Mediator Subcomplex Research Articles

Factors targeting MED12 to drive tumorigenesis?

Mediator Subcomplex 12 (MED12) is part of the transcriptional preinitiation machinery. Mutations of its gene predominantly occur in two types of highly frequent benign tumors, uterine leiomyomas and fibroadenomas of the breast, where they apparently act as driver mutations. Nevertheless, their presence is not restricted to benign tumors having been found at considerable frequencies in uterine leiomyosarcomas, malignant phyllodes tumors, and chronic lymphocytic leukemia also. Most of the mutations are located within exon 2 of the gene but in rare cases the intron 1/exon 2 boundary or exon 1 are affected. As to their type, predominantly single nucleotide exchanges with a hotspot in one codon are found, but small deletions clustering around that hotspot also are not uncommon. According to their presumed classification as gain-of-function mutations, these latter deletions are leaving the open reading frame intact. As to the types of mutations, so far no apparent differences between the tumor entities affected have emerged. Interestingly, this pattern with small deletions clustered around the hotspot of single nucleotide exchanges resembles that seen as a result of targeted gene editing. In contrast to other driver mutations the percentage of MED12-mutation positive tumors of independent clonal origin increases with the number of tumors per patient suggesting unknown etiological factors supporting site specific mutagenesis. These factors may act by inducing simultaneous site-specific double strand breaks the erroneous repair of which may lead to corresponding mutations. As inducers of DNA damage and its repair such as foreign nucleic acids of the microbiome displaying sequence homology to the putative target site might play a role. Interestingly, a 16 base pair homology of the hotspot to a putative terminator base-paired hairpin sequence of a Staphylococcus aureus tRNA gene cluster has been noted which might form R-loop like structures with its target sequence thus inducing said changes.

Cytogenetically normal uterine leiomyomas without MED12-mutations - a source to identify unknown mechanisms of the development of uterine smooth muscle tumors.

BackgroundRecent findings on genetic changes in uterine leiomyomas suggest these benign tumors being a heterogeneous group of diseases in terms of molecular pathogenesis with those showing karyotype alterations as well as those characterized only by cytogenetically invisible mutations of mediator subcomplex 12 (MED12). Herein, five uterine leiomyomas (UL) with an apparently normal karyotype that lacked MED12-mutations were investigated by copy number variation arrays along with their matching myometrium to search for small genomic imbalances.ResultsOf five tumors one showed chromothripsis-like phenomena with numerous gains and losses of small segments mainly clustered to five chromosomal regions i.e. 2p14-2pter, 2q33.1-2q37.3, 5q31.3-5qter,11q14.1-11qter, and 18p11.21-18q2.3. Apparently, these cells had escaped detection by classical cytogenetics. Histologically, the tumor presented as a cellular leiomyoma with extended hyalinization. Of the remaining four tumors, one had a small intragenic deletion of the HMGA2 gene that was lacking in the corresponding myometrium. The other three tumors did not show relevant copy number alterations at all.ConclusionsOverall, the results suggest that leiomyomas with an apparently normal karyotype based on classical cytogenetics and lacking MED12 mutations represent a heterogeneous group of diseases. While the HMGA2 deletion detected in one of the tumors likely represents the driver mutation and, due to its size, has escaped detection by classical cytogenetics, the extended genomic imbalances detected in one of the other cases cannot be overlooked by this method suggesting an inability of the affected cells to divide in vitro. Of particular interest in that case is the occurrence of so-called “chromothripsis” or “firestorms” without involvement of the loci of common chromosomal rearrangements in UL, as e.g. 12q14 ~ 15 and 6p21. While chromothripsis was initially described as a hallmark of malignancy, the etiology and significance of this phenomenon in benign tumors still remain obscure. In uterine smooth muscle tumors, these changes per se do not indicate malignancy.

Uterine fibroids: do we deal with more than one disease?

Uterine fibroids rank among the most frequent symptomatic human tumors at all. Recent data suggest that mutations of the mediator subcomplex 12 gene (MED12) and rearrangements of the gene-encoding high-mobility group protein AT-hook 2 (HMGA2) characterize major genetic subtypes of these tumors, which, for example, differ by their average size. Herein, we have investigated a total of 289 fibroids from 120 patients. Of these fibroids, 256 were fully genetically analyzed. Of the latter group, 20 (7.8%) fibroids had a chromosomal rearrangement of 12q14-15 reflecting a rearranged allele of HMGA2 and 179 (69.9%) fibroids had a mutation of MED12. The remaining tumors had either another genetic abnormality or no detectable abnormality at all. We were able to demonstrate that tumors of both groups also display striking differences of their frequency in individual patients. Whereas 70.0% (14/20) HMGA2-mutated fibroids made their appearance as solitary nodules, 85.5% (153/179) MED12-mutated fibroids occurred as multiple nodules as a rule of independent clonal origin, as reflected by different MED12 mutations. These findings are likely to point to a different pathogenesis of both types of fibroids. In the predominant of these groups so far, an unknown "mutator" may cause independent mutations of MED12, resulting in an independent clonal outgrowth of nodules. Furthermore, the low but existing risk of MED12-mutated fibroids to undergo malignant transformation after a leiomyoma-STUMP (smooth muscle tumors of uncertain malignant potential)-leiomyosarcoma sequence excludes the latter mutation as a suitable stand-alone marker for benign growth.

The CD24hi smooth muscle subpopulation is the predominant fraction in uterine fibroids

Uterine fibroids are the most common gynecological tumors affecting women in their reproductive age. Despite this high incidence the pathogenesis of fibroids is widely unsolved. Whereas formerly only imbalances in hormonal levels were considered to account for tumor development, the identification of genetic changes likely to affect myometrial stem cell reservoirs provided a novel approach to fibroid genesis. Here, we identified a certain subset of cells by the surface marker CD24 with increased abundance in fibroids compared with myometrial tissue. Fibroid cells expressing CD24 shared certain features of immature or progenitor-like cells such as quiescence, reduced expression of smooth muscle differentiation markers and elevated expression of genes involved in the wingless-type (WNT)-pathway such as beta-catenin. In addition, a positive correlation between CD24 and wingless-type family member 4 (WNT4) expression was observed in uterine fibroids with mediator subcomplex 12 gene (MED12) mutations. Our findings suggest that cells highly expressing CD24 represent a type of immature smooth muscle progenitor cells. Their accumulation might be driven by disturbed differentiation processes caused by genetic changes possibly involving MED12 mutations or high mobility group AT-hook (HMGA)2 rearrangements.

Molecular topography of the MED12-deleted region in smooth muscle tumors: a possible link between non-B DNA structures and hypermutability

BackgroundDeletions of the gene encoding mediator subcomplex 12 (MED12) in human smooth muscle tumors rank among the most frequent genomic alterations in human tumors at all. In a minority of these cases, small deletions are found. In an attempt to delineate key features of the deletions aimed at a better understanding of the molecular pathogenesis of uterine smooth muscle tumors we have analyzed 70 MED12 deletions including 46 cases from the literature and 24 own unpublished cases.ResultsThe average length of the deletions was 18.7 bp ranging between 2 bp and 43 bp. While in general multitudes of 3 clearly dominated leaving the transcript in frame, deletions of 21, 24, 30, and 33 nucleotides were clearly underrepresented. Within the DNA segment affected deletion breakpoints were not randomly distributed. Most breakpoints clustered within the center of the segment where two peaks of breakpoint clusters could be distinguished. Interestingly, one of these clusters coincides with the loop of a putative folded non-B DNA structure whereas a much lower number of breaks noted in the 5′ and 3′ stem of the structure forming an intramolecular B-helix. The second cluster mainly consisting of 3′ breaks was located in a region downstream adjacent to the stem.ConclusionThe present study describes for the first time main characteristics of MED12 deletions occurring in smooth muscle tumors. Interestingly, the non-random distribution of breakpoints within the deletion hotspot region may point to a role of non-canonical DNA structures for the occurrence of these mutations and the molecular pathogenesis of uterine smooth muscle tumors, respectively.

A conserved Mediator–CDK8 kinase module association regulates Mediator–RNA polymerase II interaction

The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) carboxy-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a preeminent complex in eukaryotic transcription regulation. We used macromolecular electron microscopy and biochemistry to investigate the subunit organization, structure, and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator’s Middle module interferes with CTD-dependent RNAPII binding to a previously unknown Middle module CTD-binding site targeted early on in a multi-step holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD, and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the Mediator mechanism.

MED12 mutations occurring in benign and malignant mammalian smooth muscle tumors

Mutations of the mediator subcomplex 12 gene (MED12) recently have been described in a large group of uterine leiomyomas (UL) but only in a single malignant uterine smooth muscle tumor. To further address the occurrence of fibroid-type MED12 mutations in smooth muscle tumors, we have analyzed samples from 34 leiomyosarcomas (LMS), 21 UL, two extrauterine leiomyomas (EL), and 10 canine genital leiomyomas for the presence of MED12 mutations of the UL-type. Interestingly, besides UL MED12 mutations were found in one uterine LMS, one EL, and two canine vaginal leiomyomas. The results confirm the occurrence of fibroid-type MED12 mutations in malignant uterine smooth muscle tumors thus suggesting a rare but existing leiomyoma-LMS sequence. In addition, for the first time MED12 mutations are reported in smooth muscle tumors in a non-primate mammalian species.

GATA-1-Dependent and -Independent Modes of Establishing the Erythroid Cell Genetic Network.

Abstract 2083The constant physiological demand to generate large numbers of red blood cells requires a complex genetic network established by the master regulatory transcription factor GATA-1, which orchestrates erythroblast survival, proliferation, and differentiation. Many questions remain regarding how GATA-1 instigates genetic networks and to what extent GATA-1-independent mechanisms regulate erythropoiesis. Med1, a component of the broadly expressed Mediator complex (Mediator), facilitates GATA-1-dependent transcriptional activation at select target genes, although its contribution to GATA-1 function in cell-based assays is considerably less than that of the cell type-specific coregulator Friend of GATA-1. Med1-nullizygous mice have hematopoietic, cardiac, and vascular defects, though the underlying mechanisms are not defined. Furthermore, whether Med1 coactivator activity is dedicated to GATA-1 in erythroid cells and whether it controls numerous or a restricted cohort of genes is also unclear. Using a genetic complementation assay in GATA-1-null erythroid cells and a functional genomics approach, we demonstrate that Med1 regulates a restricted gene ensemble in erythroid cells, consisting predominantly of genes not controlled by GATA-1. Of the 265 Med1-regulated genes and 1054 GATA-1-regulated genes, only 35 genes were regulated by Med1 and GATA-1. Given the preponderance of GATA-1-independent Med1 targets, it is attractive to propose that Med1 has important GATA-1-independent functions required to exert its crucial hematopoietic activities. Since Med1 is a Mediator subunit, it is presumed to function through Mediator to regulate target genes. However, Med1 interacts with various trans-acting factors, and therefore its gene regulatory activity may not invariably rely on Mediator or a Mediator subcomplex. As Mediator is largely unstudied in erythroid cells, we asked whether Mediator subunit expression is regulated upon primary human erythroid cell maturation ex vivo. Mining the Human Erythroblast Maturation Database revealed that Med25 is strongly up-regulated during maturation. Knockdown of Med25 significantly dysregulated all ten of the highest responding Med1 target genes. Simultaneous knockdowns of Med1 and Med25 altered expression of 9 of the 10 top Med1 target genes, resembling the individual factor knockdowns. These results support the hypothesis that Med1 and Med25 function in the erythroid Mediator complex to regulate these genes. Med1 regulated these genes in a cell type-specific manner, as 8 of the 10 top Med1 targets in G1E-ER-GATA-1 proerythroblast-like cells and Mouse Erythroleukemia Cells were not dysregulated upon Med1 knockdown in Mouse Embryonic Fibroblasts. As Med1 modulated, but was not essential for, GATA-1-dependent transcription, we reasoned that certain Med1 target genes may exert GATA-1-independent activities to control erythroid cell development and/or function. The Med1 target gene Rrad encodes a small GTPase induced during primary human erythroid cell maturation, but its regulation/function has not been described in the hematopoietic system. Loss-of-function analysis in G1E-ER-GATA-1 cells indicated that Rrad confers survival. Knocking-down Rrad increased early apoptosis 2.5 fold (p < 0.05). The Rrad requirement for survival was more pronounced when cells were deprived of Erythropoietin (Epo) and Stem Cell Factor (SCF). In cells cultured without Epo, early apoptosis increased 7.0 fold upon Rrad knockdown [from 1.0% ± 0.1% to 7.2% ± 0.5% (p < 0.05)]. Removing SCF from the media significantly increased apoptotic cells, and Rrad knockdown elevated this further from 28% ± 2.4% to 46% ± 2.8% (p < 0.01), while the number of live cells decreased 4.7 fold (p < 0.01). These studies established a dual role for Mediator in erythroid cell regulation as a context-dependent GATA-1 coregulator and a GATA-1-independent regulator of cell type-specific genes, including potentially critical regulators of erythroid cell development, survival, and function. Mechanistically, given the greater than twenty components of the canonical Mediator, it will be particularly instructive to compare our findings to that of other key Mediator components, which shall yield a comprehensive understanding of their regulation and function during the progressive transitions from erythroid precursors to the erythrocyte. Disclosures:No relevant conflicts of interest to declare.

MED12 mutations in uterine fibroids—their relationship to cytogenetic subgroups

Recurrent chromosomal alterations are found in roughly 20% of all uterine fibroids but in the majority cytogenetic changes are lacking. Recently, mutations of the gene mediator subcomplex 12 (MED12) have been detected in a majority of fibroids but no information is available whether or not they co-occur with cytogenetic subtypes as, e.g., rearrangements of the genes encoding high mobility group AT-hook (HMGA) proteins. In a total of 80 cytogenetically characterized fibroids from 50 patients, we were not only able to confirm the frequent occurrence of MED12 mutations but also to stratify two mutually exclusive pathways of leiomyomagenesis with either rearrangements of HMGA2 reflected by clonal chromosome abnormalities affecting 12q14~15 or by mutations affecting exon 2 of MED12. On average the latter mutations were associated with a significantly smaller tumor size. However, G>A transitions of nucleotides c.130 or c.131 correlate with a significantly larger size of the fibroids compared to other MED12 mutations thus explaining the high prevalence of the former mutations among clinically detectable fibroids. Interestingly, fibroids with MED12 mutations expressed significantly higher levels of the gene encoding wingless-type MMTV integration site family, member 4 (WNT4). Based on these findings and data from the literature, we hypothesize that estrogen and the mutated MED12 cooperate in activating the Wnt pathway which in turn activates β-catenin known to cause leiomyoma-like lesions in a mouse model. The occurrence of a "fibroid-type mutation" in a rare histologic subtype of endometrial polyps suggests that this mechanism is not confined to uterine leiomyomas.

Cdk8 Regulates Stability of the Transcription Factor Phd1 To Control Pseudohyphal Differentiation of Saccharomyces cerevisiae

The yeast Saccharomyces differentiates into filamentous pseudohyphae when exposed to a poor source of nitrogen in a process involving a collection of transcription factors regulated by nutrient signaling pathways. Phd1 is important for this process in that it regulates expression of most other transcription factors involved in differentiation and can induce filamentation on its own when overproduced. In this article, we show that Phd1 is an unstable protein whose degradation is initiated through phosphorylation by Cdk8 of the RNA polymerase II mediator subcomplex. Phd1 is stabilized by cdk8 disruption, and the naturally filamenting Σ1278b strain was found to have a sequence polymorphism that eliminates a Cdk8 phosphorylation site, which both stabilizes the protein and contributes to enhanced differentiation. In nitrogen-starved cells, PHD1 expression is upregulated and the Phd1 protein becomes stabilized, which causes its accumulation during differentiation. PHD1 expression is partially dependent upon Ste12, which was also previously shown to be destabilized by Cdk8-dependent phosphorylations, but to a significantly smaller extent than Phd1. These observations demonstrate the central role that Cdk8 plays in initiation of differentiation. Cdk8 activity is inhibited in cells shifted to limiting nutrient conditions, and we argue that this effect drives the initiation of differentiation through stabilization of multiple transcription factors, including Phd1, that cause activation of genes necessary for filamentous response.

Cyclin-Dependent Kinase 8 Positively Cooperates with Mediator To Promote Thyroid Hormone Receptor-Dependent Transcriptional Activation

Mediator is a multisubunit assemblage of proteins originally identified in humans as a coactivator bound to thyroid hormone receptors (TRs) and essential for thyroid hormone (T3)-dependent transcription. Cyclin-dependent kinase 8 (CDK8), cyclin C, MED12, and MED13 form a variably associated Mediator subcomplex (termed the CDK8 module) whose functional role in TR-dependent transcription remains unclear. Using in vitro and cellular approaches, we show here that Mediator complexes containing the CDK8 module are specifically recruited into preinitiation complexes at the TR target gene type I deiodinase (DioI) together with RNA polymerase II (Pol II) in a TR- and T3-dependent manner. We found that CDK8 is essential for robust T3-dependent Dio1 transcription and that CDK8 knockdown via RNA interference decreased Pol II occupancy, and also the recruitment of the Pol II kinase CDK9, at the DioI promoter. Chromatin immunoprecipitation revealed CDK8 occupancy at the DioI promoter concurrent with active transcription, thus suggesting CDK8 involvement in transcriptional reinitiation. Mutagenesis assays showed that CDK8 kinase activity is necessary for full T3-dependent DioI activation, whereas in vitro kinase studies indicated that CDK8 may contribute to Pol II phosphorylation. Collectively, our data suggest CDK8 plays an important coactivator role in TR-dependent transcription by promoting Pol II recruitment and activation at TR target gene promoters.

Mechanistic studies of the mRNA transcription cycle

We have now completed an atomic crystallographic model of the 12-subunit yeast RNA polymerase II in elongation mode, with DNA and RNA in the active-centre cleft, and the NTP substrate at the growing end of the RNA. From these studies has emerged a detailed three-dimensional view of mRNA elongation. We have extended this structural analysis to a polymerase elongation complex bound by the transcript cleavage factor TFIIS (transcription factor IIS), which is required for polymerase escape from DNA arrest sites. A detailed model of this complex reveals a single tuneable active site for RNA polymerization and cleavage, and changes in the position of the RNA and polymerase domains, reflecting the dynamic nature of the elongation complex. An additional structure of a polymerase CTD (C-terminal domain) phosphopeptide bound by the 3'-RNA processing factor Pcf11 provides insights into the coupling of transcription elongation to mRNA processing. The structure of the CTD phosphatase Scp1 trapped in an intermediary enzymatic state explains CTD dephosphorylation during recycling of the polymerase. We also recently reported the first crystal structure of a Mediator subcomplex, which reveals an extended helical fold with a conserved hinge.

DEAD-box RNA helicase Sub2 is required for expression of lacZ fusions in Saccharomycescerevisiae and is a dosage-dependent suppressor of RLR1 ( THO2)

RLR1 ( THO2) encodes a novel, phylogenetically-conserved KEKE motif protein involved in transcription and transcription-associated recombination in Saccharomyces cerevisiae. One characteristic aspect of RLR1 function is its requirement for expression of the Escherichia coli lacZ reporter gene regardless of the yeast promoter to which it is fused. rlr1-1 was originally isolated (employing lacZ as a transcriptional reporter) as a suppressor of a mutation in the gene encoding Sin4, a subunit of the Mediator subcomplex of the RNA polymerase II (PolII) transcriptional machinery. To clarify the function of Rlr1, we performed a genetic screen for dosage-dependent suppressors of the cold-sensitive phenotype of rlr1-1. From this screen we isolated SUB2, encoding a conserved DEAD-box RNA helicase family member having roles in both pre-mRNA splicing and mRNA export in yeast, flies, and humans. We demonstrate that Sub2, like Rlr1, is required for lacZ to be expressed in yeast, and that sub2 mutants manifest rlr1-like growth defects. Our results are consistent with a hypothesis where expression of lacZ fusions in yeast preferentially requires a Sub2-mediated mRNP assembly/export pathway linked to transcription via Rlr1.

The structural and functional organization of the yeast mediator complex.

The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects of transcription by RNA polymerase II (pol II). Mediator is composed of two functionally distinct subcomplexes, Rgr1 and Srb4. To identify the structures and functions of each subcomplex, we expressed recombinant proteins for each subunit and assayed their interactions with each other and with basal transcription proteins. The Rgr1 subcomplex is composed of the Gal11 module, which binds activators, and the Med9/10 module. The Med9/10 module is required for both transcriptional activation and repression, and these activities appear to be carried out by two submodules. Proteins in the Med9 submodule interact physically and genetically with Srb10/11, suggesting that the Med9 submodule mediates the repression of pol II. Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II. Both subcomplexes of Mediator interacted with a distinct set of basal transcription factors and pol II. The modular organization of Mediator and the associated functions suggest that the Mediator complex may recruit and/or stabilize the preinitiation complex through several points of contact with transcriptional regulators and basal transcription factors.

Negative regulation of transcription by the yeast global transcription factors, Gal11 and Sin4.

Gal11 and Sin4 proteins are yeast global transcription factors that regulate transcription of a variety of genes, both positively and negatively. Gal11, in a major part, functions in the activation of transcription, whereas Sin4 has an opposite role, yet they are reported to be present as a complex in the so-called RNA polymerase II holoenzyme. To reveal howthese auxiliary factors participate in switching transcription on and off, a complex formation between Gal11 and Sin4 and its effect on the negative regulation of transcription were studied. Using an artificial promoter that is negatively regulated by Gal11, it was shown that the presence of Sin4 or Pgd1/Hrs1/Med3 was required for Gal11 to repress both basal and activated transcription. Genetic and biochemical studies using a temperature-sensitive Gal11 mutant showed that the amino acid region 866-910 essential for Gal11 function was also important for repression of transcription and a complex formation with Sin4. Analysis with dam methylase accessibility to the promoter region suggested that nucleosome structure may be involved in negative regulation. Based on these results, possible mechanisms by which a mediator subcomplex regulates transcription is discussed.

Multiple signals regulate GAL transcription in yeast.

Gal4p activates transcription of the Saccharomyces GAL genes in response to galactose and is phosphorylated during interaction with the RNA polymerase II (Pol II) holoenzyme. One phosphorylation at S699 is necessary for full GAL induction and is mediated by Srb10p/CDK8 of the RNA Pol II holoenzyme mediator subcomplex. Gal4p S699 phosphorylation is necessary for sensitive response to inducer, and its requirement for GAL induction can be abrogated by high concentrations of galactose in strains expressing wild-type GAL2 and GAL3. Gal4p S699 phosphorylation occurs independently of Gal3p and is responsible for the long-term adaptation response observed in gal3 yeast. SRB10 and GAL3 are shown to represent parallel mechanisms for GAL gene induction. These results demonstrate that Gal4p activity is controlled by two independent signals: one that acts through Gal3p-galactose and a second that is mediated by the holoenzyme-associated cyclin-dependent kinase Srb10p. Since Srb10p is regulated independently of galactose, our results suggest a function for CDK8 in coordinating responses to specific inducers with the environment through the phosphorylation of gene-specific activators.

RLR1 (THO2), required for expressing lacZ fusions in yeast, is conserved from yeast to humans and is a suppressor of SIN4

We isolated a mutation (rlr1-1; required for lacZ RNA) in the Saccharomyces cerevisiae (Sc) RLR1 gene as a suppressor of sin4, a component of the Mediator subcomplex of the RNA polymerase II holoenzyme and a determinant of chromatin structure. RLR1 encodes a deduced protein found also in fission yeast, nematode worms, and humans. The presence of these orthologs suggests that Rlr1 family members comprise a class of putative KEKE motif-containing proteins, characteristic of certain chaperones as well as regulators and subunits of the mammalian 20S proteasome. A role for RLR1 (THO2) in transcription appears to occur at a step subsequent to transcription initiation (see also Piruat, J.I. and Aguilera, A., 1998. EMBO J. 17, 4859-4872); Sc genes fused to the reporter gene lacZ were expressed at a very low level, while the corresponding native chromosomal genes were expressed at approximately normal levels in rlr1 mutants. Our studies show that rlr1 mutations cause a wide range of growth defects in addition to their novel affect on lacZ.

An activator binding module of yeast RNA polymerase II holoenzyme.

The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.

Association of an activator with an RNA polymerase II holoenzyme.

RNA polymerase II holoenzymes have been described that consist of RNA polymerase II, a subset of general transcription factors, and four SRB proteins. The SRB proteins, which were identified through a selection for genes involved in transcription initiation by RNA polymerase II in vivo, are a hallmark of the holoenzyme. We report here the isolation and characterization of additional SRB genes. We show that the products of all nine SRB genes identified thus far are components of the RNA polymerase II holoenzyme and are associated with a holoenzyme subcomplex termed the mediator of activation. The holoenzyme is capable of responding to a transcriptional activator, suggesting a model in which activators function, in part, through direct interactions with the holoenzyme. Immunoprecipitation experiments with anti-SRB5 antibodies demonstrate that the acidic activating domain of VP16 specifically binds to the holoenzyme. Furthermore, the holoenzyme and the mediator subcomplex bind to a VP16 affinity column. These results provide a more complete description of the RNA polymerase II holoenzyme and suggest that this form of the transcription apparatus can be recruited to promoters via direct interactions with activators.