MED12

The kinase module gene, MED12, is located on X-chromosome (at Xq13) and encodes a 2212 aa (240kDa sized) protein. This protein can be divided into four distinct domains: leucine-rich (L) domain, leucine-serine-rich (LS) domain, proline-glutamine-leucine-rich (PQL) domain and a poly-glutamine (Opa) domain (Figure 7). MED12 is highly conserved among eukaryotes, particularly in mammals, with 96% identity between mouse and human amino acid sequences (Philibert & Madan, 2007). Med12 gene is essential for early mouse development. The null embryos are arrested at the head-fold stage (E7.5), since they cannot induce mesoderm formation (Rocha et al, 2010). The hypomorphic mutants expressing less than 10% MED12 protein die at E10.5 and have defects in neural tube closure, axis elongation, somitogenesis and heart formation. Both canonical Wnt/ -catenin and non- canonical Wnt/PCP pathways are disrupted in these hypomorphic mutants. This is expected as MED12 physically interacts with β-catenin to activate the transcription of Wnt-responsive genes (Kim et al, 2006). MED12 was also reported to interact with Nanog and coregulates its target genes. RNAi-mediated Med12 knockdown was shown to reduce the expression of Nanog and its target genes in embryonic stem (ES) cells, which then start the differentiation process (Tutter et al, 2009). In contrast to this, Med12+/+, Med12-/- and Med12hypo ES cells showed unaltered expression of Nanog and its target genes, suggesting that MED12 is not required for ES cell pluripotency (Rocha et al, 2010). Nevertheless, MED12 is involved in the transcriptional regulation of many signaling pathways. Indeed, the C. elegans ortholog of

Figure 7: Domain structure of MED12.

L  Leucine-rich domain, LS  Leucine- and Serine-rich domain, PQL  Proline-, Glutamine- and Leucine-rich domain, Opa  Opposite paired domain. FG- (R961W) and Lujan-related (N1007S) mutations are found in LS domain, the most common uterine leiomyoma-linked (G44D) mutation is found in the L domain. The methylation site (R1862) associated with cancer drug sensitivity is present in the PQL domain. The methylation site (R1899) identified in the current study is also present in the PQL domain. All known interactors bind MED12 at its PQL domain.

MED12, dpy-22, was identified to be one of the six highly connected ‘hub’ genes (all of which encode chromatin regulators) that interact with numerous components of diverse signaling pathways and serve as modifier genes in multiple mechanistically unrelated genetic diseases (Lehner et al, 2006). Sox10 is a transcription factor required for terminal differentiation of myelinating glia. MED12 interacts with Sox10 and induces genes necessary to drive the myelination of neurons. Glia-specific deletion of Med12 in mice showed defects in terminal differentiation and myelin gene expression of both Schwann cells and Oligodendrocytes (Vogl et al, 2013). Amyloid precursor protein (APP), a transmembrane protein associated with Alzheimer’s disease, is cleaved by γ-secretase to produce amyloid-β (Aβ) peptide and APP intracellular domain (AICD). Although the events of amyloid-β plaque formation and consequent neuronal death have been well studied, little is known about the mechanism by which AICD activates its target genes, which are associated with disease pathology. AICD was shown to recruit Mediator complex via interaction with MED12 to activate its target genes. Disruption of this interaction abrogates the transactivation potential of AICD, suggesting that MED12 plays a crucial role in the pathophysiology of Alzheimer’s disease (Xu et al, 2011). In addition to this, MED12 is implicated in a number of neurological disorders. Exonic polymorphisms have been associated with neuropsychiatric diseases including schizophrenia and psychosis (Philibert, 2006; Sandhu et al, 2003). Recurrent missense mutations in MED12 have been shown to cause X-linked intellectual disability (XLID) syndromes such as Optiz- Kaveggia (or FG) syndrome and Lujan-Fryns (or Lujan) syndrome (Graham & Schwartz, 2013; Risheg et al, 2007; Schwartz et al, 2007). In addition to mental retardation, these two syndromes share many overlapping characteristics like macrocephaly, dysgenesis of corpus callosum, hypotonia, craniofacial dysmorphisms, seizures and behavioural problems. To understand the molecular mechanisms by which R961W (FG) and N1007S (Lujan) mutations could contribute to XLID phenotypes, Boyer group identified MED12 binding partners and

morphogen that controls the left-right, dorso-ventral axes patterning, limb patterning and development of brain, spinal cord and most other organs. The Boyer group showed that Shh- activated Gli3 recruits Mediator complex via interaction with MED12, wherein enzymatically active CDK8 suppresses Gli3 transactivation activity. R961W and N1007S mutations in MED12 prevent recruitment of CDK-Mediator to the target gene promoters, thereby hyperactivating Gli3-dependent Shh signaling that may possibly contribute to XLID phenotypes (Zhou et al, 2006; Zhou et al, 2012). Repressor element-1 binding factor (REST) is a transcription factor that represses the expression of neuron-specific genes in terminally differentiated non-neural tissues or undifferentiated neural precursors. RE1-bound REST recruits Mediator complex via interactions with MED19 and MED26, wherein MED12 acts as a direct interface for G9a recruitment and H3K9 dimethylation, leading to epigenetic silencing of REST-target genes. FG- and Lujan-related MED12 mutations disrupt the epigenetic restrictions imposed by REST, possibly contributing to XLID phenotypes (Ding et al, 2009; Ding et al, 2008). Studies by Sheikattar group showed that Mediator interacts with ncRNA- activating (ncRNA-a), a novel class of lncRNAs that activate their neighboring genes using a cis-mediated mechanism. NcRNA-a3 and -a7 interact with MED12 to recruit Mediator and deposit CDK8-mediated H3S10 phosphorylation at the promoters of a3 and a7 target genes (SNAI1, AURKA, and TAL1) for transcriptional activation. They also showed that FG-related MED12 mutations significantly diminish interactions with ncRNA-a, which may possibly contribute to XLID (Lai et al, 2013). In addition to neurological disorders, MED12 is also frequently mutated in human cancers like uterine leiomyomas, breast and prostate cancers (Schiano et al, 2014). Uterine leiomyoma-linked mutations in MED12 were shown to disrupt its interaction with Cyclin C-CDK8 resulting in loss of Mediator-associated kinase activity (Turunen et al, 2014). Furthermore, MED12 is linked to drug resistance in different cancer types. Cytoplasmic MED12, independent of the Mediator complex, was shown to interact with the immature form of TGFβR2 and prevent it from undergoing glycosylation and plasma

membrane association. Therefore, loss of MED12 activates TGFβ signaling that further causes MEK/ERK activation, which is sufficient to confer multidrug resistance in colon and lung cancers (Huang et al, 2012). Using genome-scale CRISPR-Cas9 knockout (GeCKO) library screening, another group identified MED12 as the top hit among genes whose loss results in drug resistance in melanoma cells (Shalem et al, 2014).

Chapter 2: Materials and Methods

Contents of this chapter are partially based on Dhar S*, Vemulapalli V*, Patananan AN, Huang GL, Di Lorenzo A, Richard S, Comb MJ, Guo A, Clarke SG, Bedford MT (2013) Loss of the major Type I arginine methyltransferase PRMT1 causes substrate scavenging by other PRMTs. Sci Rep 3: 1311. (* indicates equal contribution authors)

According to Nature Scientific Reports, the author retains the ownership of copyright to reproduce the contribution or extracts from the original article.