Transcriptional repression in eukaryotes is achieved through the interaction of sequence-specific DNA-binding transcriptional repressors with corepressors – functional intermediaries that directly or indirectly serve to inhibit transcription preinitiation complex (PIC) assembly on core promoters. Corepressors have been grouped broadly into those that interface directly with RNA polymerase II and its general transcription factors to inhibit PIC assembly and those that modify chromatin and therefore facilitate nucleosome-mediated promoter occlusion. Mediator is generally considered representative of the former corepressor class, and prior studies have attributed its chromatin-independent repressive function to its resident CDK8/CycC moiety. Thus, CDK8/CycC have been shown to repress transcription through diverse effects on the basal transcription machinery, including phosphorylation and consequent inhibition of TFIIH kinase activity as well as through direct occlusion of RNA polymerase II binding (Akoulitchev et al., 2000
; Elmlund et al., 2006
). Nonetheless, whether and how Mediator physically and functionally collaborates with chromatin modifying activities to repress transcription has not previously been established. Our finding that the MED12 interface in Mediator links REST with G9a-dependent H3K9me2 establishes a direct link between Mediator and chromatin modification leading to transcriptional repression. These studies thus implicate Mediator directly in epigenetic gene silencing and further reveal a hitherto unknown role for Mediator in facilitating the imposition of repressor-driven transcriptionally restrictive higher order chromatin structure.
Our studies further serve to clarify what has heretofore remained an obscure and complex function for MED12 in transcriptional repression. Thus, while prior genetic studies in model metazoans have implicated MED12 in negative regulation of gene expression (Janody et al., 2003
; Moghal and Sternberg, 2003
; Zhang and Emmons, 2000
), it has not previously been clear whether MED12 plays a direct or indirect role in this process. Our finding that MED12 links REST with G9a in neuronal gene silencing provides a clear mechanistic basis to explain a direct repressive function for MED12 in gene control, and also provides new insight concerning the role and requirement of MED12 module components in negative regulation of gene expression. Heretofore, repression exerted by the MED12 module has been attributed to its CDK8/CycC moiety, while MED12/MED13 have been ascribed regulatory or architectural roles critical to ensure anchored kinase activity. Our delineation of a MED12-dependent repressive function for Mediator independent of CDK8/CycC is consistent with recent genetic analyses in D. melanogaster revealing functional diversification among MED12 module components and distinct roles for MED12 in developmental gene regulation independent of CycC-regulated CDK8 activity (Loncle et al., 2007
The identification herein of MED12 as a corepressor of REST has important implications for both the biological function of Mediator and the mechanism of REST-dependent neuronal gene silencing. REST occupies a central role in non-neuronal lineage restriction by virtue of its ability to silence neuronal-specific gene expression in terminally differentiated non-neuronal cells (Ooi and Wood, 2007
). Our discovery that MED12/Mediator collaborates with REST and G9a in extra-neuronal gene silencing suggests an important and heretofore unrecognized function for Mediator in targeting neuronal gene expression to the nervous system.
Mechanistically, REST-directed neuronal gene silencing is attributed to its targeted recruitment, via distinct N- and C-terminal domains, of multiple enzymatically diverse corepressors that co-ordinately function to fine-tune the histone acetylation and methylation dynamics as well as the overall epigenetic signature associated REST-repressed genes (Ooi and Wood, 2007
). Our delineation of a MED12/Mediator- and G9a-dependent internal repression domain in REST encompassing amino acids 141–600 suggests that REST itself is more complex than originally thought, and considerably expands the network of functional interactions through which REST represses neuronal gene expression. Notably, this region in REST has previously been ascribed transcriptional repressive function, although the mechanistic basis for such activity has not been established (Roopra et al., 2000
). Our finding that Mediator and G9a physically and functionally interact with REST 141–600 not only establishes a molecular basis for the repressive activity of this domain, but also extends the notion of REST as an integrative hub for the recruitment and directed deployment of functionally diverse chromatin modifiers. Further studies will be required to establish the fundamental interaction dynamics through which individual components of the broader REST corepressor network, including MED12/Mediator, coordinately function and possibly crosstalk to impose epigenetic restrictions on REST-target gene expression.
In addition to its role in modulating chromatin structure, REST has been reported to repress neuronal gene expression through direct effects on RNA polymerase II and its accessory transcription factors. For example, REST binds directly to the TATA box-binding protein to inhibit transcription preinitiation complex (PIC) formation (Murai et al., 2004
), and RNA polymerase II small CTD phosphatases to inhibit polymerase activity (Yeo et al., 2005
). Mediator, by virtue of its ability to interact directly with both REST and RNA polymerase II, could conceivably function to assist REST in PIC inhibition, or possibly even coordinate this activity with REST-directed chromatin remodelling. Future studies will be required to distinguish among these possibilities.
Among the 30 subunits comprising human Mediator, MED12 is the only one in which genetic variation has been linked to neuropsychiatric illness and cognitive dysfunction. Our discovery that the FG/R961W and Lujan/N1007S missense mutations in MED12 disrupt its REST-specific corepressor function represents the first description of a functional defect associated with these mutations. Notably, we found that both mutations impaired recruitment of Mediator to RE1 elements with little impact on the MED12/G9a interaction, providing a plausible mechanistic basis to explain how these mutations disrupt REST-imposed epigenetic restrictions on neuronal gene expression. Thus, impaired recruitment of Mediator, identified herein to be essential link between RE1-bound REST and G9a, results in reduced G9a recruitment, diminished levels of G9a-dependent H3K9me2, and de-repression of REST-target gene expression. The underlying basis by which the FG and Lujan mutations in MED12 impair Mediator recruitment to RE1 elements remains to be clarified, but could involve a conformational change in MED12/Mediator and resultant masking of a REST target subunit(s) in Mediator other than MED12, which we have thus far been unable to identify as a direct interface for REST. Further studies will be required to validate this prediction and establish the definitive basis by which the FG and Lujan mutations in MED12 impair Mediator recruitment to RE1 elements.
Our discovery that the FG/R961W and Lujan/N1007S missense mutations in MED12 disrupt REST-imposed restrictions on neuronal gene expression offers a possible epigenetic perspective to explain the role of MED12 in XLMR. Because REST and MED12 have both been implicated in neuronal development (Ballas et al., 2005
; Hong et al., 2005b; Kuwabara et al., 2004
; Rau et al., 2006
; Wang et al., 2006
), misregulation of REST target genes arising as a consequence of pathological mutations in MED12 could conceivably affect neuronal differentiation and possibly contribute to XLMR. In this regard, we note that additional members of the REST interactome have been linked to mental retardation (MR) (Kleefstra et al., 2006
; Tahiliani et al., 2007
). Haploinsufficiency arising from mutations/deletions in the gene encoding the H3K9 HMTase GLP/Eu-HMTase1 (a stoichimetric binding partner of G9a) have been shown to be causative for the 9q34 subtelomeric deletion syndrome characterized by severe MR, while mutations in the H3K4 histone demethylase SMCX/JARID1C have been linked to XLMR (Kleefstra et al., 2006
; Tahiliani et al., 2007
). However, the impact of pathological mutations in either protein on REST-dependent repressive activity is unknown. Our findings linking altered REST repressor function with XLMR-associated mutations in MED12 could provide a paradigm for how pathological defects in a broader REST corepressor network contribute to MR.
In conclusion, our work reveals that pathological mutations in MED12 associated with XLMR disrupt its newly identified role as mediator of REST-directed G9a-dependent extra-neuronal gene silencing. These findings establish a new function for Mediator in epigenetic restriction of neuronal gene expression to the nervous system, and shed new light on the mechanism of Mediator in transcriptional repression as well as the etiology of MED12-associated XLMR syndromes.