MED23 Mutation Links Intellectual Disability to Dysregulation of Immediate Early Gene Expression

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Science  26 Aug 2011:
Vol. 333, Issue 6046, pp. 1161-1163
DOI: 10.1126/science.1206638


MED23 is a subunit of the Mediator complex, a key regulator of protein-coding gene expression. Here, we report a missense mutation (p. R617Q) in MED23 that cosegregates with nonsyndromic autosomal recessive intellectual disability. This mutation specifically impaired the response of JUN and FOS immediate early genes (IEGs) to serum mitogens by altering the interaction between enhancer-bound transcription factors (TCF4 and ELK1, respectively) and Mediator. Transcriptional dysregulation of these genes was also observed in cells derived from patients presenting with other neurological disorders linked to mutations in other Mediator subunits or proteins interacting with MED. These findings highlight the crucial role of Mediator in brain development and functioning and suggest that altered IEG expression might be a common molecular hallmark of cognitive deficit.

Mammalian Mediator (MED) is an evolutionary conserved multiprotein complex composed of more than 20 subunits that form four distinct modules (fig. S1, upper scheme) (13). This large complex is a key regulator of gene expression that functions as an adaptor and conveys essential information from transcription factors (TFs) bound at upstream responsive elements to the basal RNA polymerase II (Pol II) transcription machineries (4, 5). Mediator also plays a key role in sensing developmental and environmental signals to deliver adequate outputs to the transcription machinery (6), and specific subunits are likely to be dedicated to fine-tuning of distinct expression programs. Reports linking Mediator subunits to human diseases may thus allow a deeper understanding of how cell-specific expression programs are regulated.

We ascertained a large Algerian consanguineous multiplex family in which five affected individuals presented with nonsyndromic intellectual disability (ID) (Fig. 1A). Genetic analyses identified a disease-causing mutation (c.1850G>A, p.R617Q) within the MED23 gene (NM_015979.2) that encodes one of the tail module’s Mediator subunits (Fig. 1B) [see supporting online material (SOM) text]. This variation cosegregated with the disease and was not present in dbSNP build 132, nor was it detected in any of 608 control chromosomes, including 242 chromosomes from individuals of Algerian origin. Moreover, R617 was absolutely conserved across all MED23 orthologs, from Xenopus to Drosophila and human. This mutation does not affect MED23 expression, protein stability or architecture, or composition of the whole Mediator complex (see SOM text and fig. S1).

Fig. 1

Identification of the MED23 mutation in an intellectually disabled family and characterization of MED23/R617Q mutated protein in Mediator complex. (A) Pedigree of the family. Filled symbols indicate affected individuals and slashes, deceased individuals (B) Electrophoregrams showing the c.1850G>A variation in the MED23 sequence in an affected individual and a healthy control. (C to E) Time-course analysis of EGR1 (C), JUN (D), and FOS (E) expression after serum addition to serum-starved wild-type (WT), M23/R617Q, and M23/R617Q cells transfected with MED23 cDNA. The values of three independent experiments are shown relative to serum-starved cells at time 0. *P < 0.05 and **P < 0.01.

MED23 was originally identified as a genetic suppressor of a hyperactive ras phenotype in Caenorhabditis elegans (7) and mediates the immediate early gene (IEG) response to serum mitogens (8). Gene expression analysis by microarray experiments after serum addition to serum-starved control- (wild type) and patient-derived (M23/R617Q) cultured skin fibroblasts revealed a difference in the serum response of a small number of genes (SOM and fig. S2). We thus further evaluated the effect of this mutation on JUN, FOS, and EGR1 expression by quantitative reverse transcription–polymerase chain reaction (RT-PCR). Expression of EGR1 was unchanged in response to serum addition in both wild-type and M23/R617Q cells (Fig. 1C). However, in response to serum, JUN expression was down-regulated and FOS expression was up-regulated in M23/R617Q cell compared to controls (Fig. 1, D and E). M23/R617Q cells engineered to express wild-type MED23 protein responded to serum treatment as did wild-type cells [EGR1 expression was unchanged; JUN and FOS RNA syntheses were restored to control levels (Fig. 1)]. Finally, the R617Q mutation had no effect on transcriptional activation induced by other stimuli such as ultraviolet (UV) irradiation or nuclear receptor activation, suggesting that it only causes a selective impairment of a subset of transcription activation processes (see SOM text and fig. S3).

To test whether dysregulation of IEG expression in M23/R617Q cells was the result of defective recruitment of transcriptional factors, we next performed chromatin immunoprecipitation (ChIP) experiments with antibodies directed against various proteins involved in the formation of the transcriptional preinitiation complex (PIC). ChIP showed decreased recruitment of MED proteins to the JUN promoter region in M23/R617Q cells compared with controls (Fig. 2, A to C). Reduced amounts of bound TFIIH (CDK7) and p-Tef-b complex (CDK9) were also observed at the JUN promoter in M23/R617Q cells (Fig. 2, E and F). These two complexes are responsible for the Ser2 and Ser5 phosphorylation of the Pol II C-terminal domain (CTD) and play a key role in promoting its escape into the productive elongation phase (9). Although recruitment of Pol II to the promoter appears equally effective in wild-type or M23/R617Q cells (Fig. 2D), Pol II at the JUN promoter in M23/R617Q cells showed defective phosphorylation (fig. S4, A and B). The aberrant phosphorylation of Pol II diminished its ability to initiate transcription and to further escape into the productive elongation phase (fig. S4C). Pol II at the FOS promoter was indistinguishable between wild-type and M23/R617Q fibroblasts (Fig. 2, J to O).

Fig. 2

Response of IEGs to serum in WT and M23/R617Q skin fibroblasts. ChIP analysis using primer sets specific for JUN (A to I) and FOS promoters (J to R) was performed on the chromatin fraction from WT and M23/R617Q cells that were untreated (−) or treated with serum for 10 min (+). Values from real-time PCR were normalized to the percentage of input chromatin. Error bars represent the SD of at least two independent experiments. *P < 0.05 and **P < 0.01.

Mediator couples chromatin remodeling and PIC formation (10). Acetylation of histone H3 lysine 9 (H3K9Ac) or di- and trimethylation of histone H3 lysine 4 (H3K4M) are common hallmarks of transcription activation, whereas methylation of histone H3 lysine 9 (H3K9M) has been shown to correlate with transcriptional repression (11). ChIP using antibodies raised against H3K9Ac, H3K4M, or H3K9M showed that cells carrying the R617Q mutation displayed reduced H3K9 acetylation and increased H3K9 methylation (but no change in H3K4 methylation) compared with controls (Fig. 2, G to I), suggesting impaired chromatin remodeling in the vicinity of the JUN promoter in M23/R617Q cells. No such differences were noted at the FOS promoter (Fig. 2, P to R).

We next investigated the effect of the R617Q mutation on the binding of several transcription factors on JUN and FOS promoter regions. The JUN promoter includes several AP1, SP1, and MEF2/RSRF proximal responsive elements, as well as a distal TCF regulatory element located 3 kb upstream of the transcription start site (Fig. 3, upper scheme) (12, 13). Phosphorylated JUN (JUN-P), MEF2, and SP1 proteins are normally present on JUN promoter upon serum induction of wild-type and M23/R617Q cells (Fig. 3, A to C). Serum treatment of wild-type cells also caused TCF4 recruitment at its responsive distal TCF element (Fig. 3D), and ChIP revealed incidental binding of TCF4 to the proximal promoter (Fig. 3E) owing to the formation of a chromatin loop (SOM text and fig. S5) (13, 14). By contrast, TCF4 recruitment at both distal and proximal promoters was impaired in M23/R617Q cells (Fig. 3, D and E). These results, combined with the observation that TCF4 coprecipitated with the wild-type MED23 subunit (Fig. 3I), led us to conclude that Mediator is required to link enhancer-bound TCF4 with the basal transcription machinery, a linkage that fails in the presence of the MED23/R617Q mutation.

Fig. 3

Binding of transcription activators to IEG promoter regions. ChIP experiments using primer sets specific for JUN proximal promoter (A to C and E) or distal promoters (D) and FOS promoter (F to H) were performed on the chromatin fraction from WT and M23/R617Q cells that were untreated (−) or treated with serum for 10 min (+). Values from real-time PCR were normalized to the percentage of input chromatin. Error bars represent the SD of at least two independent experiments. * P < 0.05 and ** P < 0.01. (I) Coimmunoprecipitation experiments showing interaction between MED23 and TCF4 or ELK1 in HeLa cells.

FOS transcription is also controlled by AP1 sites and is mediated by cooperative binding of serum response factor (SRF) and Ets ternary transcription factors (TCFs) [ELK1, SAP1 (ELK4), and NET (SAP2/ERP/ELK3)] to serum response elements (SREs) (15). The MED23 mutation does not affect binding of JUN-P to the AP1 sites in the FOS promoter (Fig. 3F). By contrast, we observed reduced binding of ELK1, which interacts with MED23 (Fig. 3I) (16), to the FOS promoter in M23/R617Q cells (Fig. 3G), and increased binding of its related paralog ELK3 (Fig. 3, G and H). Notably, these data are consistent with previous analyses in mouse demonstrating that ELK3 was the predominant ternary complex in mouse fibroblasts and that loss of MED23 nearly eliminated activation by ELK1 (17). Taken together, our results suggest that the dysregulation of FOS expression observed in M23/R617Q cells upon serum induction is due to altered constitution of what remains nonetheless a functional PIC.

IEG expression affects brain development and plasticity (18). We questioned whether abnormal serum-induced JUN and FOS expression could be also observed in neurological disorders caused by mutations in genes encoding other Mediator subunits or proteins interacting with MED. Mutations of MED12 are responsible for two allelic forms of X-linked ID syndromes: Opitz-Kaveggia syndrome [Mendelian Inheritance in Man (MIM) 305450] and Fryns-Lujan syndrome (MIM309520) (19, 20). Mutations in the XPD subunit of TFIIH, which interacts with Mediator (Fig. 4A) (2123), result in the autosomal recessive disorder diseases xeroderma pigmentosum (XP-D, MIM278730) and trichothiodystrophy (TTD, MIM601675) (24). Approximately 30% of XP patients have progressive neurological degeneration, and all TTD cases are mentally retarded. Similar EGR1 expression (Fig. 4, B and E) but impaired regulation of JUN and FOS expression upon serum induction was observed in lymphoblastoid cell lines derived from an Opitz-Kaveggia patient (M12-L; Fig. 4, C and D) and in skin fibroblasts derived from a XP-D case (Fig. 4, F and G). M12-L and XP-D cells also showed impaired transcriptional activation induced by other stimuli such as UV irradiation or nuclear receptor ligands (fig. S6) (25, 26). Fibroblasts derived from a TTD patient exhibited a partial up-regulation of EGR1 and a down-regulation of JUN expression (Fig. 4, E and F). As a control, no difference was observed in fibroblasts derived from a XP patient who presented with multiple skin cancers but no neurological abnormalities and who carried a mutation in the xeroderma pigmentosum group C protein (XP-C, MIM279720) (Fig. 4, H to J).

Fig. 4

Defective IEG expression in other neurological disorders. (A) Mediator subunits MED12 and MED23 were coimmunoprecipitated with TFIIH subunits CDK7 and p62 using 500-μg HeLa extracts. Induction of EGR1 (B, E, H), JUN (C, F, I), and FOS (D, G, J) expression after serum treatment of lymphoblastoid cell lines derived from a healthy subject (WT-L) or an Opitz-Kaveggia patient (M12-L), and of skin fibroblasts derived from a healthy subject (WT), a patient with xeroderma pigmentosum group D (XP-D), a patient with trichothiodystrophy (TTD), and a patient with xeroderma pigmentosum group C (XP-C). Values are shown relative to nontreated cells. Error bars represent the SD of three independent experiments.

We have shown that a single MED23 mutation may have distinct effects on transactivation complex formation and opposite consequences for gene expression owing to impaired interaction between mutated MED23 protein and cognate specific DNA binding protein (fig. S7). This study underlines, therefore, the key role of the MED23 subunit in organizing protein interactions within the transactivation complex.

The nonsyndromic ID identified here correlates with transcriptional defects in the IE genes. However, patients with syndromic conditions showed disruptions in a larger spectrum of processes, including nuclear receptor–mediated response and UV-induced gene expression.

IEGs may couple short-term neuronal activity with changes in gene transcription of neurons. Moreover, JUN and FOS are implicated in learning and consolidation of a long-term memory trace (27, 28). We thus propose that the intellectual disability observed in patients with Mediator and TFIIH mutations could be, at least in part, the result of impaired fine-tuning of IEG expression during development. We propose that altered IEG expression might provide a molecular signature for cognitive deficits.

Supporting Online Materials

Materials and methods

SOM Text

Figs. S1 to S7

Table S1

References (2936)

References and Notes

  1. Acknowledgments: We are grateful to the patients and their families for their participation in the study. We thank J. P. Jais for LOD-score calculation. Work in the J.-M. Egly’s laboratory was supported by a European Research Council Advanced Grant 2009, the French National Research Agency (no. ANR-08-GENOPAT-042), and the Association pour la Recherche sur le Cancer (SL220100601335). L. Colleaux’s laboratory was supported in part by the Centre National de la Recherche Scientifique and the French National Research Agency (no. ANR-08-MNP-010). S.H. was supported by an INSERM young investigator grant. S.B. was supported by the Région Ile-de France, the Fondation pour la Recherche Médicale, and the Fondation Jerome Lejeune. The authors declare that they have no conflicts of interest.
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