MediatorWeb: a protein-protein interaction network database for the RNA polymerase II Mediator complex.

The protein-protein interaction (PPI) network of the Mediator complex is very tightly regulated and depends on different developmental and environmental cues. Here, we present an interactive platform for comparative analysis of the Mediator subunits from humans, baker's yeast Saccharomyces cerevisiae, and model plant Arabidopsis thaliana in a user-friendly web-interface database called MediatorWeb. MediatorWeb provides an interface to visualize and analyze the PPI network of Mediator subunits. The database facilitates downloading the untargeted and unweighted network of Mediator complex, its submodules, and individual Mediator subunits to better visualize the importance of individual Mediator subunits or their submodules. Further, MediatorWeb offers network visualization of the Mediator complex and interacting proteins that are functionally annotated. This feature provides clues to understand functions of Mediator subunits in different processes. In an additional tab, MediatorWeb provides quick access to secondary and tertiary structures, as well as residue-level contact information for Mediator subunits in each of the three model organisms. Another useful feature of MediatorWeb is detection of interologs based on orthologous analyses, which can provide clues to understand the functions of Mediator complex in less explored kingdoms. Thus, MediatorWeb and its features can help the user to understand the role of Mediator complex and its subunits in the transcription regulation of gene expression.

MEDIATOR SUBUNIT17 integrates jasmonate and auxin signaling pathways to regulate thermomorphogenesis.

Plant adjustment to environmental changes involves complex crosstalk between extrinsic and intrinsic cues. In the past two decades, extensive research has elucidated the key roles of PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and the phytohormone auxin in thermomorphogenesis. In this study, we identified a previously unexplored role of jasmonate (JA) signaling components, the Mediator complex, and their integration with auxin signaling during thermomorphogenesis in Arabidopsis (Arabidopsis thaliana). Warm temperature induces expression of JA signaling genes including MYC2, but, surprisingly, this transcriptional activation is not JA dependent. Warm temperature also promotes accumulation of the JA signaling receptor CORONATINE INSENSITIVE1 (COI1) and degradation of the JA signaling repressor JASMONATE-ZIM-DOMAIN PROTEIN9, which probably leads to de-repression of MYC2, enabling it to contribute to the expression of MEDIATOR SUBUNIT17 (MED17). In response to warm temperature, MED17 occupies the promoters of thermosensory genes including PIF4, YUCCA8 (YUC8), INDOLE-3-ACETIC ACID INDUCIBLE19 (IAA19), and IAA29. Moreover, MED17 facilitates enrichment of H3K4me3 on the promoters of PIF4, YUC8, IAA19, and IAA29 genes. Interestingly, both occupancy of MED17 and enrichment of H3K4me3 on these thermomorphogenesis-related promoters are dependent on PIF4 (or PIFs). Altered accumulation of COI1 under warm temperature in the med17 mutant suggests the possibility of a feedback mechanism. Overall, this study reveals the role of the Mediator complex as an integrator of JA and auxin signaling pathways during thermomorphogenesis.

The Mediator subunit OsMED15a is a transcriptional co-regulator of seed size/weight-modulating genes in rice.

Although several transcription factors (TFs) that regulate seed size/weight in plants are known, the molecular landscape regulating this important trait is unclear. Here, we report that a Mediator subunit, OsMED15a, links rice grain size/weight-regulating TFs to their target genes. Expression analysis and high-resolution quantitative trait loci (QTL) mapping suggested that OsMED15a is involved in rice seed development. OsMED15a has an N-terminal, three-helical KIX domain. Two of these helices, α1 and α3, and three amino acids, 76LRC78, within OsMED15a helix α3 were important for its interaction with several proteins, including interactions with the transactivation domains of two NAC-type TFs, OsNAC024 and OsNAC025. Moreover, OsMED15a, OsNAC024, and OsNAC025 all exhibited increased expression during seed development, and we identified several grain size/weight-associated SNPs in these genes in 509 low- and high-grain-weight rice genotypes. RNAi-mediated repression of OsMED15a expression down-regulated the expression of the grain size/weight regulating genes GW2, GW5 and DR11 and reduced grain length, weight, and yield. Of note, both OsNAC024 and OsNAC025 bound to the promoters of these three genes. We conclude that the transactivation domains of OsNAC024 and OsNAC025 target the KIX domain of OsMED15a in the regulation of grain size/weight-associated genes such as GW2, GW5, and D11. We propose that the integrated molecular-genetics approach used here could help identify networks of functional alleles of other regulator and co-regulator genes and thereby inform efforts for marker-assisted introgression of useful alleles in rice crop improvement.

Interaction map of Arabidopsis Mediator complex expounding its topology.

Understanding of mechanistic details of Mediator functioning in plants is impeded as the knowledge of subunit organization and structure is lacking. In this study, an interaction map of Arabidopsis Mediator complex was analyzed to understand the arrangement of the subunits in the core part of the complex. Combining this interaction map with homology-based modeling, probable structural topology of core part of the Arabidopsis Mediator complex was deduced. Though the overall topology of the complex was similar to that of yeast, several differences were observed. Many interactions discovered in this study are not yet reported in other systems. AtMed14 and AtMed17 emerged as the key component providing important scaffold for the whole complex. AtMed6 and AtMed10 were found to be important for linking head with middle and middle with tail, respectively. Some Mediator subunits were found to form homodimers and some were found to possess transactivation property. Subcellular localization suggested that many of the Mediator subunits might have functions beyond the process of transcription. Overall, this study reveals role of individual subunits in the organization of the core complex, which can be an important resource for understanding the molecular mechanism of functioning of Mediator complex and its subunits in plants.

Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer.

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.

KIX domain of AtMed15a, a Mediator subunit of Arabidopsis, is required for its interaction with different proteins.

Med15 is an important subunit of Mediator Tail module and is characterized by a KIX domain present towards amino terminal. In yeast and metazoans, Med15 KIX domain has been found to interact with various transcription factors regulating several processes including carbohydrate metabolism, lipogenesis, stress response and multidrug resistance. Mechanism of Med15 functioning in Arabidopsis is largely unknown. In this study, interactome of KIX domain of Arabidopsis Med15, AtMed15a, was characterized. We found 45 proteins that interact with AtMed15a KIX domain, including 11 transcription factors, 3 single strand nucleic acid-binding proteins and 1 splicing factor. The third helix of the KIX domain was found to be involved in most of the interactions. Mapping of the regions participating in the interactions revealed that the activation domain of a transcription factor, UKTF1 interacted with AtMed15a KIX domain. Thus, our results suggest that in Arabidopsis, activation domain of transcription factors target KIX domain of AtMed15a for their transcriptional responses.

Evolution of disorder in Mediator complex and its functional relevance.

Mediator, an important component of eukaryotic transcriptional machinery, is a huge multisubunit complex. Though the complex is known to be conserved across all the eukaryotic kingdoms, the evolutionary topology of its subunits has never been studied. In this study, we profiled disorder in the Mediator subunits of 146 eukaryotes belonging to three kingdoms viz., metazoans, plants and fungi, and attempted to find correlation between the evolution of Mediator complex and its disorder. Our analysis suggests that disorder in Mediator complex have played a crucial role in the evolutionary diversification of complexity of eukaryotic organisms. Conserved intrinsic disordered regions (IDRs) were identified in only six subunits in the three kingdoms whereas unique patterns of IDRs were identified in other Mediator subunits. Acquisition of novel molecular recognition features (MoRFs) through evolution of new subunits or through elongation of the existing subunits was evident in metazoans and plants. A new concept of 'junction-MoRF' has been introduced. Evolutionary link between CBP and Med15 has been provided which explain the evolution of extended-IDR in CBP from Med15 KIX-IDR junction-MoRF suggesting role of junction-MoRF in evolution and modulation of protein-protein interaction repertoire. This study can be informative and helpful in understanding the conserved and flexible nature of Mediator complex across eukaryotic kingdoms.