COMMENTARY

Yin and yang of function revealed by mutants

Arnold J. Berk1 Department of Microbiology, Immunology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095

ediator is an ∼30-subunit multiprotein complex ap- M proximately twice the size of the 12-subunit RNA poly- merase (i.e., RNA polymerase II [Pol II]) that transcribes most human . Me- diator functions as a molecular bridge between Pol II bound at start sites (TSSs) and activation and repression domains of regulatory sequence-specific DNA binding transcription factors (TFs) bound to enhancers and promoter proxi- mal transcription control sequences (1–5) (Fig. 1). In PNAS, Zhou et al. (6) report a significant advance in understanding how mutations in one Mediator subunit, MED12, result in two types of X-linked intellectual disability known as FG and Lujan syndromes. Studies of the mutant Mediators reveal how MED12 interactions with different components of the tran- scriptional machine produce opposing ac- fl tivating and inhibitory in uences necessary Fig. 1. Diagram representing simultaneous binding of several regulatory TF activation domains to for the proper level of transcription re- different surfaces of the Mediator. dsDNA is represented by a thin blue line and histone octamers by quired for normal development. orange discs. Regulatory TFs bind to specific DNA sequences at promoter proximal regions (bottom left Interactions between the Mediator of Mediator) and distal enhancers (top of Mediator) via their DNA-binding domains (blue), which are complex and regulatory TFs control linked through flexible regions of polypeptide to activation domains (green). In response to interactions several processes that regulate tran- with activation domains, Mediator binds to RNA Pol II and promotes binding of the polymerase and its scription, including chromatin modifica- general TFs to the TSS. [Reprinted with permission from W. H. Freeman and Company (21).] tion in the region of the promoter, which fl – in uences binding of TFs to DNA (7 9) The importance of Mediator function common with the unicellular ancestor of (interconversion between condensed and for transcription control raises the expec- all eukaryotic cells that are needed to decondensed chromatin, Fig. 1), the as- tation that mutations in genes encoding sustain cellular life. Rather, they are re- sembly at the TSS of a preinitiation com- fi fi human Mediator subunits might have quired for the ne control of gene ex- plex composed of Pol II and its ve profound effects on embryonic develop- pression needed to execute the genetic initiation factors (called general TFs) (10– ment. Accordingly, complete KO of mouse program that underlies embryological de- 12), the frequency of reinitiation by Pol II Mediator subunits results in embryonic velopment. KO of Med1 leads to profound and its general TFs (1–5), and, at a large lethality. In the earliest study in mammals, defects in embryonic development, in- fraction of promoters in multicellular ani- KO of the mouse Mediator subunit cluding an abnormally functioning heart, mals, release from a pause in transcription resulting in embryonic death (17). Med23 by elongating Pol II in the promoter Med22, which is highly conserved in Me- ∼ diator complexes from all eukaryotes (14), KO embryos have less profound devel- proximal region 50 bp from the TSS (13). opmental defects, but defects in remodel- In current models, the Mediator complex and essential for viability of yeast (15), caused embryonic lethality at the early ing of the vasculature prevent the normal integrates positive and negative signals of flow of red blood cells, probably account- varying strength from multiple regulatory blastocyst stage, consistent with a pro- fi found defect in transcription (16). In ing for death of the embryo (18). TFs bound to speci c sites in enhancer and Although these extreme mutations in promoter-proximal DNA control elements contrast, KO of subunits such as Med1 (17) and Med23 (18), which have diverged Mediator subunits result in embryonic le- to determine the rate of initiation by RNA thality, more subtle mutations in Pol II, and hence the amount of mRNA considerably in sequence during the evo- have been discovered because they allow expressed and translated into . In lution of multicellular organisms (14), development of viable fetuses to term, the simplest model, regulatory domains of were not incompatible with cell viability but present clinically after birth as a result TFs bind simultaneously to various surfa- because KO embryos grew to tens of of developmental abnormalities (19) ces of the large mediator complex to in- thousands of cells. However, the mutant fluence the rate of transcription (Fig. 1). embryos die at about embryonic day 9.5, However, it is equally possible that signals when they become large enough to require from sequential interactions between Me- a functioning circulatory system to provide Author contributions: A.J.B. wrote the paper. diator and activators and/or repressors are oxygen and nutrients throughout the em- The author declares no conflict of interest. integrated to control transcription initia- bryo (17, 18). These Mediator subunits are See companion article on page 19763. tion and elongation. not required for expression of genes in 1E-mail: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1217267109 PNAS | November 27, 2012 | vol. 109 | no. 48 | 19519–19520 Downloaded by guest on September 24, 2021 Table 1. Human Mediator subunit mutations linked to developmental disorders callosum defects, are also observed in pa- Disorder Associated mutation tients with mutations in GLI3. Conse- quently, they analyzed the impact of the X-linked mental retardation syndromes FG and Lujan MED12 mutations on FG syndrome MED12 R961W transcription of genes activated by SHH Lujan syndrome MED12 N1007S signaling and on the association of CDK8 Infantile cerebral and cerebellar atrophy MED17 L371P with several promoters through ChIP as- Autosomal recessive axonal MED25 A335V says. They find that GLI3 target genes, but Charcot–Marie–Tooth disease not genes regulated by several other signal Congenital retinal folds, microcephaly, CDK19 haploinsufficiency transduction pathways, are greatly over- and mental retardation expressed in response to SHH signaling in Transposition of the great arteries MED13L haploinsufficiency; MED13L cells from patients with FG and Lujan (E251G; R1872H; S2023G) syndrome (6). ChIP assays showed that References and in-depth discussion are provided by Spaeth et al. (19). CDK8 was depleted at GLI3-regulated promoters, but not at other promoters. Moreover, in experiments with cells in (Table 1). Zhou et al. (6) report on ex- plasticity of synaptic connections between which the endogenous CDK8 or MED12 periments with cells cultured from patients differentiated neurons. Earlier research were depleted by siRNAs, CDK8 enzy- with FG syndrome or Lujan syndrome by Ding et al (8) had shown that the FG matic kinase activity and the WT MED12 that begin to explain the transcriptional and Lujan MED12 mutations disrupt re- sequence were required to constrain tran- abnormalities in these patients and also pression of genes by the TF REST through scriptional activation by GLI3 in response reveal how an interplay between positive dimethylation, followed by trimethylation to SHH signaling. These observations lead and negative inputs to the Mediator result of histone H3 lysine 9 (H3K9me3). to a model in which the proper level of in the appropriate level of transcription H3K9me3 is a binding site for hetero- transcriptional activation by SHH signal- required for normal development. chromatin (e.g., HP1s), and gen- ing results from a balance of activating FG and Lujan syndromes are rare syn- erates a chromatin structure that blocks influences from the binding of the GLI3 dromes characterized by mental retar- access of TFs to their binding sites in DNA activation domain to MED12 and in- dation, dysgenesis of the corpus callosum, (Fig. 1, “Condensed chromatin”). As a hibition by CDK8 phosphorylation of deafness, seizures, and behavioral dis- consequence, REST is a master regulator some component of the multiprotein turbances, and are often associated with of neuronal cell differentiation by inhibit- transcription machine. The MED12 anorectal and urogenital malformations ing expression of genes that induce neu- mutations in FG and Lujan syndromes and congenital heart defects in FG syn- ronal differentiation in proliferating greatly diminish the inhibitory influence drome. Genetic analysis of the recessive neural progenitor cells and in differenti- of CDK8, resulting in abnormally high mutations revealed that FG and Lujan ated nonneuronal cells. transcriptional activation of genes syndromes are associated with amino acid Zhou et al (6) report that the MED12 targeted by the SHH signal transduction substitutions R961W and N1007S, re- amino acid substitutions associated with spectively, in Mediator subunit MED12. FG and Lujan syndromes also lead to pathway. This overactivation of SHH The MED12 subunit is required for nor- misregulation by the GLI3 TF in response signaling likely contributes to the de- mal gene control in response to signaling to SHH signaling. MED12 is a component velopmental abnormalities observed in from neighboring cells by Notch, Wnt, and of a four-subunit Mediator “kinase mod- these syndromes. Sonic hedgehog (SHH) protein ligands. ule” including MED12, MED13, CDK8, We can hope that further mapping of These ligands bind to regulatory receptors and cyclin C. CDK8, the protein kinase human mutations resulting in develop- that span the cell membrane and elicit subunit of the module, interacts with mental abnormalities will uncover addi- intracellular responses to the extracellular MED12. GLI3, a TF whose activity is tional mutations in Mediator subunits, and signals. Notch, Wnt, and SHH have critical regulated by SHH signaling, was shown that careful analysis of the mutant Medi- functions in central nervous system de- earlier by the same group to bind directly ators will be as revealing about unexpected velopment, from controlling the develop- to MED12 (20). Zhou et al. (6) realized mechanisms of Mediator function as the ment of brain morphology and neuronal that several of the phenotypes of the FG FG and Lujan syndrome MED12 mutants cell differentiation to regulation of the and Lujan syndromes, such as corpus have been (6, 8).

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