemic for Mongolia. One strain from the Bayan-Ulegeiskii aimak had the rare genovariant 4.ANT. None of the strains of the biovar antiqua belonged to its ancient 0.ANT branch, which is inconsistent with the commonly accepted idea that ancient marmot's plague agent race originates from Mongolia. Six out of eight strains of the minor subspecies belonged to the ulegeica subspecies, which are endemic to Mongolia, one strain belonged to the microtus group, and the last belonged to a previously uncharacterized variant of the minor subspecies.

Renin cells are crucial for survival - they control fluid-electrolyte and blood pressure homeostasis, vascular development, regeneration, and oxygen delivery to tissues. During embryonic development, renin cells are progenitors for multiple cell types that retain the memory of the renin phenotype. When there is a threat to survival, those descendants are transformed and reenact the renin phenotype to restore homeostasis. We tested the hypothesis that the molecular memory of the renin phenotype resides in unique regions and states of these cells' chromatin. Using renin cells at various stages of stimulation, we identified regions in the genome where the chromatin is open for transcription, mapped histone modifications characteristic of active enhancers such as H3K27ac, and tracked deposition of transcriptional activators such as Med1, whose deletion results in ablation of renin expression and low blood pressure. Using the rank ordering of super-enhancers, epigenetic rewriting, and enhancer deletion analysis, we found that renin cells harbor a unique set of super-enhancers that determine their identity. The most prominent renin super-enhancer may act as a chromatin sensor of signals that convey the physiologic status of the organism, and is responsible for the transformation of renin cell descendants to the renin phenotype, a fundamental process to ensure homeostasis.

Mediator complex has been extensively shown to regulate the levels of several protein-coding genes; however, its role in the regulation of miRNAs in humans remains unstudied so far. Here we show that MED1, a Mediator subunit in the Middle module of Mediator complex, is overexpressed in breast cancer and is a negative prognostic factor. The levels of several miRNAs (miR-100-5p, -191-5p, -193b-3p, -205-5p, -326, -422a and -425-5p) were found to be regulated by MED1. MED1 induces miR-191/425 cluster in an estrogen receptor-alpha (ER-α) dependent manner. Occupancy of MED1 on estrogen response elements (EREs) upstream of miR-191/425 cluster is estrogen and ER-α-dependent and ER-α-induced expression of these miRNAs is MED1-dependent. MED1 mediates induction of cell proliferation and migration and the genes associated with it (JUN, FOS, EGFR, VEGF, MMP1, and ERBB4) in breast cancer, which is abrogated when used together with miR-191-inhibition. Additionally, we show that MED1 also regulates the levels of direct miR-191 target genes such as SATB1, CDK6 and BDNF. Overall, the results show that MED1/ER-α/miR-191 axis promotes breast cancer cell proliferation and migration and may serve as a novel target for therapy.

The MED1 subunit of the Mediator transcriptional coregulator complex coactivates GATA1 and induces erythropoiesis. Here, we show the dual mechanism of GATA1- and MED1-mediated transcription. MED1 expression levels in K562 erythroleukemia cells paralleled the levels of GATA1-targeted gene transcription and erythroid differentiation. An N-terminal fragment of MED1, MED1(1-602), which is incapable of interacting with GATA1, enhanced GATA1-targeted gene transcription and erythroid differentiation, and introduction of MED1(1-602) into Med1(-/-) mouse embryonic fibroblasts (MEFs) partially rescued GATA1-mediated transcription. The C-terminal zinc-finger domain of GATA1 interacts with the MED1(1-602)-interacting coactivator CCAR1, CoCoA and MED1(681-715). CCAR1 and CoCoA synergistically enhanced GATA1-mediated transcription from the γ-globin promoter in MEFs. Recombinant GATA1, CCAR1, CoCoA and MED1(1-602) formed a complex in vitro, and GATA1, CCAR1, CoCoA and MED1 were recruited to the γ-globin promoter in K562 cells during erythroid differentiation. Therefore, in addition to the direct interaction between GATA1 and MED1, CoCoA and CCAR1 appear to relay the GATA1 signal to MED1, and multiple modes of the GATA1-MED1 axis may help to fine-tune GATA1 function during GATA1-mediated homeostasis events.

Emerging evidence has demonstrated that alternative splicing has a vital role in regulating protein function, but how alternative splicing factors can be regulated remains unclear. We showed that the PPM1G, a protein phosphatase, regulated the phosphorylation of SRSF3 in hepatocellular carcinoma (HCC) and contributed to the proliferation, invasion, and metastasis of HCC. PPM1G was highly expressed in HCC tissues compared to adjacent normal tissues, and higher levels of PPM1G were observed in adverse staged HCCs. The higher levels of PPM1G were highly correlated with poor prognosis, which was further validated in the TCGA cohort. The knockdown of PPM1G inhibited the cell growth and invasion of HCC cell lines. Further studies showed that the knockdown of PPM1G inhibited tumor growth in vivo. The mechanistic analysis showed that the PPM1G interacted with proteins related to alternative splicing, including SRSF3. Overexpression of PPM1G promoted the dephosphorylation of SRSF3 and changed the alternative splicing patterns of genes related to the cell cycle, the transcriptional regulation in HCC cells. In addition, we also demonstrated that the promoter of PPM1G was activated by multiple transcription factors and co-activators, including MYC/MAX and EP300, MED1, and ELF1. Our study highlighted the essential role of PPM1G in HCC and shed new light on unveiling the regulation of alternative splicing in malignant transformation.

Super-enhancers (SEs) are unique areas of the genome which drive high-level of transcription and play a pivotal role in the cell physiology. Previous studies have established several important genes in cancer as SE-driven oncogenes. It is likely that oncogenes may hack the resident tissue regenerative program and interfere with SE-driven repair networks, leading to the specific pancreatic ductal adenocarcinoma (PDAC) phenotype. Here, we used ChIP-Seq to identify the presence of SE in PDAC cell lines. Differential H3K27AC marks were identified at enhancer regions of genes including c-MYC, MED1, OCT-4, NANOG, and SOX2 that can act as SE in non-cancerous, cancerous and metastatic PDAC cell lines. GZ17-6.02 affects acetylation of the genes, reduces transcription of major transcription factors, sonic hedgehog pathway proteins, and stem cell markers. In accordance with the decrease in Oct-4 expression, ChIP-Seq revealed a significant decrease in the occupancy of OCT-4 in the entire genome after GZ17-6.02 treatment suggesting the possible inhibitory effect of GZ17-6.02 on PDAC. Hence, SE genes are associated with PDAC and targeting their regulation with GZ17-6.02 offers a novel approach for treatment.

The base excision repair DNA N-glycosylase MBD4 (also known as MED1), an interactor of the DNA mismatch repair protein MLH1, plays a central role in the maintenance of genomic stability of CpG sites by removing thymine and uracil from G:T and G:U mismatches, respectively. MBD4 is also involved in DNA damage response and transcriptional regulation. The interaction with other proteins is likely critical for understanding MBD4 functions. To identify novel proteins that interact with MBD4, we used tandem affinity purification (TAP) from HEK-293 cells. The MBD4-TAP fusion and its co-associated proteins were purified sequentially on IgG and calmodulin affinity columns; the final eluate was shown to contain MLH1 by western blotting, and MBD4-associated proteins were identified by mass spectrometry. Bands with molecular weight higher than that expected for MBD4 (˜66 kD) yielded peptides corresponding to MBD4 itself and the small ubiquitin-like molecule-1 (SUMO1), suggesting that MBD4 is sumoylated in vivo. MBD4 sumoylation was validated by co-immunoprecipitation in HEK-293 and MCF7 cells, and by an in vitrosumoylation assay. Sequence and mutation analysis identified three main sumoylation sites: MBD4 is sumoylated preferentially on K137, with additional sumoylation at K215 and K377. Patterns of MBD4 sumoylation were altered, in a DNA damage-specific way, by the anti-metabolite 5-fluorouracil, the alkylating agent N-Methyl-N-nitrosourea and the crosslinking agent cisplatin. MCF7 extract expressing sumoylated MBD4 displays higher thymine glycosylase activity than the unmodified species. Of the 67 MBD4 missense mutations reported in The Cancer Genome Atlas, 14 (20.9%) map near sumoylation sites. These results indicate that MBD4 is sumoylated in vivo in a DNA damage-specific manner, and suggest that sumoylation serves to regulate its repair activity and could be compromised in cancer. This study expands the role played by sumoylation in fine-tuning DNA damage response and repair.

Tnk1/Kos1 is a non-receptor protein tyrosine kinase found to be a tumor suppressor. It negatively regulates cell growth by indirectly suppressing Ras activity. We identified and characterized the critical cis-elements required for Tnk1/Kos1's promoter activity. Results indicate that the murine Tnk1 promoter lacks a conventional TATA, CAAT or initiator element (Inr) but contains multiple transcription start sites. Transcription is initiated by a TATA-like element composed of an AT rich sequence at -30 (30 bp upstream) from the major transcription start site and an Inr-like element that overlaps the multiple start sites. Deletion analysis of the m-Tnk1 promoter reveals the presence of both positive (-25 to -151) and negative (-151 to -1201) regulatory regions. The three GC boxes which bind Sp1 and Sp3 with high affinity, an AP2 site (that overlaps with an AML1 site) and a MED1 site comprise the necessary cis-elements of the proximal promoter required for both constitutive and inducible Tnk1/Kos1 expression. Importantly, results reveal that cellular stress reverses the repression of Tnk1/Kos1 and induces its expression through increased high affinity interactions between nuclear proteins Sp1, Sp3, AP2 and MED1 for the m-Tnk1 promoter. These findings provide a mechanism by which the m-Tnk1 promoter can be dynamically regulated during normal growth.

The peroxisome proliferator-activated receptor-α (PPARα) plays a key role in lipid metabolism and energy combustion. Chronic activation of PPARα in rodents leads to the development of hepatocellular carcinomas. The ability of PPARα to induce expression of its target genes depends on Mediator, an evolutionarily conserved complex of cofactors and, in particular, the subunit 1 (Med1) of this complex. Here, we report the identification and characterization of PPARα-interacting cofactor (PRIC)-295 (PRIC295), a novel coactivator protein, and show that it interacts with the Med1 and Med24 subunits of the Mediator complex. PRIC295 contains 10 LXXLL signature motifs that facilitate nuclear receptor binding and interacts with PPARα and five other members of the nuclear receptor superfamily in a ligand-dependent manner. PRIC295 enhances the transactivation function of PPARα, PPARγ, and ERα. These data demonstrate that PRIC295 interacts with nuclear receptors such as PPARα and functions as a transcription coactivator under in vitro conditions and may play an important role in mediating the effects in vivo as a member of the PRIC complex with Med1 and Med24.

[This corrects the article DOI: 10.1371/journal.pone.0160755.].

Background: Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease.

Methods: Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate.

Results: We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes.

Conclusions: Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies.

Keywords: DNA methylation; epigenetics; gene regulation; myeloma.

In this issue of Cancer Discovery, Rasool and colleagues show that TF11H/CDK7 phosphorylates the MED1 component of the Mediator complex, which enhances its interaction with androgen receptor (AR), and that this phosphorylation is increased in prostate cancer that is resistant to castration and enzalutamide. A covalent CDK7-specific inhibitor (THZ1) impairs AR-mediated MED1 recruitment to chromatin, and can suppress enzalutamide resistance in vitro and induce tumor regression in a castration-resistant prostate cancer xenograft model, suggesting a novel therapeutic approach for advanced prostate cancer.See related article by Rasool et al., p. 1538.

Breast cancer, one of the most frequent cancer types, is a leading cause of death in women worldwide. Estrogen receptor (ER) α is a nuclear hormone receptor that plays key roles in mammary gland development and breast cancer. About 75% of breast cancer cases are diagnosed as ER-positive; however, nearly half of these cancers are either intrinsically or inherently resistant to the current anti-estrogen therapies. Recent studies have identified an ER coactivator, Mediator Subunit 1 (MED1), as a unique, tissue-specific cofactor that mediates breast cancer metastasis and treatment resistance. MED1 is overexpressed in over 50% of human breast cancer cases and co-amplifies with another important breast cancer gene, receptor tyrosine kinase HER2. Clinically, MED1 expression highly correlates with poor disease-free survival of breast cancer patients, and recent studies have reported an increased frequency of MED1 mutations in the circulating tumor cells of patients after treatment. In this review, we discuss the biochemical characterization of MED1 and its associated MED1/Mediator complex, its crosstalk with HER2 in anti-estrogen resistance, breast cancer stem cell formation, and metastasis both in vitro and in vivo. Furthermore, we elaborate on the current advancements in targeting MED1 using state-of-the-art RNA nanotechnology and discuss the future perspectives as well.

Hepatic macroautophagy/autophagy and fatty acid metabolism are transcriptionally regulated by nuclear receptors (NRs); however, it is not known whether their transcriptional co-activators are involved in autophagy. We thus examined MED1 (mediator complex subunit 1), a key component of the Mediator Complex that directly interacts with NRs, on these processes. We found that MED1 knockdown (KD) in cultured hepatic cells decreased autophagy and mitochondrial activity that was accompanied by decreased transcription of genes involved in these processes. Lipophagy and fatty acid β-oxidation also were impaired. These effects also occurred after thyroid hormone stimulation, nutrient-replete or -deplete conditions, and in liver-specific Med1 KD (Med1 LKD) mice under fed and fasting conditions. Together, these findings showed that Med1 played a key role in hepatic autophagy, mitochondria function, and lipid metabolism under these conditions. Additionally, we identified downregulated hepatic genes in Med1 LKD mice, and subjected them to ChIP Enrichment Analysis. Our findings showed that the transcriptional activity of several NRs and transcription factors (TFs), including PPARA and FOXO1, likely were affected by Med1 LKD. Finally, Med1 expression and autophagy also were decreased in two mouse models of nonalcoholic fatty liver disease (NAFLD) suggesting that decreased Med1 may contribute to hepatosteatosis. In summary, MED1 plays an essential role in regulating hepatic autophagy and lipid oxidation during different hormonal and nutrient conditions. Thus, MED1 may serve as an integrator of multiple transcriptional pathways involved in these metabolic processes.Abbreviations: BAF: bafilomycin A₁; db/db mice; Lepr^db/db mice; ECAR: extracellular acidification rate; KD: knockdown; MED1: mediator complex subunit 1; NAFLD: nonalcoholic fatty liver disease; OCR: oxygen consumption rate; PPARA/PPARα: peroxisomal proliferator activated receptor alpha; TF: transcription factor; TFEB: transcription factor EB; tf-LC3: tandem fluorescence RFP-GFP-LC3; TG: triglyceride; TH: Thyroid hormone; TR: thyroid hormone receptors; V-ATPase: vacuolar-type H⁺-ATPase; WDF: Western diet with 15% fructose in drinking water.

Keywords: Autophagy; lipid oxidation; liver; med1; starvation.

Spermatogenesis is a highly coordinated process. Signaling from nuclear hormone receptors, like those for retinoic acid (RA), is important for normal spermatogenesis. However, the mechanisms regulating these signals are poorly understood. Mediator complex subunit 1 (MED1) is a transcriptional enhancer that directly modulates transcription from nuclear hormone receptors. MED1 is present in male germ cells throughout mammalian development, but its function during spermatogenesis is unknown. To determine its role, we generated mice lacking Med1 specifically in their germ cells beginning just before birth. Conditional Med1 knockout males are fertile, exhibiting normal testis weights and siring ordinary numbers of offspring. RA-responsive gene products stimulated by RA gene 8 (Stra8) and synaptonemal complex protein 3 (Sycp3) are first detected in knockout spermatogonia at the expected time points during the first wave of spermatogenesis, and persist with normal patterns of cellular distribution in adult knockout testes. Meiotic progression, however, is altered in the absence of Med1. At postnatal day 7 (P7), zygotene-stage knockout spermatocytes are already detected, unlike in control testes, with fewer pre-leptotene-stage cells and more leptotene spermatocytes observed in the knockouts. At P9, Med1 knockout spermatocytes prematurely enter pachynema. Once formed, greater numbers of knockout spermatocytes remain in pachynema relative to the other stages of meiosis throughout testis development and its maintenance in the adult. Meiotic exit is not inhibited. We conclude that MED1 regulates the temporal progression of primary spermatocytes through meiosis, with its absence resulting in abbreviated pre-leptotene, leptotene, and zygotene stages, and a prolonged pachytene stage.

Gene amplification is a common event in breast cancer, and identifies actual and potential targets of molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded invasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a total of 906 gene loci judged as 'gain' or 'amplified' was further confirmed to have been amplified based on fluorescence in situ hybridization (FISH). The results showed that 109 of 322 tumors (34%) displayed gene amplification of at least one of the 22 genes. The frequencies of the amplification of four regions containing known driver oncogenes were as follows: 8p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q24 (MYC), 11q13 (CCND1, C11ORF30), and 17q11-21 (CPD, MED1, ERBB2, CDC6, TOP2A, MAPT) exhibited amplification in 9.6%, 9.6%, 12.4%, and 12.1% of the tumors, respectively. In addition to homogeneously staining region- or double-minute chromosome-type amplifications, a centromere-associated-type amplification was found in nine tumors. Co-localization of the amplicon on 8p11 and the amplicon on 11q13 in single cells was found in 10 tumors, and in six of those tumors the two amplicons constituted single amplification units. Similarly, an amplicon consisting of ERBB2 and its flanking genes on 17q12-21 co-localized with an amplicon on 8p11 in 10 tumors and with the amplicon on 11q13 in five tumors. Thus, precise and feasible analysis of gene amplification status can be obtained using a combination of MLPA and FISH.

Tooth enamel is mineralized through the differentiation of multiple dental epithelia including ameloblasts and the stratum intermedium (SI), and this differentiation is controlled by several signaling pathways. Previously, we demonstrated that the transcriptional coactivator Mediator 1 (MED1) plays a critical role in enamel formation. For instance, conditional ablation of Med1 in dental epithelia causes functional changes in incisor-specific dental epithelial stem cells, resulting in mineralization defects in the adult incisors. However, the molecular mechanism by which Med1 deficiency causes these abnormalities is not clear. Here, we demonstrated that Med1 ablation causes early SI differentiation defects resulting in enamel hypoplasia of the Med1-deficient molars. Med1 deletion prevented Notch1-mediated differentiation of the SI cells resulting in decreased alkaline phosphatase (ALPL), which is essential for mineralization. However, it does not affect the ability of ameloblasts to produce enamel matrix proteins. Using the dental epithelial SF2 cell line, we demonstrated that MED1 directly activates transcription of the Alpl gene through the stimulation of Notch1 signaling by forming a complex with cleaved Notch1-RBP-Jk on the Alpl promoter. These results suggest that MED1 may be essential for enamel matrix mineralization by serving as a coactivator for Notch1 signaling regulating transcription of the Alpl gene.

Regnase-1 is not only a key component in maintaining intracellular homeostasis but also a critical negative regulator in preventing autoimmune diseases and cancer development. To keep homeostatic state, Regnase-1 has to be maintained at a desired level in multiple cell types. However, the molecular mechanism of keeping a certain transcriptional level of Reganase-1 is largely unknown. In this study, we found a super-enhancer (Reg-1-SE) around Regnase-1 gene is able to control the homeostatic expression of Regnase-1. Functional inhibition of super-enhancers through BRD4 inhibitors or genetic silence of key components such as BRD4 and MED1 significantly downregulates Regnase-1 expression at multiple cell types. Consistently, treatment of JQ1 or I-BET-762 dramatically decreases the protein level of Regnase-1. By analyzing Regnase-1 gene, the distribution of H3K27Ac is highly enriched at a 8 kb DNA region around the second intron. Several DNA elements at the second intron are highly conserved between different species. Deletion of the second intron by CRISPR-Cas9 technology significantly reduces the expression of Regnase-1. JQ1 or I-BET-762 failed to further downregulate the expression of Regnase-1 in cells without the second intron. Our result reveals a novel molecular mechanism by which a super-enhancer around the second intron regulates the expression of Regnase-1, and in turn maintains a desired level of Regnase-1.

The 15th natural plague focus in China, the Junggar Basin plague focus, is located near an important communication route connecting China and Central Asia and was discovered after 2005. To characterize the phenotypic and genetic diversity of the Yersinia pestis population in this newly established focus, we collected 25 Y. pestis strains from six counties across Junggar Basin in 2005-2006, and determined their biochemical features and genotypes based on multiple-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindromic repeats analysis. We inferred the phylogenetic positions and possible sources of the Junggar strains by comparing their genotypes with the genetic diversity for known representative Y. pestis strains. Our results indicate that the major genotype of Junggar strains belongs to 2.MED1, a lineage of biovar Medievalis with identical biochemical characters and high virulence in mice. Although share a similar ecology, the 2.MED1 in Junggar Basin are not descended from known strains in the neighboring Central Asian Desert plague foci. Therefore, the emergence of the Junggar Basin plague focus is not attributable to the recent clonal spread of Y. pestis from Central Asia. We also identified two distinct minor genotypes in Junggar Basin, one of which clusters genetically with the 0.ANT1 strains of the Tianshan Mountain natural plague focus and another belongs to a 1.IN lineage not previously reported. Our study clarifies the phenotypic and genetic characters of Junggar Y. pestis strains. These findings extend our knowledge of the population diversity of Y. pestis and will facilitate future plague surveillance and prevention in Junggar Basin and adjacent regions.

DNA-bound transcription factors (TFs) governing developmental gene regulation have been proposed to recruit polymerase II machinery at gene promoters through specific interactions with dedicated subunits of the evolutionarily conserved Mediator (MED) complex. However, whether such MED subunit-specific functions and partnerships have been conserved during evolution has been poorly investigated. To address this issue, we generated the first Drosophila melanogaster loss-of-function mutants for Med1, known as a specific cofactor for GATA TFs and hormone nuclear receptors in mammals. We show that Med1 is required for cell proliferation and hematopoietic differentiation depending on the GATA TF Serpent (Srp). Med1 physically binds Srp in cultured cells and in vitro through its conserved GATA zinc finger DNA-binding domain and the divergent Med1 C terminus. Interestingly, GATA-Srp interaction occurs through the longest Med1 isoform, suggesting a functional diversity of MED complex populations. Furthermore, we show that Med1 acts as a coactivator for the GATA factor Pannier during thoracic development. In conclusion, the Med1 requirement for GATA-dependent regulatory processes is a common feature in insects and mammals, although binding interfaces have diverged. Further work in Drosophila should bring valuable insights to fully understand GATA-MED functional partnerships, which probably involve other MED subunits depending on the cellular context.