The Long Road to Understanding RNAPII Transcription Initiation and Related Syndromes.

In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.

Nucleotide Excision Repair and Transcriptional Regulation: TFIIH and Beyond.

Transcription factor IIH (TFIIH) is a multiprotein complex involved in both transcription and DNA repair, revealing a striking functional link between these two processes. Some of its subunits also belong to complexes involved in other cellular processes, such as chromosome segregation and cell cycle regulation, emphasizing the multitasking capabilities of this factor. This review aims to depict the structure of TFIIH and to dissect the roles of its subunits in different cellular mechanisms. Our understanding of the biochemistry of TFIIH has greatly benefited from studies focused on diseases related to TFIIH mutations. We address the etiology of these disorders and underline the fact that TFIIH can be considered a promising target for therapeutic strategies.

Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression.

Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.

Cockayne's Syndrome A and B Proteins Regulate Transcription Arrest after Genotoxic Stress by Promoting ATF3 Degradation.

Cockayne syndrome (CS) is caused by mutations in CSA and CSB. The CSA and CSB proteins have been linked to both promoting transcription-coupled repair and restoring transcription following DNA damage. We show that UV stress arrests transcription of approximately 70% of genes in CSA- or CSB-deficient cells due to the constitutive presence of ATF3 at CRE/ATF sites. We found that CSB, CSA/DDB1/CUL4A, and MDM2 were essential for ATF3 ubiquitination and degradation by the proteasome. ATF3 removal was concomitant with the recruitment of RNA polymerase II and the restart of transcription. Preventing ATF3 ubiquitination by mutating target lysines prevented recovery of transcription and increased cell death following UV treatment. Our data suggest that the coordinate action of CSA and CSB, as part of the ubiquitin/proteasome machinery, regulates the recruitment timing of DNA-binding factors and provide explanations about the mechanism of transcription arrest following genotoxic stress.

TFIIH: when transcription met DNA repair.

The transcription initiation factor TFIIH is a remarkable protein complex that has a fundamental role in the transcription of protein-coding genes as well as during the DNA nucleotide excision repair pathway. The detailed understanding of how TFIIH functions to coordinate these two processes is also providing an explanation for the phenotypes observed in patients who bear mutations in some of the TFIIH subunits. In this way, studies of TFIIH have revealed tight molecular connections between transcription and DNA repair and have helped to define the concept of 'transcription diseases'.

CDK7 and MITF repress a transcription program involved in survival and drug tolerance in melanoma.

Melanoma cell phenotype switching between differentiated melanocytic and undifferentiated mesenchymal-like states drives metastasis and drug resistance. CDK7 is the serine/threonine kinase of the basal transcription factor TFIIH. We show that dedifferentiation of melanocytic-type melanoma cells into mesenchymal-like cells and acquisition of tolerance to targeted therapies is achieved through chronic inhibition of CDK7. In addition to emergence of a mesenchymal-type signature, we identify a GATA6-dependent gene expression program comprising genes such as AMIGO2 or ABCG2 involved in melanoma survival or targeted drug tolerance, respectively. Mechanistically, we show that CDK7 drives expression of the melanocyte lineage transcription factor MITF that in turn binds to an intronic region of GATA6 to repress its expression in melanocytic-type cells. We show that GATA6 expression is activated in MITF-low melanoma cells of patient-derived xenografts. Taken together, our data show how the poorly characterized repressive function of MITF in melanoma participates in a molecular cascade regulating activation of a transcriptional program involved in survival and drug resistance in melanoma.

Revisiting the Function of CDK7 in Transcription by Virtue of a Recently Described TFIIH Kinase Inhibitor.

In this issue of Molecular Cell, Nilson et al. (2015) took advantage of THZ1, a recently described covalent inhibitor of the TFIIH kinase CDK7, to further characterize the role of this enzyme in the early stages of transcription and postprocessing events. They unveiled an unexpected function of CDK7 in RNA polymerase II pausing and mRNA capping.

The Cockayne syndrome group A and B proteins are part of a ubiquitin-proteasome degradation complex regulating cell division.

Cytokinesis is monitored by a molecular machinery that promotes the degradation of the intercellular bridge, a transient protein structure connecting the two daughter cells. Here, we found that CSA and CSB, primarily defined as DNA repair factors, are located at the midbody, a transient structure in the middle of the intercellular bridge, where they recruit CUL4 and MDM2 ubiquitin ligases and the proteasome. As a part of this molecular machinery, CSA and CSB contribute to the ubiquitination and the degradation of proteins such as PRC1, the Protein Regulator of Cytokinesis, to ensure the correct separation of the two daughter cells. Defects in CSA or CSB result in perturbation of the abscission leading to the formation of long intercellular bridges and multinucleated cells, which might explain part of the Cockayne syndrome phenotypes. Our results enlighten the role played by CSA and CSB as part of a ubiquitin/proteasome degradation process involved in transcription, DNA repair, and cell division.

XPG and XPF endonucleases trigger chromatin looping and DNA demethylation for accurate expression of activated genes.

Nucleotide excision repair factors, initially characterized as part of DNA repair, have been shown to participate in the transcriptional process in the absence of genotoxic attack. However, their molecular function when recruited at the promoters of activated genes together with the transcription machinery remained obscure. Here we show that the NER factors XPG and XPF are essential for establishing CTCF-dependent chromatin looping between the promoter and terminator of the activated RARß2 gene. Silencing XPG and/or XPF endonucleases, or mutations in their catalytic sites, prevents CTCF recruitment, chromatin loop formation, and optimal transcription of RARß2. We demonstrated that XPG endonuclease promotes DNA breaks and DNA demethylation at promoters allowing the recruitment of CTCF and gene looping, which is further stabilized by XPF. Our results highlight a timely orchestrated activity of the NER factors XPG and XPF in the formation of the active chromatin hub that controls gene expression.

Poly (ADP-ribose) glycohydrolase regulates retinoic acid receptor-mediated gene expression.

Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.

MED12-related XLID disorders are dose-dependent of immediate early genes (IEGs) expression.

Mediator occupies a key role in protein coding genes expression in mediating the contacts between gene specific factors and the basal transcription machinery but little is known regarding the role of each Mediator subunits. Mutations in MED12 are linked with a broad spectrum of genetic disorders with X-linked intellectual disability that are difficult to range as Lujan, Opitz-Kaveggia or Ohdo syndromes. Here, we investigated several MED12 patients mutations (p.R206Q, p.N898D, p.R961W, p.N1007S, p.R1148H, p.S1165P and p.R1295H) and show that each MED12 mutations cause specific expression patterns of JUN, FOS and EGR1 immediate early genes (IEGs), reflected by the presence or absence of MED12 containing complex at their respective promoters. Moreover, the effect of MED12 mutations has cell-type specificity on IEG expression. As a consequence, the expression of late responsive genes such as the matrix metalloproteinase-3 and the RE1 silencing transcription factor implicated respectively in neural plasticity and the specific expression of neuronal genes is disturbed as documented for MED12/p.R1295H mutation. In such case, JUN and FOS failed to be properly recruited at their AP1-binding site. Our results suggest that the differences between MED12-related phenotypes are essentially the result of distinct IEGs expression patterns, the later ones depending on the accurate formation of the transcription initiation complex. This might challenge clinicians to rethink the traditional syndromes boundaries and to include genetic criterion in patients' diagnostic.

Variants in MED12L, encoding a subunit of the mediator kinase module, are responsible for intellectual disability associated with transcriptional defect.

PURPOSE: Mediator is a multiprotein complex that allows the transfer of genetic information from DNA binding proteins to the RNA polymerase II during transcription initiation. MED12L is a subunit of the kinase module, which is one of the four subcomplexes of the mediator complex. Other subunits of the kinase module have been already implicated in intellectual disability, namely MED12, MED13L, MED13, and CDK19. METHODS: We describe an international cohort of seven affected individuals harboring variants involving MED12L identified by array CGH, exome or genome sequencing. RESULTS: All affected individuals presented with intellectual disability and/or developmental delay, including speech impairment. Other features included autism spectrum disorder, aggressive behavior, corpus callosum abnormality, and mild facial morphological features. Three individuals had a MED12L deletion or duplication. The other four individuals harbored single-nucleotide variants (one nonsense, one frameshift, and two splicing variants). Functional analysis confirmed a moderate and significant alteration of RNA synthesis in two individuals. CONCLUSION: Overall data suggest that MED12L haploinsufficiency is responsible for intellectual disability and transcriptional defect. Our findings confirm that the integrity of this kinase module is a critical factor for neurological development.

Correction: Variants in MED12L, encoding a subunit of the Mediator kinase module, are responsible for intellectual disability associated with transcriptional defect.

In the Acknowledgements section of the paper the authors neglected to mention that the study was supported by a grant from the National Human Genome Research Institute (NHGRI) UM1HG007301 (S.H., M.L.T.). In addition, the award of MD was associated with the authors Michelle L. Thompson and Susan Hiatt instead of PhD. The PDF and HTML versions of the Article have been modified accordingly.

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals.

NER factors are recruited to active promoters and facilitate chromatin modification for transcription in the absence of exogenous genotoxic attack.

Upon gene activation, we found that RNA polymerase II transcription machinery assembles sequentially with the nucleotide excision repair (NER) factors at the promoter. This recruitment occurs in absence of exogenous genotoxic attack, is sensitive to transcription inhibitors, and depends on the XPC protein. The presence of these repair proteins at the promoter of activated genes is necessary in order to achieve optimal DNA demethylation and histone posttranslational modifications (H3K4/H3K9 methylation, H3K9/14 acetylation) and thus efficient RNA synthesis. Deficiencies in some NER factors impede the recruitment of others and affect nuclear receptor transactivation. Our data suggest that there is a functional difference between the presence of the NER factors at the promoters (which requires XPC) and the NER factors at the distal regions of the gene (which requires CSB). While the latter may be a repair function, the former is a function with respect to transcription unveiled in the current study.

A ubiquitin-binding domain in Cockayne syndrome B required for transcription-coupled nucleotide excision repair.

Transcription-coupled nucleotide excision repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSB(del)) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSB(del) remains capable of assembling nucleotide excision repair factors and repair synthesis proteins around damage-stalled RNAPII, but such repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process.

TFIIH-dependent MMP-1 overexpression in trichothiodystrophy leads to extracellular matrix alterations in patient skin.

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP.

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes.

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.

In TFIIH, XPD helicase is exclusively devoted to DNA repair.

The eukaryotic XPD helicase is an essential subunit of TFIIH involved in both transcription and nucleotide excision repair (NER). Mutations in human XPD are associated with several inherited diseases such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. We performed a comparative analysis of XPD from Homo sapiens and Chaetomium thermophilum (a closely related thermostable fungal orthologue) to decipher the different molecular prerequisites necessary for either transcription or DNA repair. In vitro and in vivo assays demonstrate that mutations in the 4Fe4S cluster domain of XPD abrogate the NER function of TFIIH and do not affect its transcriptional activity. We show that the p44-dependent activation of XPD is promoted by the stimulation of its ATPase activity. Furthermore, we clearly demonstrate that XPD requires DNA binding, ATPase, and helicase activity to function in NER. In contrast, these enzymatic properties are dispensable for transcription initiation. XPD helicase is thus exclusively devoted to NER and merely acts as a structural scaffold to maintain TFIIH integrity during transcription.

Dynamic partnership between TFIIH, PGC-1α and SIRT1 is impaired in trichothiodystrophy.

The expression of protein-coding genes requires the selective role of many transcription factors, whose coordinated actions remain poorly understood. To further grasp the molecular mechanisms that govern transcription, we focused our attention on the general transcription factor TFIIH, which gives rise, once mutated, to Trichothiodystrophy (TTD), a rare autosomal premature-ageing disease causing inter alia, metabolic dysfunctions. Since this syndrome could be connected to transcriptional defects, we investigated the ability of a TTD mouse model to cope with food deprivation, knowing that energy homeostasis during fasting involves an accurate regulation of the gluconeogenic genes in the liver. Abnormal amounts of gluconeogenic enzymes were thus observed in TTD hepatic parenchyma, which was related to the dysregulation of the corresponding genes. Strikingly, such gene expression defects resulted from the inability of PGC1-α to fulfill its role of coactivator. Indeed, extensive molecular analyses unveiled that wild-type TFIIH cooperated in an ATP-dependent manner with PGC1-α as well as with the deacetylase SIRT1, thereby contributing to the PGC1-α deacetylation by SIRT1. Such dynamic partnership was, however, impaired when TFIIH was mutated, having as a consequence the disruption of PGC1-α recruitment to the promoter of target genes. Therefore, besides a better understanding of the etiology of TFIIH-related disease, our results shed light on the synergistic relationship that exist between different types of transcription factors, which is necessary to properly regulate the expression of protein coding genes.