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The unifying theme of our research program is to understand the function and regulation of the multiprotein Mediator of transcription, and further clarify how Mediator dysfunction as a consequence of mutation or misexpression of its constituent subunits contributes to human disease. Mediator is a conserved multisubunit signal-processor through which regulatory information conveyed by gene-specific transcription factors is transduced to RNA polymerase II. In this capacity, Mediator serves to channel regulatory signals from activator and repressor proteins to affect changes in gene expression programs that control diverse physiological processes, including cell growth and homeostasis, development, and differentiation. Structurally, Mediator is assembled from a set of core subunits into three distinct modules termed “head”, “middle”, and “tail” that bind tightly to RNA Polymerase II. MED12, along with MED13, CDK8, and Cyclin C (CycC), comprise a fourth “kinase” module that resides in variable association with core Mediator. Notably, we and others have shown that MED12-dependent CDK8 activation within Mediator is required for nuclear transduction of signals instigated by multiple developmental and oncogenic pathways with which MED12 is biochemically and genetically linked. Our primary research interest over the past several years has centered on the Mediator kinase module and its role as an endpoint in signal transduction pathways. In this regard, we aim understand how physiological cell signals that converge on the kinase module in Mediator inform proper development of the brain, uterus, and intestine, and further clarify how pathololgical dysregulation of these signals elicit X-linked intellectual disability and neurodegenerative disease, uterine leiomyomata, and colorectal cancer. We expect these studies to reveal important mechanistic insight concerning the function of Mediator in developmental gene control that might be leveraged to advantage in the development of molecularly targeted therapies.
1. Molecular basis of Mediator subunit 12 in uterine leiomyomagenesis
Uterine leiomyomas (UL; or fibroids) are monoclonal neoplasms of the myometrium and represent the most frequent tumors among women worldwide. Although benign, they nonetheless account for significant morbidity. They are the primary indicator for hysterectomy, and a major cause of gynecologic and reproductive dysfunction, ranging from profuse menstrual bleeding and pelvic pain to infertility, recurrent miscarriage, and pre-term labor. Current treatment options are primarily surgical and often only precursory to hysterectomy. Newer medical therapies, designed to target the tumor-promoting actions of estrogen and progesterone, have shown early promise in clinical trials, but are otherwise fraught with long-term safety and other concerns. The development of alternative molecularly targeted therapies will progress only as fast our acquired insight concerning the molecular etiology of UL. In this regard, somatic mutations in exons 1 and 2 of the gene encoding the transcriptional Mediator subunit MED12 were recently found to occur at high frequency (~70%) in UL, suggesting a likely etiological basis previously lacking for the vast majority of these clinically significant lesions.
Mediator is a conserved multisubunit signal processor through which regulatory information conveyed by gene-specific transcription factors is transduced to RNA polymerase II (Pol II). MED12, along with MED13, Cyclin C (CycC) and CDK8, comprise a structurally distinct 4-subunit “kinase” module within Mediator. Notably, we and others have shown that MED12-dependent CDK8 activation is required for nuclear transduction of signals instigated by multiple oncogenic pathways with which MED12 is biochemically and genetically linked. In addition, MED12 itself is a target of oncogenic mutation, including exons 1 and 2 mutations linked to UL. However, the impact of these mutations on MED12 function and the molecular basis for their tumorigenic potential are unknown. Recently, we showed that UL-linked mutations in in MED12 disrupt its ability to activate CDK8 in Mediator, leading to reduced site-specific RNA Pol II phosphorylation and global dysregulation of gene expression. Our current studies are focused on how these defects drive fibrotic transformation of uterine smooth muscle cells and whether correction of these defects can delay or reverse the fibrotic phenotype. We expect these studies to have important implications for personalized treatment of women with UL.
2.Molecular basis of Mediator subunit 12 in syndromic X-linked intellectual disability
X-linked intellectual disability (XLID) affects 1–2 of every 1,000 males and accounts for ∼10% of all intellectual disability (ID). Approximately one-third of XLID cases are associated with sufficiently coincident somatic, neurobehavioral, or metabolic features to permit diagnostic designation and are therefore classified as “syndromal” in nature. To date, more than 140 syndromal XLID conditions have been identified, almost half of which are attributable to mutations in single genes. The identification of these genes has been a dominant force in linking syndromes previously considered separate entities based on clinical criteria alone. Notable among the XLID syndromes so linked are FG (or Opitz-Kaveggia) syndrome and Lujan (or Lujan-Fryns) syndrome which arise from different missense mutations in the Xq13 gene encoding MED12, a subunit of the RNA polymerase II transcriptional Mediator. In addition to ID, FG and Lujan syndromes share several overlapping clinical manifestations, including agenesis/dysgenesis of the corpus callosum, macrocephaly, hypotonia, distinct craniofacial dysmorphisms, seizures, and behavioral disturbances. Our long-term goal is to decipher the molecular mechanisms by which mutations in MED12 elicit ID as well as the broad spectrum of overlapping and unique clinical phenotypes associated with these allelic disorders.
In this regard, we showed that FG/R961W and Lujan/N1007S missense mutations in MED12 disrupt epigenetic repression of neuronal gene expression imposed by the RE1 silencer transcription factor (REST; also known as neuron restrictive silencer factor, NRSF), a master regulator of neuronal fate. In this regard, we discovered a protein interaction network required for REST-directed neuronal gene silencing comprising REST, Mediator, and G9a histone methyltransferase, a dominant euchromatic histone 3-lysine 9 (H3K9) mono- and di-methylase in mammalian cells. We showed that the MED12 interface in Mediator links REST with G9a-dependent histone H3K9 dimethylation to suppress neuronal genes in nonneuronal cells. Importantly, we found that the FG and Lujan mutations in MED12 disrupt its REST corepressor function through impaired recruitment of Mediator to RE1 (REST repressor) elements. Impaired recruitment of Mediator, an essential link between RE1-bound REST and G9a, leads to reduced G9a recruitment, diminished levels of G9a-dependent H3K9me2, and derepression of REST target gene expression. Together, these findings implicate Mediator in epigenetic restriction of neuronal gene expression to the nervous system and suggest a pathologic basis for MED12-associated XLMR involving impaired REST-dependent neuronal gene regulation
More recently, we reported that the FG and Lujan mutations in MED12 disrupt a Mediator-imposed constraint on GLI3-dependent Sonic Hedgehog (SHH) signaling. In this regard, it is notable that several phenotypes associated with FG and/or Lujan syndromes including macrocephaly, corpus callosal defects, hypertolerism, syndactyly, and cognitive impairment, overlap with a subset of those variously appearing in Greig cephalopolysyndactyly syndrome (GCPS) and/or Pallister-Hall syndrome (PHS) arising from mutations in GLI3, a Sonic Hedgehog (SHH) signaling effector that we had previously shown physically and functionally interacts with MED12. Given the physical and functional interaction between GLI3 and MED12 coupled with the fact that mutations in each of these interacting proteins elicit congenital anomaly syndromes with overlapping phenotypes, we hypothesized, and recently confirmed, that pathogenic mutations in MED12 leading to FG and Lujan syndromes elicit dysregulated GLI3-dependent SHH signaling. We showed that the FG/R961W and Lujan/N1007S mutations disrupt the gene-specific association of MED12 with CDK8, an additional Mediator subunit that we identified to be a suppressor of GLI3 transactivation activity. In FG/R961W and Lujan/N1007S patient-derived cells, we documented enhanced SHH pathway activation and GLI3-target gene induction coincident with impaired recruitment of CDK8 onto promoters of GLI3-target genes, but not non–GLI3-target genes. Our findings that XLID mutations in MED12 elicit aberrant GLI3-dependent SHH signaling not only suggest an additional basis for cognitive dysfunction through altered brain development, but may further explain a broad range of clinically diverse non-CNS phenotypes associated with these syndromal disorders, including digit, craniofacial, corpus callosal, and anorectal malformations. Collectively, these findings suggest that dysregulated GLI3-dependent SHH signaling contributes to phenotypes of individuals with FG and Lujan syndromes and further reveal a basis for the gene-specific manifestation of pathogenic mutations in a global transcriptional coregulator.
Dysregulation of the REST and SHH pathways as a consequence of XLID mutations in MED12 could impact neuronal differentiation and function, since both pathways have been implicated in neural progenitor cell (NPC) maintenance. Specifically, REST has been implicated in the regulation of genes that promote neuronal differentiation and migration, while SHH is an established mitogen that stimulates NPC proliferation and cell cycle kinetics. We are currently exploring the role of MED12/Mediator in neural NPC biology, with an emphasis on its coordinate regulation REST- and SHH-dependent gene expression programs. We expect these studies to have important human health implications for cell replacement therapy in neurological disease as well as the etiology of XLID.
3. Mediator as a transducer of amyloid precursor protein-dependent nuclear signaling
Alzheimer’s disease (AD) is a profoundly debilitating neurodegenerative condition and the leading cause of dementia among the elderly. Current treatment options are ineffective and no determinative antemortem diagnositic is presently available. Overcoming these barriers will require a better understanding of the molecular processes that drive AD onset and progression, leading to improvements in diagnosis and treatment. The prevailing model to explain AD pathogenesis posits that neuronal degeneration and clinical demise are precipitated by the gradual accumulation, in brain regions serving memory and cognition, of amyloid- (A) peptide, a catabolite of the amyloid precursor protein (APP). However, recent studies imply a complex etiology for AD, particularly in its idiopathic late onset form, that may also involve amyloid-independent pathways. In this regard, amyloidogenic processing of APP is known to produce, along with A, a small APP intracellular domain (AICD) that traffics to the nucleus and transcriptionally activates genes implicated in the pathophsyiology of AD. Although amyloid-independent, this APP-dependent nuclear signaling pathway is nonetheless tightly linked to A production, suggesting a possible synergistic route to disease onset.
Heretofore, the mechanistic basis by which the AICD stimulates target gene transcription in the nucleus has remained obscure. Recently, however, we discovered that the AICD activates transcription by targeting the MED12 interface in Mediator, a multisubunit signal processor through which regulatory information conveyed by gene-specific transcription factors is transduced to RNA polymerase II. These findings identify the MED12 interface in Mediator as a crucial missing link in APP-dependent nuclear signaling and implicate Mediator in a broad range of developmental and pathological processes driven by APP. Our findings reveal a significant contribution of Mediator to AICD-dependent transcription control genome-wide, and further implicate the AICD and Mediator in the coordinate regulation of a neural gene network with established links to AD. Because of their common proteolytic derivation from APP, pathological changes in A levels could be paralleled by changes in concentration-dependent AICD-mediated target gene induction. Accordingly, AICD-target genes could represent early biological indicators of A-induced pathology. Our current studies are focused on the mechanistic basis of coordinate transcriptional control by the AICD and Mediator as well as the identification of AICD/Mediator-target genes as possible biomarkers in AD.
4. Mediator as a transducer and therapeutic target in oncogenic Wnt/catenin signaling
Worldwide, colorectal cancer (CRC) is the second and third most commonly diagnosed cancer in women and men, respectively, and a leading cause of cancer-related deaths. Although early surgical excision of non-invasive tumors is essentially curative, few effective treatment options are available for advanced stage disease, rendering the development of alternative molecularly targeted therapies crucial to improve CRC treatment. CRC arises from intestinal crypts, wherein progenitor-derived epithelial cells commence differentiation as they initiate their ascent up intestinal villi. Maintenance of the crypt progenitor phenotype is dependent upon the expression of genes programmed by the canonical Wnt/catenin pathway, and constitutive pathway activation arising through mutations in the APC tumor suppressor or catenin is a driving force in >90% of CRCs. Accordingly, strategies designed to inhibit the transcriptional activity of catenin have long been considered a promising therapeutic recourse to block oncogenic Wnt signaling in CRC. Heretofore, progress in this area has been limited by lack of mechanistic insight concerning how -catenin activates gene transcription in the nucleus.
In this regard, we recently showed that -catenin activates transcription through its direct physical and functional interaction with the MED12 subunit in Mediator, a conserved multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Within Mediator, MED12 nucleates the assembly of a discrete “kinase” module that also includes MED13, CyclinC (CycC), and CDK8, a newly identified oncoprotein required for -catenin-dependent gene activation and CRC cell growth. Notably, CDK8 amplification occurs in 20-40% of CRCs and identifies those with poor prognosis, while missense mutations in MED12 exon 2 were recently identified at low frequency in CRC. These findings identify the Mediator kinase module as a target of oncogenic activation leading to dysregulated Wnt/catenin signaling. Our current studies are focused on the mechanism by which the Mediator kinase module transduces oncogenic information conveyed by constitutively activated catenin. We expect these studies to have important basic and translational implications for Mediator biology and Wnt/-catenin-driven cancers, including CRC.
- Turunen, M., Spaeth, J.M, Keskitalo, S., Park, M.J., Kivioja, T., Clark, A.D., Makinen, N., Gao, F., Vaharautio, A., Aavlkko, M., Kampjarvi, K., Nurkkala, H., Vahteristo, P., Kim, C.A., Aaltonen, L.A., Varjosalo, M., Taipaile, J., and Boyer, T.G. (2014) Uterine-leiomyoma-linked MED12 mutations disrupt Mediator-associated CDK activity. Cell Rep 7: 654-660. PMCID: PMC4041330
*Featured Highlight: Alderton, G.K. (2014) Transcription: Mediating tumorigenesis. Nat Rev Cancer 14: 382. PMID: 24854073
- Kampjarvi, K., Park, M.J., Mehine, M., Kim, N.H., Clark, A.D., Butzow, R., Bohling, T., Bohm, J., Mecklin, J.-P., Jarvinen, H., Tomlinson, I.P.M., van de Spuy, Z.M., Sjoberg, J., Boyer, T.G., and Vahteristo, P. (2014) Mutations in exon 1 highlight the role of MED12 in uterine leiomyomas. Hum Mut 35: 1136-1141.
- Ding, N., Zhou, H., Esteve, P.-E., 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. PMCID: PMC2583939
*Featured Previewed Article: Malik, S. and Roeder, R.G. (2008) Epigenetics? Mediator does that too! Mol Cell 31: 305-306. PMID: 18691961
- Zhou, H., Spaeth, J.M., Kim, N.H., Xu, X., Friez, M.J., Schwartz, C.E., and Boyer, T.G. (2012) MED12 mutations link intellectual disability syndromes with dysregulated GLI3-dependent Sonic hedgehog signaling. Proc Natl Acad Sci, USA 109: 19763-19768. PMCID: PMC3511715
*Featured Previewed Article: Berk, A.J. (2012) Yin and yang of mediator function revealed by human mutants. Proc Natl Acad Sci, USA 109: 19519-19520 PMCID: PMC3511750
- Vulto-van Silfhout, A.T., de Vries, B.B, van Bon B.W., Hoischen, A., Ruiterdamp-Versteeg, M., Gilissen, C., Gao, G., van Zwam, M., Harteveld, C.L., van Essen, A.J., Hamel, B.C., Kleefstra, T., Willemsen, M.A, Yntema, H.G., van Bokhoven, H., Brunner, H.G., Boyer, T.G, and de Brouwer, A.P. (2013) Mutations in MED12 cause X-linked Ohdo syndrome. Am J Hum Genet 92: 401-406. PMCID: PMC3591845
- Xu, X., Zhou, H., and Boyer T.G. (2011) Mediator is a transducer of amyloid-precursor-protein-dependent nuclear signaling. EMBO Rep12: 216-222. PMCID: PMC3059912
*Featured Previewed Article: <>Turner, A.J. et al. (2011) Mediator: the missing link in amyloid precursor protein signaling EMBO Rep 12: 180-181. PMCID: PMC3059916
- Kim, S., Xu, X., Hecht, A, and Boyer, T.G. (2006) Mediator is a transducer of Wnt/-catenin signaling. J Biol Chem 281: 14066-14075. PMID: 16565090
Other relevant publications
Boyer, T.G., Martin, M.E.D., Lees, E., Ricciardi, R.P., and Berk, A.J. (1999) Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein. Nature 399: 276-279. PMID: 10353252
*Featured Previewed Article: Kingston, R.E. (1999) A shared but complex bridge. Nature 399: 199-200.
Spaeth, J.M., Kim, N.H., and Boyer T.G. (2011) Mediator and human disease. Semin Cell Dev Biol 22: 776-787. PMID: 21840410