Super-enhancer-driven expression of BAHCC1 promotes melanoma cell proliferation and genome stability.

Super-enhancers (SEs) are stretches of enhancers ensuring a high level of expression of key genes associated with cell function. The identification of cancer-specific SE-driven genes is a powerful means for the development of innovative therapeutic strategies. Here, we identify a MITF/SOX10/TFIIH-dependent SE promoting the expression of BAHCC1 in a broad panel of melanoma cells. BAHCC1 is highly expressed in metastatic melanoma and is required for tumor engraftment, growth, and dissemination. Integrative genomics analyses reveal that BAHCC1 is a transcriptional regulator controlling expression of E2F/KLF-dependent cell-cycle and DNA-repair genes. BAHCC1 associates with BRG1-containing remodeling complexes at the promoters of these genes. BAHCC1 silencing leads to decreased cell proliferation and delayed DNA repair. Consequently, BAHCC1 deficiency cooperates with PARP inhibition to induce melanoma cell death. Our study identifies BAHCC1 as an SE-driven gene expressed in melanoma and demonstrates how its inhibition can be exploited as a therapeutic target.

Active mRNA degradation by EXD2 nuclease elicits recovery of transcription after genotoxic stress.

The transcriptional response to genotoxic stress involves gene expression arrest, followed by recovery of mRNA synthesis (RRS) after DNA repair. We find that the lack of the EXD2 nuclease impairs RRS and decreases cell survival after UV irradiation, without affecting DNA repair. Overexpression of wild-type, but not nuclease-dead EXD2, restores RRS and cell survival. We observe that UV irradiation triggers the relocation of EXD2 from mitochondria to the nucleus. There, EXD2 is recruited to chromatin where it transiently interacts with RNA Polymerase II (RNAPII) to promote the degradation of nascent mRNAs synthesized at the time of genotoxic attack. Reconstitution of the EXD2-RNAPII partnership on a transcribed DNA template in vitro shows that EXD2 primarily interacts with an elongation-blocked RNAPII and efficiently digests mRNA. Overall, our data highlight a crucial step in the transcriptional response to genotoxic attack in which EXD2 interacts with elongation-stalled RNAPII on chromatin to potentially degrade the associated nascent mRNA, allowing transcription restart after DNA repair.

Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions.

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.

Promoters of ASCL1- and NEUROD1-dependent genes are specific targets of lurbinectedin in SCLC cells.

Small-Cell Lung Cancer (SCLC) is an aggressive neuroendocrine malignancy with a poor prognosis. Here, we focus on the neuroendocrine SCLC subtypes, SCLC-A and SCLC-N, whose transcription addiction was driven by ASCL1 and NEUROD1 transcription factors which target E-box motifs to activate up to 40% of total genes, the promoters of which are maintained in a steadily open chromatin environment according to ATAC and H3K27Ac signatures. This leverage is used by the marine agent lurbinectedin, which preferentially targets the CpG islands located downstream of the transcription start site, thus arresting elongating RNAPII and promoting its degradation. This abrogates the expression of ASCL1 and NEUROD1 and of their dependent genes, such as BCL2, INSM1, MYC, and AURKA, which are responsible for relevant SCLC tumorigenic properties such as inhibition of apoptosis and cell survival, as well as for a part of its neuroendocrine features. In summary, we show how the transcription addiction of these cells becomes their Achilles's heel, and how this is effectively exploited by lurbinectedin as a novel SCLC therapeutic endeavor.

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.

Defective transcription of ATF3 responsive genes, a marker for Cockayne Syndrome.

Cockayne syndrome (CS) is a rare genetic disorder caused by mutations (dysfunction) in CSA and CSB. CS patients exhibit mild photosensitivity and severe neurological problems. Currently, CS diagnosis is based on the inefficiency of CS cells to recover RNA synthesis upon genotoxic (UV) stress. Indeed, upon genotoxic stress, ATF3, an immediate early gene is activated to repress up to 5000 genes encompassing its responsive element for a short period of time. On the contrary in CS cells, CSA and CSB dysfunction impairs the degradation of the chromatin-bound ATF3, leading to a permanent transcriptional arrest as observed by immunofluorescence and ChIP followed by RT-PCR. We analysed ChIP-seq of Pol II and ATF3 promoter occupation analysis and RNA sequencing-based gene expression profiling in CS cells, as well as performed immunofluorescence study of ATF3 protein stability and quantitative RT-PCR screening in 64 patient cell lines. We show that the analysis of few amount (as for example CDK5RAP2, NIPBL and NRG1) of ATF3 dependent genes, could serve as prominent molecular markers to discriminate between CS and non-CS patient's cells. Such assay can significantly simplify the timing and the complexity of the CS diagnostic procedure in comparison to the currently available methods.

A chromatin scaffold for DNA damage recognition: how histone methyltransferases prime nucleosomes for repair of ultraviolet light-induced lesions.

The excision of mutagenic DNA adducts by the nucleotide excision repair (NER) pathway is essential for genome stability, which is key to avoiding genetic diseases, premature aging, cancer and neurologic disorders. Due to the need to process an extraordinarily high damage density embedded in the nucleosome landscape of chromatin, NER activity provides a unique functional caliper to understand how histone modifiers modulate DNA damage responses. At least three distinct lysine methyltransferases (KMTs) targeting histones have been shown to facilitate the detection of ultraviolet (UV) light-induced DNA lesions in the difficult to access DNA wrapped around histones in nucleosomes. By methylating core histones, these KMTs generate docking sites for DNA damage recognition factors before the chromatin structure is ultimately relaxed and the offending lesions are effectively excised. In view of their function in priming nucleosomes for DNA repair, mutations of genes coding for these KMTs are expected to cause the accumulation of DNA damage promoting cancer and other chronic diseases. Research on the question of how KMTs modulate DNA repair might pave the way to the development of pharmacologic agents for novel therapeutic strategies.

TFIIE orchestrates the recruitment of the TFIIH kinase module at promoter before release during transcription.

In eukaryotes, the general transcription factors TFIIE and TFIIH assemble at the transcription start site with RNA Polymerase II. However, the mechanism by which these transcription factors incorporate the preinitiation complex and coordinate their action during RNA polymerase II transcription remains elusive. Here we show that the TFIIEα and TFIIEß subunits anchor the TFIIH kinase module (CAK) within the preinitiation complex. In addition, we show that while RNA polymerase II phosphorylation and DNA opening occur, CAK and TFIIEα are released from the promoter. This dissociation is impeded by either ATP-γS or CDK7 inhibitor THZ1, but still occurs when XPB activity is abrogated. Finally, we show that the Core-TFIIH and TFIIEß are subsequently removed, while elongation factors such as DSIF are recruited. Remarkably, these early transcriptional events are affected by TFIIE and TFIIH mutations associated with the developmental disorder, trichothiodystrophy.

Activation of P-TEFb by RBM7: To Live or Let Die.

In this issue of Molecular Cell, Bugai et al. (2019) unveil that a key step of the pro-survival cellular response to a genotoxic attack is the activation of P-TEFb by RBM7. This crucial step triggers RNA polymerase II release from promoter-proximal pausing and expression of DNA damage response genes.

Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression.

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.

SerpinB2 is involved in cellular response upon UV irradiation.

Ultraviolet light induced pyrimidine dimer is a helix distortion DNA damage type, which recruits repair complexes. However, proteins of these complexes that take part in both DNA damage recognition and repair have been well-described, the regulation of the downstream steps of nucleotide excision repair (NER) have not been clearly clarified yet. In a high-throughput screen, we identified SerpinB2 (SPB2) as one of the most dramatically upregulated gene in keratinocytes following UV irradiation. We found that both the mRNA and the protein levels of SPB2 were increased upon UV irradiation in various cell lines. Additionally, UV damage induced translocation of SPB2 from the cytoplasm to the nucleus as well as the damage induced foci formation of it. Here we show that SPB2 co-localizes with XPB involved in the NER pathway at UV-induced repair foci. Finally, we demonstrated that UV irradiation promoted the association of SPB2 with ubiquitylated proteins. In basal cell carcinoma tumour cells, we identified changes in the subcellular localization of SPB2. Based on our results, we conclude that SPB2 protein has a novel role in UV-induced NER pathway, since it regulates the removal of the repair complex from the damaged site leading to cancerous malformation.

Is TFIIH the new Achilles heel of cancer cells?

TFIIH is a 10-subunit complex involved in transcription and DNA repair. It contains several enzymatic activities including a ATP-dependent DNA translocase in XPB and a cyclin-dependent kinase in CDK7. Recently the discovery of several XPB and CDK7 inhibitors with specific impact on the transcriptional addiction of many tumors pinpointed these activities as potential target in cancer chemotherapy. Unexpectedly a basal transcription factor involved in global mRNA expression now emerges a one of the most clinically promising Achilles heels of cancerous cells. These inhibitors also proved to be useful tools to unveil new functions of TFIIH in gene expression.

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.

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.

Lurbinectedin Specifically Triggers the Degradation of Phosphorylated RNA Polymerase II and the Formation of DNA Breaks in Cancer Cells.

We have defined the mechanism of action of lurbinectedin, a marine-derived drug exhibiting a potent antitumor activity across several cancer cell lines and tumor xenografts. This drug, currently undergoing clinical evaluation in ovarian, breast, and small cell lung cancer patients, inhibits the transcription process through (i) its binding to CG-rich sequences, mainly located around promoters of protein-coding genes; (ii) the irreversible stalling of elongating RNA polymerase II (Pol II) on the DNA template and its specific degradation by the ubiquitin/proteasome machinery; and (iii) the generation of DNA breaks and subsequent apoptosis. The finding that inhibition of Pol II phosphorylation prevents its degradation and the formation of DNA breaks after drug treatment underscores the connection between transcription elongation and DNA repair. Our results not only help to better understand the high specificity of this drug in cancer therapy but also improve our understanding of an important transcription regulation mechanism. Mol Cancer Ther; 15(10); 2399-412. ©2016 AACR.