InteractiveFly: GeneBrief

Mediator complex subunit 19: Biological Overview | References

Hox genes in species across the metazoa encode transcription factors (TFs) containing highly-conserved homeodomains that bind target DNA sequences to regulate batteries of developmental target genes. DNA-bound Hox proteins, together with other TF partners, induce an appropriate transcriptional response by RNA Polymerase II (PolII) and its associated general transcription factors. How the evolutionarily conserved Hox TFs interface with this general machinery to generate finely regulated transcriptional responses remains obscure. One major component of the PolII machinery, the Mediator (MED) transcription complex, is composed of roughly 30 protein subunits xized in modules that bridge the PolII enzyme to DNA-bound TFs. This study investigate the physical and functional interplay between Drosophila melanogaster Hox developmental TFs and MED complex proteins. The Med19 subunit was found to directly bind Hox homeodomains, in vitro and in vivo. Loss-of-function Med19 mutations act as dose-sensitive genetic modifiers that synergistically modulate Hox-directed developmental outcomes. Using clonal analysis, a role was identified for Med19 in Hox-dependent target gene activation. A conserved, animal-specific motif was found that is required for Med19 homeodomain binding, and for activation of a specific Ultrabithorax target. These results provide the first direct molecular link between Hox homeodomain proteins and the general PolII machinery. They support a role for Med19 as a PolII holoenzyme-embedded 'co-factor' that acts together with Hox proteins through their homeodomains in regulated developmental transcription (Boube, 2014).

The finely regulated gene transcription permitting development of pluricellular organisms involves the action of transcription factors (TFs) that bind DNA targets and convey this information to RNA polymerase II (PolII). Hox TFs, discovered through iconic mutations of the Drosophila melanogaster Bithorax and Antennapedia Complexes, play a central role in the development of a wide spectrum of animal species. Hox proteins orchestrate the differentiation of morphologically distinct segments by regulating PolII-dependent transcription of complex batteries of downstream target genes whose composition and nature are now emerging. The conserved 60 amino acid (a.a.) homeodomain (HD), a motif used for direct binding to DNA target sequences, is central to this activity. Animal orthologs of the Drosophila proteins make use of their homeodomains to play widespread and crucial roles in differentiation programs yielding the very different forms of sea urchins, worms, flies or humans. They do so by binding simple TAAT-based sequences within regulatory DNA of developmental target genes. One crucial aspect of understanding how Hox proteins transform their versatile but low-specificity DNA binding into an exquisite functional specificity involves the identification of functional partners. Known examples include the TALE HD proteins encoded by extradenticle (exd)/Pbx and homothorax (hth)/Meis, which assist Hox proteins to form stable ternary DNA-protein complexes with much-enhanced specificity. This involves contacts with the conserved Hox Hexapeptide (HX) motif near the HD N-terminus, or alternatively, with the paralog-specific UBD-A motif detected in Ubx and Abdominal-A (Abd-A) proteins. Other TFs that can serve as positional Hox partners include the segment-polarity gene products Engrailed (En) and Sloppy paired, that collaborate with Ubx and Abd-A to repress abdominal expression of Distal-less. Finally, specific a.a. residues in the HX motif, the HD and the linker separating them play a distinctive role in DNA target specificity, allowing one Hox HD region to select paralog-specific targets (Boube, 2014).

Contrasting with knowledge of collaborations involving Hox and partner TFs, virtually nothing is known of what transpires at the interface with the RNA Polymerase II (PolII) machinery itself to generate an appropriate transcriptional response. The lone evidence directly linking Hox TFs to the PolII machine comes from the observation that the Drosophila TFIID component BIP2 binds the Antp HX motif (Boube, 2014).

Another key component of the PolII machinery is the Mediator (MED) complex conserved from amoebae to man that serves as an interface between DNA-bound TFs and PolII. MED possesses a conserved, modular architecture characterized by the presence of head, middle, tail and optional CDK8 modules. Some of the 30 subunits composing MED appear to play a general structural role in the complex while others interact with DNA-bound TFs bridging them to PolII. Together, these subunits and the MED modules they form associate with PolII, TFs and chromatin to regulate PolII-dependent transcription (Boube, 2014).

The analysis of a Drosophila skuld/Med13 mutation isolated by dose-sensitive genetic interactions with homeotic proboscipedia (pb) and Sex combs reduced (Scr) genes led to a view that MED is a Hox co-factor (Boube, 2000). However, how MED might act with Hox TFs in developmental processes has not been explored. This work pursues the hypothesis that Hox TFs modulate PolII activity through direct binding to one or more MED subunits. Starting from molecular assays, Med19 was identified as a subunit that binds to the homeodomain of representative Hox proteins through an animal-specific motif. Loss-of-function (lof) Med19 mutations isolated in this work reveal that Med19 affects Hox developmental activity and target gene regulation. Taken together, these results provide the first molecular link between Hox TFs and the general transcription machinery, showing how Med19 can act as an embedded functional partner, or 'co-factor', that directly links DNA-bound Hox homeoproteins to the PolII machinery (Boube, 2014).

Hox homeodomain proteins are well-known for their roles in the control of transcription during development. Further, much is known about the composition and action of the PolII transcription machine. However, virtually nothing is known of how the information of DNA-bound Hox factors is conveyed to PolII in gene transcription. The Drosophila Ultrabithorax-like mutant affecting the large subunit of RNA PolII provokes phenotypes reminiscent of Ubx mutants (Mortin, 1992), but the molecular basis of this remains unknown. The lone direct evidence linking Hox TFs to the PolII machine is binding of the Antp HX motif to the TFIID component BIP2 (Prince, 2008). This study undertook to identify physical and functional links between Drosophila Hox developmental TFs and the MED transcription complex. The results unveil a novel aspect of the evolutionary Hox gene success story, extending the large repertory of proteins able to interact with the HD to include the Drosophila MED subunit Med19. HD binding to Med19 via the conserved HIM suggests this subunit is an ancient Hox collaborator. Accordingly, loss-of-function mutants reveal that Med19 contributes to normal Hox developmental function and does so at least in part via its HIM element. Thus this analysis reveals a previously unsuspected importance for Med19 in Hox-affiliated developmental functions (Boube, 2014).

A fundamental property of the modular MED complex is its great flexibility that allows it to wrap around PolII and to change form substantially in response to contact with specific TFs (Tsai, 2011). Recent work in the yeast S. cerevisiae places Med19 at the interfaces of the head, middle and CDK8 kinase modules (Tsai, 2013). Med19 is thus well-positioned to play a pivotal regulatory role in governing MED conformation (see Model for the role of Med19 at the interface of Hox and MED). The results raise the intriguing possibility that MED structural regulation and physical contacts with DNA-bound TFs can pass through the same subunit. In agreement with this idea, recent work identified direct binding between mouse Med19 (and Med26) and RE1 Silencing Transcription Factor (REST) (Ding, 2009). This binding involves a 460 a.a. region of REST encompassing its DNA-binding Zn fingers. The present work goes further, in identifying a direct link between the conserved Hox homeodomain and Med19 HIM that is the first instance for a direct, functionally relevant contact of MED with a DNA-binding motif rather than an activation domain (Boube, 2014).

Med19 contributes to developmental processes with Antp (spiracle eversion), Dfd (Mx palp), and Ubx (haltere differentiation). Other phenotypes identified indicate further, non-Hox related roles for Med19. As shown in this study, complete loss of Med19 function leads to cell lethality that can be conditionally alleviated when surrounded by weakened, Minute mutation-bearing cells. These observations, that uncouple HIM-dependent functions from the role of Med19 in cell survival/proliferation, are compatible with reports correlating over-expression of human Med19/Lung Cancer Metastasis-Related Protein 1 (LCMR1) in lung cancer cells with clinical outcome (Chen, 2011). Further, RNAi-mediated knock-down of Med19 in cultured human tumor cells can reduce proliferation, and tumorigenicity when injected into nude mice (Zhang, 2012). A recent whole-genome, RNAi-based screen identified Med19 as an important element of Androgen Receptor activity in prostate cancer cells where gene expression levels also correlated with clinical outcome (Imberg-Kazdan, 2013). It will be of clear interest to examine how, and with what partners, Med19 carries out its roles in cell proliferation/survival (Boube, 2014).

The role played by mammalian Med19 and Med26 in binding the REST TF, involved in inhibiting neuronal gene expression in non-neuronal cells (Ding, 2008; Ding, 2009), provides an instance of repressive Med19 regulatory function. This study found that Med19 activity is required in the Drosophila haltere disc for transcriptional activation of CG13222/edge and bab2, but is dispensable for Ubx-mediated repression of five negatively-regulated target genes. Ubx can choose to activate or it can repress, at least in part through an identified repression domain at the C-terminus just outside its homeodomain. Conversely Med19, which binds the Ubx homeodomain, appears to have much to do with activation (Boube, 2014).

Concerning the mechanisms of Ubx-mediated repression, one illuminating example comes from analyses of regulated embryonic Distal-less expression. Ubx can associate combinatorially with Exd and Hth, plus the spatially restricted co-factors Engrailed or Sloppy-paired in repressing Distal-less . Engrailed in turn is able to recruit Groucho co-repressor, suggesting that localized repression involves DNA-bound Ubx/Exd/Hth/Engrailed, plus Engrailed-bound Groucho. Groucho has been proposed to function as a co-repressor that actively associates with regulatory proteins and organizes chromatin to block transcription. The yeast Groucho homolog Tup1 interacts with DNA-binding factors to mask their activation domains, thereby preventing recruitment of co-activators (including MED) necessary for activated transcription. The number of targets remains too small to be sure Med19 is consecrated to activation. Nonetheless, it will be of interest to determine whether Groucho can play a role in blocking MED/Ubx interactions that could provide an economical means for distinguishing gene activation from repression (Boube, 2014).

The conserved Hox proteins and the gene complexes that encode them are well-known and widely used to study development and evolution. As to the evolutionary conservation of the Mediator transcription complex, the presence of MED constituents in far-flung eukaryotic species from unicellular parasites to humans indicates that this complex existed well before the emergence of the modern animal Hox protein complexes. The DNA-binding domains are often the most conserved elements of TF primary sequence, and in the case of the Hox HD, recent forays into 'synthetic biology' agree that this was the functional heart of the ancestral proto-Hox proteins. Indeed, Scr, Antp and Ubx mini-Hox peptides containing HX, linker and HD motifs behave to a good approximation like the full-length forms, directing appropriate gene activation and repression resulting in genetic transformations. The current results showing direct HD binding to Med19 HIM, and thus access to the PolII machinery, allow the activity of these mini-Hox proteins to be rationalized. It is surmised that at the time when the Hox HD emerged to become a major developmental transcription player, its capacity to connect with MED through specific existing sequences was a prerequisite for functional success. One expected consequence of this presumed initial encounter with Med19 (a selective pressure on both partners and subsequent refinement of binding sequences) is in agreement with the well-known conservation of Hox homeodomains, and with the observed conservation of the newly-identified HIM element in Hox-containing eumetazoans. It is imagined that subsequent evolution over the several hundred million years separating flies and mammals will have allowed this initial contact to be consolidated through subsequent binding to other MED subunits, ensuring versatile but reliable interactions at the MED-TF interface (Boube, 2014).

Hox homeodomain proteins are traditionally referred to as selector or 'master' genes that determine developmental transcription programs. The low sequence specificity of Hox HD transcription factors is enhanced by their joint action with other TFs, of which prominent examples, the TALE homeodomain proteins Extradenticle/Pbx and Homothorax/Meis are considered to be Hox co-factors. However, a Hox TF in the company of Exd and Hth could still not be expected to shoulder all the regulatory tasks necessary to make a segment with all the coordinated cell-types it is made up of, and collaboration with cell-type specific TFs appears to be requisite. A useful alternative conception visualizes Hox proteins not as 'master-selectors' that act with co-factors, but as highly versatile co-factors in their own right that can act with diverse cell-specific identity factors to generate the cell types of a functional segment. A model is envisaged where a Hox protein would be central to assembling cell-specific transcription factors into TF complexes that interface with MED (Boube, 2014).

Such Hox-anchored TF complexes could make use of selective HD binding to Med19 as a beach-head for more extensive access to MED, such that loss of the Hox protein would incapacitate the complex: in the case of Ubx^- cells, inactivating bab2 or de-repressing sal. Accordingly, three observations suggest that binding of Hox-centered TF complexes involves additional MED subunits surrounding Med19: (1) bab2 target gene expression is entirely lost in Ubx-deficient cells but can persist in some Med19^- cells; (2) edge-GFP in Med19^- cells expressing Med19ΔHIM-VC was not altogether refractory to Ubx-activated edge-GFP expression; and (3) Med19ΔHIM-VC is not entirely impaired for Ubx binding, as seen in co-immunoprecipitations. Thus Hox protein input conveyed through Med19-HIM at the head-middle-Cdk8 module hinge might provide an economical contribution toward organizing TF complexes that influence overall MED conformation and hence transcriptional output. Decoding how the information-rich MED interface including Med19 accomplishes this will be an important part of understanding transcriptional specificity in evolution, development and pathology (Boube, 2014).

Search PubMed for articles about Drosophila Med19

Boube, M., Faucher, C., Joulia, L., Cribbs, D. L. and Bourbon, H. M. (2000). Drosophila homologs of transcriptional mediator complex subunits are required for adult cell and segment identity specification. Genes Dev 14: 2906-2917. PubMed ID: 11090137

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: e1004303. PubMed ID: 24786462

Chen, L., Liang, Z., Tian, Q., Li, C., Ma, X., Zhang, Y., Yang, Z., Wang, P. and Li, Y. (2011). Overexpression of LCMR1 is significantly associated with clinical stage in human NSCLC. J Exp Clin Cancer Res 30: 18. PubMed ID: 21306606

Ding, N., Zhou, H., Esteve, P. O., Chin, H. G., Kim, S., Xu, X., Joseph, S. M., Friez, M. J., Schwartz, C. E., Pradhan, S. and Boyer, T. G. (2008). Mediator links epigenetic silencing of neuronal gene expression with x-linked mental retardation. Mol Cell 31: 347-359. PubMed ID: 18691967

Ding, N., Tomomori-Sato, C., Sato, S., Conaway, R. C., Conaway, J. W. and Boyer, T. G. (2009). MED19 and MED26 are synergistic functional targets of the RE1 silencing transcription factor in epigenetic silencing of neuronal gene expression. J Biol Chem 284: 2648-2656. PubMed ID: 19049968

Imberg-Kazdan, K., Ha, S., Greenfield, A., Poultney, C. S., Bonneau, R., Logan, S. K. and Garabedian, M. J. (2013). A genome-wide RNA interference screen identifies new regulators of androgen receptor function in prostate cancer cells. Genome Res 23: 581-591. PubMed ID: 23403032

Mortin, M. A., Zuerner, R., Berger, S. and Hamilton, B. J. (1992). Mutations in the second-largest subunit of Drosophila RNA polymerase II interact with Ubx. Genetics 131: 895-903. PubMed ID: 1516820

Prince, F., Katsuyama, T., Oshima, Y., Plaza, S., Resendez-Perez, D., Berry, M., Kurata, S. and Gehring, W. J. (2008). The YPWM motif links Antennapedia to the basal transcriptional machinery. Development 135: 1669-1679. PubMed ID: 18367556

Tsai, C. J. and Nussinov, R. (2011). Gene-specific transcription activation via long-range allosteric shape-shifting. Biochem J 439: 15-25. PubMed ID: 21916844

Tsai, K. L., Sato, S., Tomomori-Sato, C., Conaway, R. C., Conaway, J. W. and Asturias, F. J. (2013). A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction. Nat Struct Mol Biol 20: 611-619. PubMed ID: 23563140

Zhang, H., Jiang, H., Wang, W., Gong, J., Zhang, L., Chen, Z. and Ding, Q. (2012). Expression of Med19 in bladder cancer tissues and its role on bladder cancer cell growth. Urol Oncol 30: 920-927. PubMed ID: 21478038

date revised: 18 May 2014

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