InteractiveFly: GeneBrief

Mediator complex subunit 1: Biological Overview | References

Gene name - Mediator complex subunit 1

Synonyms -

Cytological map position - 78E1-78E1

Function - miscellaneous transcription factor

Keywords - Mediator complex component - a specific cofactor for GATA TFs - coactivator for the GATA factor Pannier during thoracic development - required for cell proliferation and hematopoietic differentiation dependent on the GATA TF Serpent development - blood and heart development

Symbol - MED1

FlyBase ID: FBgn0037109

Genetic map position - chr3L:21,628,325-21,634,446

Classification - Mediator of RNA polymerase II transcription subunit 1

Cellular location - nuclear

NCBI links: EntrezGene, Nucleotide, Protein

Med1 orthologs: Biolitmine

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 complex (MED). However, whether such MED subunit specific functions and partnerships have been conserved during evolution has been poorly investigated. To address this issue, the first Drosophila loss-of-function mutants were generated for Med1, known as a specific cofactor for GATA TFs and hormone nuclear receptors in mammals. Med1 was shown to be required for cell proliferation and hematopoietic differentiation dependent on the GATA TF Serpent (Srp). Med1 binds Srp in cultured cells and in vitro through its conserved GATA Zinc Finger DNA-binding domain and the divergent Med1 C-terminal. Interestingly, GATA/Srp interaction occurs through the longest Med1 isoform, suggesting a functional diversity of MED complex populations. Furthermore, it was shown 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 (Immarigeon, 2019).

Precise temporal and spatial regulation of gene transcription by RNA polymerase II (Pol II) is crucial to ensure the coordinated cell fate specification in multicellular organisms. To precisely control Pol II activity, metazoans have evolved an elaborate protein machinery, including the conserved multiprotein Mediator (MED) complex, which serves as a malleable interface between DNA-bound transcription factors (TFs) and the Pol II machinery. Dedicated MED subunits have been proposed to mediate specific TF activities. Whether these specific partnerships and binding interfaces have been conserved during evolution remains an open question (Immarigeon, 2019).

The MED complex, conserved from yeast to human, contains 25 to 30 subunits organized into the head, middle, and tail modules as well as a dissociable cyclin-dependent kinase 8 (CDK8) module. The core MED, interacting directly with Pol II and its associated general transcription factors, contains essential head and middle module subunits. Conversely, more peripheral MED subunits belonging to the tail (e.g., Med15), CDK8 (e.g., Med12), and middle (e.g., Med1) modules are not required for cell viability and display more specific functions during cell differentiation. It is generally assumed that MED subunit specificity comes from their ability to interact directly with specific TFs, allowing Mediator recruitment to gene regulatory elements. For example, it has been shown that Med12 interacts directly with Sox9 and Sox10, whereas Med15 binds SMADs (3) and Med19 binds HOX (4) TFs. Another example is Med1, identified for its role as a major cofactor of hormone nuclear receptors (NRs) that directly bind its LXXL domain. Mammalian Med1 also mediates transcriptional activity of the GATA zinc finger (ZF) TF family. Physically interacting with at least five of the six mammalian GATAs, Med1 is required for GATA1, GATA2, and GATA6 target gene expression in several developmental contexts, including erythropoiesis, and is recruited to specific GATA1 and GATA2 target genes. Whereas several MED subunit-TF partnerships have been characterized in mammals, it is not known to what extent these MED subunit-specific functions have been conserved in other species (Immarigeon, 2019).

Drosophila melanogaster is an ideal model to analyze MED subunit-specific functions given that homologs of the 33 human subunits are encoded by single-copy genes and that overall MED complex structure has been conserved during evolution. Furthermore, several transcription factor families are strongly conserved both structurally and functionally in Drosophila. A good example is the GATA zinc finger factor family. In mice and humans, the GATA1/2/3 subfamily is required for blood cell lineage differentiation, and the GATA4/5/6 subfamily is involved in the meso-endoderm lineage, notably in cardiac development. In Drosophila, the GATA factor Serpent (Srp) is a central regulator of hematopoietic cell differentiation, controlling the formation of the two embryonic populations of blood cells (plasmatocytes and crystal cells), and the GATA factor Pannier (Pnr) is involved in embryonic heart development, dorsal thoracic closure, and sensory organ precursor development, revealing a functional conservation during bilaterian evolution (Immarigeon, 2019).

Mammalian GATA factors generally contain two highly conserved Cys4-type ZFs. The C-terminal ZF (C-ZF) is both necessary and sufficient for sequence-specific DNA binding at [(A/T)GATA(G/A)] genomic sites, while the N-terminal ZF (N-ZF) appears only to modulate DNA binding affinity, notably at palindromic double sites. Whereas Drosophila Pnr also displays two ZFs, srp encodes different isoforms containing either only a C-ZF (SrpC) or both a C- and an N-ZF (SrpNC), with the N-finger stabilizing the interaction of Srp with palindromic GATA sites (Immarigeon, 2019).

The GATA N-ZF also mediates interactions with key coregulators, such as Friend-of-GATA (FOG) proteins, the LIM-only protein LMO2, and the basic helix-loop-helix (bHLH) factor SCL/TAL1. GATA1 forms a pentameric transactivation complex with LMO2, the LIM-binding protein Ldb1, and the bHLH factors SCL and E1A, binding a composite E box/GATA enhancer sequence to transactivate erythroid gene expression. An equivalent pentameric complex has been characterized during Drosophila sensory organ precursor development, where the Achaete (Ac) bHLH protein and its obligatory cofactor, Daughterless (Da), associate with GATA/Pnr, dLMO, and the Lbd protein Chip for ac gene autoregulation. Drosophila Srp also interacts with orthologues of mammalian GATA cofactors. Indeed, GATA/Srp associates with the RUNX cofactor Lozenge (Lz) or the FOG factor U-shaped (Ush) to induce or repress crystal cell differentiation, respectively (Immarigeon, 2019).

Thus, GATA factor functions, DNA binding interfaces, and transcriptional cofactors appear conserved in Drosophila, but less is known about how GATA factors contact the Mediator complex to activate their target genes. By a genome-wide RNA interference screen in cultured Drosophila blood cells, previous work has identified several MED subunits (including Med1, Med12, and Med13) as modulators of GATA/Srp-induced transactivation (Gobert, 2010). It was further shown that Med12 and Med13 are indeed required in vivo for Srp-dependent crystal cell differentiation. Furthermore, a genome-wide expression profiling from Drosophila GATA/Srp- or Med12- or Med13-depleted cells revealed a significant overlap, notably concerning the innate immunity genes (Kuuluvainen, 2014). Nevertheless, no direct physical interaction in vitro was detected between Srp and Med12 or Med13 (Gobert, 2010), suggesting that GATA/Srp recruits the MED complex by contacting another subunit (Immarigeon, 2019).

This work addresses the issue of the conservation of Mediator subunit-specific functions across bilaterian evolution using as a model the Drosophila Med1 subunit whose mammalian orthologue is known as a GATA and NR cofactor. The generation of the first Med1 mutants in an insect reveals defects in GATA/Srp-dependent embryonic hematopoiesis. it was further shown that Srp forms a complex with Med1's longest isoform in Drosophila cultured cells. Furthermore, the divergent, isoform-specific C terminus of Med1 interacts with the conserved zinc finger-containing domain of Srp in vitro. The generation of Med1 mutant clones indicates a Med1 requirement for cell proliferation control and for the expression of a GATA/Pnr target gene in larval imaginal tissues. Finally, this study shows a Med1 role in Pnr-dependent transactivation and a direct interaction between the GATA/Pnr ZF-containing domain and the Drosophila-specific Med1 C terminus. Taken together, these data reveal that the Med1 Mediator subunit has conserved GATA TF coactivator functions during bilaterian evolution through divergent binding interfaces (Immarigeon, 2019).

This work reported the first Med1 loss-of-function mutants in an insect. Med1 is shown to be an essential gene which is not strictly required for cell viability but is clearly involved in context-dependent proliferation or cell survival processes. As opposed to Drosophila, mammalian Med1 is not essential for cell viability given that primary embryonic fibroblasts can be derived from null Med1 mutants in mice. Nevertheless, mouse Med1 mutant cells display impaired cell cycle regulation, suggesting a conserved Med1 function in cell proliferation control that deserves to be analyzed in the future using Drosophila as a model (Immarigeon, 2019).

The take-home message of this work is the functional partnership between Drosophila Med1 and GATA transcription factors (see MED1-GATA molecular partnership). Med1 is shown to be involved in at least two developmental processes, embryonic crystal cell differentiation and larval thoracic development, depending on two GATA transcription factors, Serpent and Pannier, respectively. Med1 is required for the expression of at least one Srp and one Pnr target gene in vivo. The partial loss of crystal cells observed in Med1 zygotic mutant embryos could reflect a partial requirement for Med1 in this differentiation process or could be due to partial rescue by the Med1 maternal contributions. Furthermore, this study showed that Med1 interacts physically with Srp and Pnr both in vitro and in cultured cells. Taken together, these results reveal evolutionarily conserved functions of Drosophila Med1 as a GATA cofactor involving a divergent Med1 region binding the conserved GATA zinc fingers. This Med1-GATA partnership appears restricted to one Med1 isoform, suggesting a new layer of regulation by the Mediator complex through a diversity of MED populations. Finally, Med1 is not required for all GATA/Pannier activity, suggesting the use of alternative MED subunits depending on the cellular context (Immarigeon, 2019).

This study shows that Med1 activity as a GATA cofactor is not restricted to vertebrates, since it also acts in Drosophila for at least two different GATA factors, suggesting an ancient GATA-Med1 partnership among bilaterians. The MED-TF partnership conservation along evolution is not the rule, since the LXXL motif of mammalian Med1 interacting with hormone nuclear receptors is not present in Drosophila Med1, whereas it is in two other Drosophila Mediator subunits, Cdk8 and Med14, which have been shown to be necessary for ecdysone nuclear receptor activity (Immarigeon, 2019).

What about the conservation of binding interfaces between Med1 and GATA factors? The results indicate that two Drosophila GATAs, like mammalian GATA1, -2, -3, and -6 and chicken GATA1, -2, and -3, bind Med1 at least through their ZF-containing domains. Nevertheless, some discrepancies exist concerning N- or C-ZF involvement in mammals. In Drosophila, this study shows that either N-ZF or C-ZF interacts with Med1ex4 in vitro and that Srp N-ZF is dispensable for binding in cultured cells. More diversity is observed for GATA-interacting domains within the Med1 protein. Med1 primary sequence conservation lies essentially within the N-terminal part, corresponding to the entire yeast orthologue, whereas the long additional C-terminal part diverges in each metazoan species. In Drosophila, this study shows that both Srp and Pnr interact with the Med1 isoform A-specific domain lacking sequence homology with mammalian Med1. In mammals, GATA-interacting domains differ depending on the GATA paralogue considered and are distributed throughout the Med1 sequence. In conclusion, despite functional conservation of the Med1-GATA partnership, the GATA-binding interface within Med1 has not been fixed during bilaterian evolution, even among paralogs. Such versatility is clearly favored by the enrichment of intrinsically disordered regions (IDRs) within many metazoan MED subunits. Indeed, IDRs, which do not fold into stable three-dimensional globular structures, would be a natural way for the Mediator complex to adapt to the increasing diversity of transcriptional regulators during evolution. Shown to interact with many TFs, Med1 has the largest IDR among MED subunits. Interestingly, partially conserved IDRs lie within a specific fragment of the longest Drosophila isoform, which this study identified as the Srp and Pnr interacting domain (Nagulapalli, 2016). It has been proposed recently that mammalian Med1 IDRs can form phase-separated droplets that compartmentalize and concentrate the transcription apparatus at superenhancers to drive robust gene expression (Sabari, 2018). Thus, it is proposed that GATA interaction with Med1 IDRs concentrates MED-Pol II clusters at GATA-bound enhancers to activate transcription (Nagulapalli, 2016; Immarigeon, 2019 and references therein).

This work reveals for the first time a TF partnership apparently restricted to one isoform of a Mediator subunit. Indeed, it was shown that (1) GATA/Srp only coprecipitates with the longest Med1A isoform in cultured cells, (2) a domain specific to isoform A is sufficient to bind both Srp and Pnr in vitro, and (3) a transgene ubiquitously expressing the Med1A isoform fully rescues the crystal cell differentiation defects of Med1-depleted embryos. More work is required to determine whether all of the GATA transactivation function of Med1 resides within the longest isoform or whether the shorter ones can partially fulfill this role or display antagonizing activity. With an antibody directed against a common protein portion, it was shown that all three Drosophila Med1 isoforms are incorporated into the MED complex (4) and that Med1 is ubiquitously expressed in imaginal tissues, but it was not possible to evaluate whether or not Med1 isoforms are differentially expressed. Nevertheless, this hypothesis is supported by the fact that the ratio between Med1 transcripts, as well as their relative levels, has been shown to change during development and in different cell lines. These data strongly suggest a physiological relevance of alternative transcript production for Med1 and pave the way for future research. If MED complexes containing different Med1 isoforms are functionally distinct, the view of MED as a unique ubiquitous entity would be challenged. Mediator would then be the name of a heterogeneous population of complexes with different regulatory specificities (Immarigeon, 2019).

Med1 is not an obligatory partner of GATA factors. Drosophila Med1 acts as a cofactor for the GATA factors Pnr and Srp, physically binding both TFs and GATA-type ZFs and mediating their target gene's transactivation. This suggests that Med1 is an obligatory partner of GATA factors necessary to recruit the Pol II transcription machinery to GATA-activated promoters, as was proposed in mammals in earlier studies. Nevertheless, it was found that Med1 is critical for achaete- but not for wingless-induced transactivation by Pnr. Similarly, it was shown that mammalian Med1 regulates only a limited subset of GATA1-dependent genes in erythroid cells (Pope, 2013) and that GATA1 recruits Med1 at activated genes but not at repressed loci (Pope, 2010). In addition, the analysis of different blood cell types produced from conditional Med1 knockout mice showed that Med1 participates in GATA1-dependent erythropoiesis but is dispensable for other GATA-dependent processes (Stumpf, 2010). This paper suggested that GATA factors, despite binding Med1 in vitro, contact other MED subunits to regulate their target genes in vivo. Along these lines, Med14, Med17, and Med25 have also been proposed as GATA1 interactors (Immarigeon, 2019).

It is now clear that the view of MED action as a binary partnership, i.e., one subunit to one TF, is too simplistic. It has been postulated that MED subunits act in a concerted manner, in a positive or negative way, by interacting simultaneously with one or several transcription factors and cofactors bound at gene enhancers, as well as promoters, to finely regulate gene expression in response to TFs. This attractive view of MED action as an integrative molecular hub device, transforming complex combinatorial inputs (TFs, cofactors, chromatin modifiers, etc.) into a simple transcriptional output, has rarely been tackled experimentally, particularly in metazoans. The use of Drosophila and the GATA-MED paradigm should allow exploration of this view in vivo (Immarigeon, 2019).

Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? Previous work established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. This study observed mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. It is further shown that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp- dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, this study shows a direct interaction between Med19 and Med1 by GST-pull-down experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit - TF partnerships are probably oversimplified models. Several mechanisms are proposed to account for the transcriptional regulation of GATAs-targeted genes (Immarigeon, 2020).

Using molecular, cellular and genetic analyses this work establishes that Drosophila GATA factors' transcriptional activity depends on the Mediator complex subunit Med19, in addition to the previously characterized Med1 cofactor. Four main conclusions that are discussed below can be drawn from these results. (1) Med19 interacts with the GATA C-ZF domain which also serves as the GATA DNA- binding domain. (2) Med19 and GATA interacting domains are evolutionary conserved suggesting conserved Med19 cofactor functions in higher metazoans. (3) Comparative analysis of Med19 and Med1 depletion indicates that Med19 but not Med1 is systematically required for GATA target gene expression suggesting a differential use of MED subunits by GATAs depending on the target gene. (4) Med1 and Med19 interact in vitro. Taken together these data allow proposal of new models of Mediator complex mechanism of action (Immarigeon, 2020).

TFs minimally contain two domains: the DNA binding domains (DBD), which have been extensively studied and allowed to define different TF families, and transcriptional activation domains (TAD), which link TFs to the RNA polymerase II machinery, and whose structure and characteristics are less well defined. GATA TFs are characterised by the presence of two ZFs which were, so far, thought to play distinct roles. While the C-ZF appeared to be dedicated to DNA binding, the N-ZF was shown to bind co-activators such as dLMO and FOG. The present data show that Med19 interacts specifically with the Pnr C-ZF. Full interaction requires both the zinc finger and its adjacent basic tail which also contributes to DNA binding. It is the first evidence that the Drosophila GATA C-ZF may play a dual role, in DNA binding and as an interface with MED subunit(s). Interestingly, the analysis of GATA ZF evolutionary conservation indicates that N- and C-ZF domains comes from a duplication event of the C-ZF with its basic tail. Thus, this transactivation function of the GATA DBD might represent an ancestral GATA function allowing minimal primitive GATAs, essentially composed of the DBD, to connect the MED complex and thus recruit the transcriptional machinery to regulate its targetgenes. They provide rationale why slightly-extended GATA ZF domains are in some cases sufficient for transcriptional activities in vivo (Immarigeon, 2020).

This dual activity of DBD is not restricted to GATA factors. It was previously shown that HOX TFs also contact Med19 through their DNA-binding homeodomain (Boube, 2014). The current data also corroborate results from a recent high- throughput approach, looking for trans- activation domains of Drosophila transcription factors. This work shows that trans-activation domains of several zinc-finger- (ZF-) and basic Helix-Loop-Helix- (bHLH-) TFs overlap structured DNA-binding domains. Altogether, these results identify a novel class of TF characterized by overlapping TAD and DBD and suggest an emerging Med19 property as a dedicated cofactor directly connecting these TFs DNA-binding domains to the general PolII transcriptional machinery (Immarigeon, 2020).

How is this dual function of DBDs achieved? Do DNA binding and transactivation functions use distinct or shared molecular determinants? Recent improvements of electron microscopy analyses could allow characterizing GATA molecular residues involved in MED- versus DNA-binding to try to separate the GATA DNA binding- from GATA transactivation functions (Immarigeon, 2020).

While Med1 is a known GATA cofactor both in mammals and in Drosophila, the role of Med19 in mediating GATA transcription regulatory properties had never been investigated until now. This study shows that Drosophila Med19 binds GATA factors, via motifs lying within the evolutionary-conserved Med19 CORE and HIM domains. Both of these domains bind to the C-ZF domain of GATAs, which is a hallmark of GATA TF family suggesting that interaction with Med19 is likely to be conserved in mammals. Yet, Med1 depletion experiments in mammalian cultured cells induces defects in only a subset of GATA1-activated genes and does not prevent GATA1-dependent repression. Furthermore, in studies of the different blood cell types produced by conditional Med1 knock-out mice, Med1 appears to be critical for erythroid lineages which depend upon GATA1-function but is dispensable for hematopoietic stem cell production and T-cell development which require GATA2 and GATA3, respectively. Thus, despite being capable of binding all GATA factors in vitro, Med1 is not critical for all GATA functions, which suggests that (an) other MED subunit(s) also bind(s) GATAs to relay their regulatory signals to the PolII machinery. Considering the evolutionary-conservation of interaction motifs within both GATAs and Med19, it is argued that Med19 is a strong candidate as a GATA cofactor in mammals (Immarigeon, 2020).

This data show that most Drosophila GATA target genes require both Med19 and Med1. How does this work? Med19 only interacts with the C-ZF domain, but Med1 can bind both GATA zinc-finger domains, suggesting that Med1 and Med19 can simultaneously bind GATA factors. It is thus proposed that in the majority of cases where GATA-driven gene expression requires both Med19 and Med1, enhancer- bound GATAs must directly contact both Med1 and Med19 subunits to recruit the Mediator complex and thus the PolII machinery at GATA target genes. Some genes (e.g. wingless) require Med19 but not Med1. How does this kind of gene specificity occur mechanistically? It is hypothesized that for these Med1-independant genes, other transcription factors might be involved in recruiting the MED (through other subunits) and hence overcome the necessity for Med1-GATA interaction (Immarigeon, 2020).

A future challenge will be to test these models by site-directed mutagenesis in vivo to assess the functional contribution of each GATA- MED contact. Nevertheless, this task is complicated because of overlapping DNA- and MED- interacting domains within GATAs. On the other hand, Med19 CORE domain has also a dual function of MED anchorage and interaction with GATA. It thus requires prior structural analysis of molecular contacts to specifically target GATA-MED interaction without affecting essential DNA-binding activity of GATAs or Med19 ability to incorporate the MED-complex (Immarigeon, 2020).

Another interpretation of these results could be that other subunits necessary for GATA target gene expression fall off from the complex when Med19 or Med1 are deleted or knocked down. However, structural analyses of MED complexes from yeast and mammalian cells lacking Med19 or Med1 indicate that global MED organization is unchanged and it is therefore considered very unlikely. Other lines of evidence indicate that complexes missing only Med19 can be isolated from Med19 depleted mammalian or yeast cells. Altogether, these results suggest that Drosophila MED subunit loss is unlikely in Med19 or Med1 depletion conditions. Since a direct physical interaction was found between GATAs and Med1/Med19, the simplest explanation for these results is considered to be that the loss of either of these subunits is enough to abolish GATA regulatory signals (Immarigeon, 2020).

Previous models of core MED structure-function analysis suggested that the middle and head modules contact the PolII enzyme and associated general transcription factors (GTFs) while the tail module interacts with sequence- specific TFs. The data show that two MED subunits of the middle module, Med1 and Med19, are able to bind GATA factors and are required for their function. They emphasize that MED should be viewed as a much more complex interface using multiple MED subunits to contact different TF combinations thus mediating specific transcriptional responses (Immarigeon, 2020).

Modelization of MED spatial organization indicate that Med1 and Med19 are most likely located at two opposite ends of the middle module, Med1 near the tail module and Med19 within the so called 'hook' domain proposed to anchor the separable CDK8 module (CKM). Nevertheless, the data indicate that Med1 and Med19 interact in vitro. Furthermore, this interaction occurs via the highly-conserved, N-terminal, MED-addressing domain of Med1 suggesting an evolutionary conservation. How, then, is it possible to reconcile the proposed MED architecture with the results showing a direct interaction between Med1 and Med19 subunits in vitro (Immarigeon, 2020)?

Two non-exclusive hypotheses are proposed: first, MED complexes could adopt different conformations, which would differ from the 'canonical' architecture of the MED complex in isolation. This is supported by observations that the MED complex changes its overall shape when engaged in interactions with either TF, CKM or PolII. Perhaps when MED is recruited by GATA, Med1 - Med19 contacts within the MED complex could stabilize one of these 'alternative' conformations (Immarigeon, 2020).

A second possibility is that Med1-Med19 interactions do not occur within but between MED complexes and could thus stabilize 'multi-MED' structures. It has been shown that master TFs control gene expression programs by establishing clusters of enhancers called super-enhancers, at genes with prominent roles in cell identity. Recent studies have revealed that, at super-enhancers, master TFs and the Mediator coactivator form phase-separated condensates, which compartmentalize and concentrate the PolII machinery to specific nuclear foci, to ensure high level of transcription. Interestingly, mammalian Med1 can form such phase-separated droplets that concentrate the transcription machinery at super-enhancers. Bringing together several MED complexes associated with TFs via Med1-Med19 trans-interaction might thus help phase-separated droplet formation at clustered gene enhancers and ensure high transcriptional level (Immarigeon, 2020).

In conclusion, this work shows that 2 MED subunits physically bind GATAs and are required to relay the regulatory signals from common TFs. This argues against the generally admitted view of binary interaction between one MED subunit and one TF, which appears as an oversimplified model for MED action. The Mediator should be viewed as a complex interface allowing fine-tuned gene regulation by TFs through specific contacts with different MED subunit combinations. This study highlights the unexpected role of Drosophila Med19 as a GATA cofactor and Med1 interactor. This work sheds new light on the GATA-MED paradigm and suggests novel means by which several MED subunits might collaborate to regulate gene transcription (Immarigeon, 2020).

Functions of Med1 orthologs in other species

Deletion of Mediator 1 suppresses TGFbeta signaling leading to changes in epidermal lineages and regeneration
Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. A molecular mechanism by which Med1 facilitates these processes was studied, in particular, by focusing on TGFbeta signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfbeta1/beta2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFbeta1/beta2) and diminishes activity of TGFbeta signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFbeta components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFbeta ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFbeta signaling pathway. Finally, this study showed that pharmacological inhibition of TGFbeta signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration (Oda, 2020).

USP22 controls iNKT immunity through MED1 suppression of histone H2A monoubiquitination

Invariant NKT (iNKT) cells constitute a distinct lymphocyte subset, and upon activation, iNKT cells modulate the function of a wide variety of other immune cells including anti-tumor effector cells in both a direct and indirect manner. The ubiquitin pathway has been shown to regulate iNKT cell immunity, but the deubiquitinase involved in this process has not been identified. This study 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-2Rbeta and T-bet (T-box transcription factor 21) gene expression through deubiquitinating histone H2A but not H2B monoubiquitination. Therefore, this study revealed USP22-mediated histone H2A deubiquitination fine-tunes MED1 transcriptional activation as a previously unappreciated molecular mechanism to control iNKT development and functions (Zang, 2020).

Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor

The thyroid hormone receptors (TRs) mediate thyroid hormone (T3)-dependent gene expression. The nuclear import and export signals that direct TR shuttling are well characterized, but little is known about factors modulating nuclear retention. This study used fluorescence-based nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells to investigate whether Mediator subunits MED1 and MED13 play a role in nuclear retention of TR. When MED1 was overexpressed, there was a striking shift towards a greater nuclear localization of TRbeta1 and the oncoprotein v-ErbA, subtypes with cytosolic populations at steady-state, and TRbeta1 intranuclear mobility was reduced. For TRalpha1, there was no observable change in its predominantly nuclear distribution pattern or mobility. Consistent with a role for MED1 in nuclear retention, the cytosolic TRalpha1 and TRbeta1 population were significantly greater in MED1(-/-) cells, compared with MED1(+/+) cells. Exposure to T3 and epidermal growth factor, which induces MED1 phosphorylation, also altered TR intranuclear dynamics. Overexpression of miR-208a, which downregulates MED13, led to a more cytosolic distribution of nuclear-localized TRalpha1; however, overexpression of MED13 had no effect on TRbeta1 localization. The known binding site of MED1 overlaps with a transactivation domain and nuclear export signal in helix 12 of TR's ligand-binding domain (LBD). Coimmunoprecipitation assays demonstrated that TR's LBD interacts directly with exportins 5 and 7, suggesting that binding of exportins and MED1 to TR may be mutually exclusive. Collectively, these data provide evidence that MED1 promotes nuclear retention of TR, and highlight the dual functionality of helix 12 in TR transactivation and nuclear export (Femia, 2020).

Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. This study demonstrates that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. This study also shows that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid-liquid phase-separation condensate properties. It is proposed that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci (Pessina, 2019).

A Non-canonical role of YAP/TEAD is required for activation of estrogen-regulated enhancers in breast cancer

YAP/TEAD are nuclear effectors of the Hippo pathway, regulating organ size and tumorigenesis largely through promoter-associated function. However, their function as enhancer regulators remains poorly understood. Through an in vivo proximity-dependent labeling (BioID) technique, this study identified YAP1 and TEAD4 protein as co-regulators of ERalpha on enhancers. The binding of YAP1/TEAD4 to ERalpha-bound enhancers is augmented upon E2 stimulation and is required for the induction of E2/ERalpha target genes and E2-induced oncogenic cell growth. Furthermore, their enhancer binding is a prerequisite for enhancer activation marked by eRNA transcription and for the recruitment of the enhancer activation machinery component MED1. The binding of TEAD4 on active ERE-containing enhancers is independent of its DNA-binding behavior, and instead, occurs through protein-tethering trans-binding. These data reveal a non-canonical function of YAP1 and TEAD4 as ERalpha cofactors in regulating cancer growth, highlighting the potential of YAP/TEAD as possible actionable drug targets for ERalpha(+) breast cancer (Zhu, 2019).

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. This study shows 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-alpha) dependent manner. Occupancy of MED1 on estrogen response elements (EREs) upstream of miR-191/425 cluster is estrogen and ER-alpha-dependent and ER-alpha-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, this study shows 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-alpha/miR-191 axis promotes breast cancer cell proliferation and migration and may serve as a novel target for therapy (Nagpal, 2018).

Coactivator condensation at super-enhancers links phase separation and gene control

Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. this study demonstrates that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes (Sabari, 2018).

Establishment of a cell-type-specific genetic network by the mediator complex component Med1

The intense physiologic demand to generate vast numbers of red blood cells requires the establishment of a complex genetic network by the master regulatory transcription factor GATA-1 and its coregulators. This network dictates the genesis of enucleated erythrocytes by orchestrating the survival, proliferation, and differentiation of progenitor cells. In addition to the crucial GATA-1 coregulator Friend of GATA-1 (FOG-1), a component of the Mediator complex, Med1, facilitates GATA-1-dependent transcription at select target genes and controls erythropoiesis. It is not known to what extent Med1 contributes to GATA-1 function or whether Med1 controls a large or restricted cohort of genes that are not regulated by GATA-1. Using a genetic complementation assay in GATA-1-null erythroid cells, this study demonstrates that Med1 and another Mediator component, Med25, regulate a restricted cohort of genes that are predominantly not controlled by GATA-1. Most of these genes were not regulated by Med1 in fibroblasts. Loss-of-function analyses with GATA-1-independent Med1 target genes indicate that Rrad, which encodes a small GTPase induced during human erythropoiesis, conferred erythroid cell survival. Thus, while Med1 is a context-dependent GATA-1 coregulator, it also exerts specialized functions in erythroid cells to control GATA-1-independent, cell-type-specific genes, which include candidate regulators of erythroid cell development and function (Pope, 2013).


Search PubMed for articles about Drosophila Mediator complex subunit 1

Boube, M., Hudry, B., Immarigeon, C., Carrier, Y., Bernat-Fabre, S., Merabet, S., Graba, Y., Bourbon, H. M. and Cribbs, D. L. (2014). Drosophila melanogaster Hox transcription factors access the RNA polymerase II machinery through direct homeodomain binding to a conserved motif of mediator subunit Med19. PLoS Genet 10(5): e1004303. PubMed ID: 24786462

Femia, M. R., Evans, R. M., Zhang, J., Sun, X., Lebegue, C. J., Roggero, V. R. and Allison, L. A. (2020). Mediator subunit MED1 modulates intranuclear dynamics of the thyroid hormone receptor. J Cell Biochem 121(4): 2909-2926. PubMed ID: 31692077

Gobert, V., Osman, D., Bras, S., Auge, B., Boube, M., Bourbon, H. M., Horn, T., Boutros, M., Haenlin, M. and Waltzer, L. (2010). A genome-wide RNA interference screen identifies a differential role of the mediator CDK8 module subunits for GATA/ RUNX-activated transcription in Drosophila. Mol Cell Biol 30(11): 2837-2848. PubMed ID: 20368357

Immarigeon, C., Bernat-Fabre, S., Auge, B., Faucher, C., Gobert, V., Haenlin, M., Waltzer, L., Payet, A., Cribbs, D. L., Bourbon, H. G. and Boube, M. (2019). Drosophila Mediator subunit Med1 is required for GATA-dependent developmental processes: divergent binding interfaces for conserved coactivator functions. Mol Cell Biol. PubMed ID: 30670567

Immarigeon, C., Bernat-Fabre, S., Guillou, E., Verger, A., Prince, E., Benmedjahed, M. A., Payet, A., Couralet, M., Monte, D., Villeret, V., Bourbon, H. M. and Boube, M. (2020). Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo. J Biol Chem. PubMed ID: 32737196

Kuuluvainen, E., Hakala, H., Havula, E., Sahal Estime, M., Ramet, M., Hietakangas, V. and Makela, T. P. (2014). Cyclin-dependent kinase 8 module expression profiling reveals requirement of mediator subunits 12 and 13 for transcription of Serpent-dependent innate immunity genes in Drosophila. J Biol Chem 289(23): 16252-16261. PubMed ID: 24778181

Nagpal, N., Sharma, S., Maji, S., Durante, G., Ferracin, M., Thakur, J. K. and Kulshreshtha, R. (2018). Essential role of MED1 in the transcriptional regulation of ER-dependent oncogenic miRNAs in breast cancer. Sci Rep 8(1): 11805. PubMed ID: 30087366

Nagulapalli, M., Maji, S., Dwivedi, N., Dahiya, P. and Thakur, J. K. (2016). Evolution of disorder in Mediator complex and its functional relevance. Nucleic Acids Res 44(4): 1591-1612. PubMed ID: 26590257

Oda, Y., Nguyen, T., Hata, A., Meyer, M. B., Pike, J. W. and Bikle, D. D. (2020). Deletion of Mediator 1 suppresses TGFbeta signaling leading to changes in epidermal lineages and regeneration. PLoS One 15(8): e0238076. PubMed ID: 32857768

Pessina, F., Giavazzi, F., Yin, Y., Gioia, U., Vitelli, V., Galbiati, A., Barozzi, S., Garre, M., Oldani, A., Flaus, A., Cerbino, R., Parazzoli, D., Rothenberg, E. and d'Adda di Fagagna, F. (2019). Functional transcription promoters at DNA double-strand breaks mediate RNA-driven phase separation of damage-response factors. Nat Cell Biol 21(10): 1286-1299. PubMed ID: 31570834

Pope, N. J. and Bresnick, E. H. (2013). Establishment of a cell-type-specific genetic network by the mediator complex component Med1. Mol Cell Biol 33(10): 1938-1955. PubMed ID: 23459945

Sabari, B. R., Dall'Agnese, A., Boija, A., Klein, I. A., Coffey, E. L., Shrinivas, K., Abraham, B. J., Hannett, N. M., Zamudio, A. V., Manteiga, J. C., Li, C. H., Guo, Y. E., Day, D. S., Schuijers, J., Vasile, E., Malik, S., Hnisz, D., Lee, T. I., Cisse, II, Roeder, R. G., Sharp, P. A., Chakraborty, A. K. and Young, R. A. (2018). Coactivator condensation at super-enhancers links phase separation and gene control. Science 361(6400). PubMed ID: 29930091

Stumpf, M., Yue, X., Schmitz, S., Luche, H., Reddy, J. K. and Borggrefe, T. (2010). Specific erythroid-lineage defect in mice conditionally deficient for Mediator subunit Med1. Proc Natl Acad Sci U S A 107(50): 21541-21546. PubMed ID: 21098667

Zhu, C., Li, L., Zhang, Z., Bi, M., Wang, H., Su, W., Hernandez, K., Liu, P., Chen, J., Chen, M., Huang, T. H., Chen, L. and Liu, Z. (2019). A non-canonical role of YAP/TEAD is required for activation of estrogen-regulated enhancers in breast cancer. Mol Cell 75(4): 791-806 e798. PubMed ID: 31303470

Biological Overview

date revised: 25 September 2020

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