Linking Activators and Basals in Transcription

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Linking Activators and Basals in Transcription: It Is All in One Family

Analysis of nuclear hormone receptor function in cells derived from xeroderma pigmentosum patients reveals novel links between DNA repair and transcription and points to novel mechanisms of regulating transcriptional regulators. The central principle of biology is that there are very few principles which last over the years. This is particularly true for the study of eukaryotic transcription regulation. Until recently, mRNA synthesis by RNA polymerase II (pol II) was considered to be a linear process. The activator binds to a specific promoter sequence and recruits coactivator complexes to this promoter. These complexes collaborate to activate the chromatinized promoter to receive basal transcription components including pol II itself. Transcription commences once the basal transcription factor TFIIH opens the DNA template and propels pol II into the production of mRNA. This principle allowed grouping of transcription factors into three functional families with separated tasks: genespecific activators, activator-recruited coactivators, and basal pol II machinery. The linearity of this pathway has been challenged by functional links between nuclear receptor function and the cdk7 subunit of TFIIH (Rochette-Egly et al., 1997). A study published in the April 5 issue of Cell (Keriel et al., 2002) reveals that the positive feedback loop between members of two families, the nuclear receptor for retinoic acid and the kinase component of TFIIH, is disrupted in cells from a particular group of xeroderma pigmentosum (XP) patients. The connection between transcription and DNA repair continues to surprise us. XP is an inherited disease with defects in nucleotideexcision repair to correct DNA damage. While the characteristic phenotype of XP is an increased risk to skin cancers, some patients also suffer from additional developmental disorders unrelated to defective DNA repair. Identification of two XP genes (XPD and XPB) as components of the basal transcription factor TFIIH suggested that developmental disorders resulted from a malfunctioning of the transcription apparatus (Frit et al., 1999). TFIIH is the last basal factor to join the pol II transcription initiation complex. ATP hydrolysis by the XPB subunit, which resembles a DNA helicase, drives promoter opening and promoter clearance (Dvir et al., 2001; Fiedler and Timmers, 2000). While essential for the DNA-repair activity of TFIIH, the XPD DNA helicase serves a structural rather than an enzymatic role in transcription. It links the Cdk activating kinase (CAK) complex to core TFIIH. CAK is a trimeric complex of cdk7, cyclin H, and MAT1 (Frit et al., 1999), which can also use the repeated CTD structure of pol II as a substrate.

Not surprisingly, TFIIH has been found as a target for interactions with gene-specific activators. These interactions can lead both to activated as well as to repressed levels of gene transcription (Liu et al., 2001). Nuclear hormone receptors (NHRs) are gene-specific activator proteins, which mediate the effects of lipophilic hormones like retinoids, vitamin D, or steroid hormones. These activators bind as dimers to their DNA target, and typically they contain two separate transcription activation domains (AF-1 and AF-2). AF-2 also carries the ligand binding domain, and its activation function is hormone dependent. Many NHRs are subjected to modification by a variety of different kinases, and this can have different effects on NHR function (Shao and Lazar, 1999). The ␣-isoform of the retinoic acid receptor (RAR␣) can be phosphorylated within AF-1 on Ser77 by the cdk7-subunit of CAK (Rochette-Egly et al., 1997). Involvement of other TFIIH subunits in NHR function remained obscure until the new study of Egly and coworkers, which investigates XP cells (HD2) carrying a mutation in XPD (Keriel et al., 2002). They report that activity of several NHRs is diminished in HD2 cells but can be restored by exogenous expression of wild-type XPD. Isolation and characterization of the mutant TFIIH complex from HD2 cells indicates that its in vitro basal transcription function is not affected but that CAK association is sensitive to conditions of moderate ionic strength. A link to RAR␣ phosphorylation comes from the observation that Ser77 of RAR␣ is hypophosphorylated in HD2 cells and that this can be restored by introduction of wild-type protein. Substitution of the serine for glutamic acid restores RAR␣ function, which remains hormone dependent. Defective RAR␣ function is observed in two independent XPD cells carrying different alleles. Common to both XPD mutants (R683W and R722W) is a weakened anchoring of CAK into TFIIH. These new findings clearly implicate the whole TFIIHcomplex in the phosphorylation and activation function of RAR␣. Although they offer an explanation for the developmental disorders observed in some XPD patients, a caveat is that HD2 cells are derived from a patient who displayed a classical XP phenotype without additional abnormalities. However, the R722W mutation was isolated from a patient with trichothiodystrophy, which is a developmental disorder. Many mutations in XPD have been isolated, but a genotype-phenotype correlation has turned out to be very complicated (de Boer and Hoeijmakers, 2000). The Egly paper provides a basis for the analysis of additional XPD alleles. Exact knowledge of their transcriptional defects may even point to intervention strategies based on hormone therapy. What could be the mechanism by which Ser77 phosphorylation by TFIIH adds to the transcriptional output of RAR␣? Assuming that promoter binding of the liganded NHR precedes TFIIH association to the promoter, several possibilities come to mind. RAR␣ phosphorylation could serve to stabilize the preinitiation complex or to allow association of additional components, which render the first rounds of transcription more efficient. It is also possible that cdk7-mediated phosphorylation stim-

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ulates subsequent rounds of initiation by a more efficient recruitment of the pol II machinery (e.g., TFIIH itself; Rochette-Egly et al., 1997). In this respect, it is noteworthy that activation of the estrogen-receptor (ER) results in two subsequent waves of ER binding and of transcription of estrogen-responsive genes (Shang et al., 2000). Another intriguing possibility is that TFIIH phosphorylation of NHRs serves to mark these receptors for ubiquitination. Ligand binding increases turnover of these proteins. An increased turnover of transcriptional activators has been linked to an increased activation, and activators can be marked by phosphorylation for ubiquitindependent proteosomal degradation (Tansey, 2001). In this scheme, TFIIH-mediated phosphorylation could result in degradation of NHR bound to promoters in the first wave of transcription. One could even speculate that degradation of a liganded NHR is necessary for subsequent rounds of transcription. Whichever model is true, it is clear that linearity of the transcription activation process is under siege and that the convenient classification of transcription factors into functional families proves deceiving. Many of the steps in mRNA synthesis are connected (Orphanides and Reinberg, 2002), and transcription regulation should be viewed as an integrated and dynamic process.

RfaH, a Bacterial Transcription Antiterminator The bacterial transcription antiterminator RfaH has been shown to act, in a purified biochemical system, by binding both RNA polymerase and the nontemplate strand of DNA at the regulatory site ops (Artsimovitch and Landick, 2002). Although most transcription regulators determine whether RNA polymerase can initiate RNA synthesis at a promoter, critical modifications also control the persistence of polymerase during elongation through genes. Thus, an essential component of eukaryotic RNA polymerase II activation provides escape from pauses or terminators at promoter-proximal regions and likely facilitates elongation through downstream barriers (Conaway et al., 2000). For the bacterial RNA polymerase, both general and gene-specific regulatory factors modify its elongation properties. The former include the nearly universally conserved and essential transcription factors NusA and NusG, which modify the rate of transcription, possibly serving to coordinate translation with transcription; in addition, the starvation regulatory nucleotide ppGpp, best known as the effector of stringent regulation at stable RNA promoters, also generally decreases the rate of elongation (Vogel and Jensen, 1997). More specific regulators act to drive transcription through terminators and other barriers (see Table): these factors include the bacteriophage ␭ antiterminators Q

H.Th. Marc Timmers Laboratory for Physiological Chemistry University Medical Centre-Utrecht P.O. Box 80560 3508 AB Utrecht The Netherlands

Selected Reading de Boer, J., and Hoeijmakers, J.H.J. (2000). Carcinogenesis 21, 453–460. Dvir, A., Conaway, J.W., and Conaway, R.C. (2001). Curr. Opin. Genet. Dev. 11, 209–214. Fiedler, U., and Timmers, H.T.M. (2000). Bioessays 22, 316–326. Frit, P., Bergmann, E., and Egly, J.-M. (1999). Biochimie 81, 27–38. Keriel, A., Stary, A., Sarasin, A., Rochette-Egly, C., and Egly, J.-M. (2002). Cell 109, 125–135. Liu, J., Akoulitchev, S., Weber, A., Ge, H., Chuikov, S., Libutti, D., Wang, X.W., Conaway, J.W., Harris, C.C., Conaway, R.C., et al. (2001). Cell 104, 353–363. Orphanides, G., and Reinberg, D. (2002). Cell 108, 439–451. Rochette-Egly, C., Adam, S., Rossignol, M., Egly, J.-M., and Chambon, P. (1997). Cell 90, 97–107. Shang, Y., Hu, X., DiRenzo, J., Lazar, M.A., and Brown, M. (2000). Cell 103, 843–852. Shao, D., and Lazar, M.A. (1999). J. Clin. Invest. 103, 1617–1618. Tansey, W.P. (2001). Genes Dev. 15, 1045–1050.

(Roberts et al., 1998) and N (DeVito and Das, 1994; Greenblatt et al., 1998), a bacterial ribosomal RNA antitermination system related to N (Condon et al., 1995), a unique RNA-based antiterminator of the bacteriophage HK022 named put (Banik-Maiti et al., 1997), and the RfaH protein (Bailey et al., 1997)—the subject of a new report in the April 19 issue of Cell by Artsimovitch and Landick (2002). The relative simplicity of the bacterial systems and the new atomic structures of RNA polymerase raise expectations for understanding how antiterminators work at the molecular level—partly fulfilled for RfaH in this new analysis that reveals important steps in its mechanism of action. RfaH and its homologs promote expression of operons encoding proteins targeted to the cell surface or membrane, including the hemolysin operon of pathogenic E. coli and the tra operon responsible for assembly of the F pilus (Bailey et al., 1997). Mutations in rfaH decrease promoter distal but not promoter proximal gene expression, a property diagnostic of elongation regulation. Like the protein antiterminators (Q, N, and the ribosomal RNA system), RfaH has an genetic site essential for its activity, a ⵑ12 base pair conserved sequence (5⬘-RGGCGGTAGYnT-3⬘) in the promoter-proximal 5⬘ untranslated region of the operon termed ops (operon polarity suppressor); deletion of ops leads to the same polar expression pattern seen with rfaH⫺ strains. ops induces a transcriptional pause both in vivo (Leeds and Welch, 1997) and in vitro (Artsimovitch and Landick, 2000), suggesting that RfaH engages RNAP at this pause by binding a specific signal in DNA or RNA—reminiscent of the action of ␭ Q protein. In the recent paper, Artsimovitch and Landick (2002) present