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Largely non-overlapping sets of cyclin-dependent kinases (CDKs) regulate cell division and RNA polymerase II (Pol II)-dependent transcription. Here we review the molecular mechanisms by which specific CDKs are thought to act at discrete steps in the transcription cycle and describe the recent emergence of transcriptional CDKs as promising drug targets in cancer. We emphasize recent advances in understanding the transcriptional CDK network that were facilitated by development and deployment of small-molecule inhibitors with increased selectivity for individual CDKs. Unexpectedly, several of these compounds have also shown selectivity in killing cancer cells, despite the seemingly universal involvement of their target CDKs during transcription in all cells. Finally, we describe remaining and emerging challenges in defining functions of individual CDKs in transcription and co-transcriptional processes and in leveraging CDK inhibition for therapeutic purposes. This Review provides insights into transcriptional regulation, and vulnerabilities of cancer cells to disruption of cyclin-dependent kinase (CDK)-mediated regulation of Pol II transcription, revealed with small-molecule CDK inhibitors.

Genomic deletion of the tumour suppressor TP53 frequently includes other neighbouring genes, such as the POLR2A housekeeping gene that encodes a crucial RNA polymerase II subunit; suppression of POLR2A with α-amanitin or by RNA interference selectively inhibits the tumorigenic potential of cancer cells, and in mouse models of cancer, tumours can be selectively targeted with α-amanitin coupled to antibodies, suggesting new therapeutic approaches for human cancers. Indirect targeting of TP53 The tumour suppressor gene TP53 is inactivated by mutation or deletion in a majority of human tumours. So far, attempts to restore the activity of its product, p53, have had little success owing to the complexity of p53 signalling. This paper suggests a new approach to targeting TP53 indirectly. Genomic deletion of TP53 frequently includes other neighbouring genes, such as the POLR2A housekeeping gene that encodes a crucial RNA polymerase II subunit. Xionbin Lu and colleagues show that loss of one copy of POLR2A renders cancer cells highly sensitive to inhibitors of RNA polymerase II, such as α-amanitin. In mouse models of cancer, tumours containing the POLR2A / TP53 co-deletion can be selectively targeted with α-amanitin conjugated to antibodies that target the cancer cells. Exploiting similar selective vulnerabilies for other genomic deletions that affect essential housekeeping in addition to tumour suppressor genes may pave a way towards selective therapies for a broad range of cancers. TP53 , a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours 1 , 2 . A tremendous effort has been made to restore p53 activity in cancer therapies 3 , 4 , 5 , 6 , 7 . However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin 8 , 9 . Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity 10 . However, we found that α-amanitin-based antibody–drug conjugates are highly effective therapeutic agents with reduced toxicity 11 . Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A . We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

Background: Retrotransposons play a central role in plant evolution and could be a powerful endogenous source of genetic and epigenetic variability for crop breeding. To ensure genome integrity several silencing mechanisms have evolved to repress retrotransposon mobility. Even though retrotransposons fully depend on transcriptional activity of the host RNA polymerase II (Pol II) for their mobility, it was so far unclear whether Pol II is directly involved in repressing their activity. Results: Here we show that plants defective in Pol II activity lose DNA methylation at repeat sequences and produce more extrachromosomal retrotransposon DNA upon stress in Arabidopsis and rice. We demonstrate that combined inhibition of both DNA methylation and Pol II activity leads to a strong stress-dependent mobilization of the heat responsive ONSEN retrotransposon in Arabidopsis seedlings. The progenies of these treated plants contain up to 75 new ONSEN insertions in their genome which are stably inherited over three generations of selfing. Repeated application of heat stress in progeny plants containing increased numbers of ONSEN copies does not result in increased activation of this transposon compared to control lines. Progenies with additional ONSEN copies show a broad panel of environment-dependent phenotypic diversity. Conclusions: We demonstrate that Pol II acts at the root of transposon silencing. This is important because it suggests that Pol II can regulate the speed of plant evolution by fine-tuning the amplitude of transposon mobility. Our findings show that it is now possible to study induced transposon bursts in plants and unlock their use to induce epigenetic and genetic diversity for crop breeding.

The centromere is a specialized chromatin region marked by the histone H3 variant CENP-A. Although active centromeric transcription has been documented for over a decade, the role of centromeric transcription or transcripts has been elusive. Here, we report that centromeric α-satellite transcription is dependent on RNA Polymerase II and occurs at late mitosis into early G1, concurrent with the timing of new CENP-A assembly. Inhibition of RNA Polymerase II-dependent transcription abrogates the recruitment of CENP-A and its chaperone HJURP to native human centromeres. Biochemical characterization of CENP-A associated RNAs reveals a 1.3 kb molecule that originates from centromeres, which physically interacts with the soluble pre-assembly HJURP/CENP-A complex in vivo, and whose down-regulation leads to the loss of CENP-A and HJURP at centromeres. This study describes a novel function for human centromeric long non-coding RNAs in the recruitment of HJURP and CENP-A, implicating RNA-based chaperone targeting in histone variant assembly. Before a cell divides, it copies its chromosomes. Initially, the two copies of each chromosome remain linked via their centromeres. These regions also serve as the attachment sites for the proteins that pull these two copies apart, and eventually segregate the chromosomes equally between the two newly formed cells. Chromosome segregation is the main function of centromeres; and in most organisms, the DNA in these regions is highly repetitive and is not thought to encode any proteins. However, it has been observed that cells need enzymes called RNA polymeraseswhich transcribe stretches of DNA into RNA moleculesto be able to separate the copies of their chromosomes correctly. This suggests that RNAs transcribed from centromeres might be required for cell division, but the identity and function of these RNAs remained elusive. Quénet and Dalal have now discovered that an RNA polymerase localizes to the DNA in human centromeres and produces RNA molecules during the early stages of the cell cycle. Two proteins–one called CENP-A and another that functions as its chaperone–that normally bind to the centromere and determine its structure were found less often in this region of the chromosome if the activity of the RNA polymerase was inhibited. Qunet and Dalal identified a specific RNA molecule that is transcribed from the centromeric DNA, which directly binds to the CENP-A protein and its chaperone before CENP-A is assembled onto the centromeric DNA. Reducing the levels of this RNA within the cells made them unable to separate their chromosomes correctly during cell divisions. Qunet and Dalal also demonstrated that this centromeric RNA is needed to specifically target both the CENP-A protein, via its chaperone, to the centromere. The findings of Qunet and Dalal demonstrate that RNAs produced from a specific part of the chromosome can help target DNA-binding proteins back to that region's DNA sequence. Following on from this work, the next challenge will be to determine if other RNA molecules are used for the same purpose in humans and other species.

NRDE-2 active in transcription regulation Small RNAs function in both the cytoplasm, inhibiting expression from messenger RNAs, and in the nucleus, to silence heterochromatin and prevent genome rearrangement. In this study, Guang et al . characterize a new protein involved in RNA interference in the nucleus. NRDE-2 associates with the Argonaute protein NRDE-3 and siRNAs on nascent transcripts. This association prevents elongation by RNA polymerase II, thereby making this a co-transcriptional form of gene silencing. Small regulatory RNAs function both in the cytoplasm, inhibiting expression from messenger RNAs, and in the nucleus, silencing heterochromatin and preventing genome rearrangement. Now a new protein involved in RNA interference in the nucleus has been characterized. This protein, NRDE-2, associates with NRDE-3 and short interfering RNAs on nascent transcripts. This association prevents elongation of the transcripts by RNA polymerase II, making this a co-transcriptional form of gene silencing. Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defence against parasitic nucleic acids and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei 1 , 2 . For instance, in plants and fungi, short interfering RNA (siRNAs) associate with nascent transcripts and direct chromatin and/or DNA modifications 1 , 2 . To understand further the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in Caenorhabditis elegans nuclei 3 . Here we show that the gene nuclear RNAi defective-2 ( nrde-2 ) encodes an evolutionarily conserved protein that is required for siRNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct an NRDE-2-dependent silencing of pre-messenger RNAs (pre-mRNAs) 3′ to sites of RNAi, an NRDE-2-dependent accumulation of RNA polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3′ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation: siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.

Transcription-replication conflicts frequently occur at repetitive DNA elements involved in genome maintenance functions. The KSHV terminal repeats (TR) function as the viral episome maintenance element when bound by the viral encoded nuclear antigen LANA. Here, we show that transcription-replication conflicts occur at or near LANA binding sites in the TR. We show by proximity ligation assay (PLA) that PCNA and RNAPII colocalize with LANA-nuclear bodies (LANA-NBs). Using DNA-RNA-IP (DRIP) assays with S9.6 antibody, we demonstrate that R-loops form at the TR. We find that these R-loops are also associated with histone H3pS10 a marker for R-loops associated with transcription-replication conflicts. Inhibitors of RNAPII eliminated R-loop formation at TR and reduced active histone modifications H3K4me3 and H3K27ac, with a corresponding increase in heterochromatic H3K9me3. RNAPII inhibitors also disrupted LANA binding to the TR, but did not eliminate LANA-NBs. We show that LANA can induce R-loops on a plasmid containing 8, but not 2 copies of the TR, and that the N-terminal histone binding function of LANA is required for this activity. RNaseH treatment eliminated R-loops and reduced LANA binding to the TR. Taken together, our study indicates that LANA induces histone modifications associated with RNA and DNA polymerase activity and the formation of R-loops that correlate with episome maintenance function. These findings provide new insights into mechanisms of KSHV episome maintenance during latency and more generally for genome maintenance of repetitive DNA.

Transcription of the eukaryotic genomes is carried out by three distinct RNA polymerases I, II, and III, whereby each polymerase is thought to independently transcribe a distinct set of genes. To investigate a possible relationship of RNA polymerases II and III, we mapped their in vivo binding sites throughout the human genome by using ChIP-Seq in two different cell lines, GM12878 and K562 cells. Pol III was found to bind near many known genes as well as several previously unidentified target genes. RNA-Seq studies indicate that a majority of the bound genes are expressed, although a subset are not suggestive of stalling by RNA polymerase III. Pol II was found to bind near many known Pol III genes, including tRNA, U6, HVG, hY, 7SK and previously unidentified Pol III target genes. Similarly, in vivo binding studies also reveal that a number of transcription factors normally associated with Pol II transcription, including c-Fos, c-Jun and c-Myc, also tightly associate with most Pol III-transcribed genes. Inhibition of Pol II activity using α-amanitin reduced expression of a number of Pol III genes (e.g., U6, hY, HVG), suggesting that Pol II plays an important role in regulating their transcription. These results indicate that, contrary to previous expectations, polymerases can often work with one another to globally coordinate gene expression.

Many cancer therapeutics target DNA and exert cytotoxicity through the induction of DNA damage and inhibition of transcription. We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Polyamide treatment activates p53 signaling in LNCaP prostate cancer cells without detectable DNA damage. Genome-wide mapping of RNAP2 binding shows reduction of occupancy, preferentially at transcription start sites, but occupancy at enhancer sites is unchanged. Polyamide treatment results in a time- and dose-dependent depletion of the RNAP2 large subunit RPB1 that is preventable with proteasome inhibition. This polyamide demonstrates antitumor activity in a prostate tumor xenograft model with limited host toxicity.

The Herpes Simplex Virus 1 (HSV-1)-encoded ICP22 protein plays an important role in viral infection and affects expression of host cell genes. ICP22 is known to reduce the global level of serine (Ser)2 phosphorylation of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptapeptide repeats comprising the carboxy-terminal domain (CTD) of the large subunit of RNA polymerase (pol) II. Accordingly, ICP22 is thought to associate with and inhibit the activity of the positive-transcription elongation factor b (P-TEFb) pol II CTD Ser2 kinase. We show here that ICP22 causes loss of CTD Ser2 phosphorylation from pol II engaged in transcription of protein-coding genes following ectopic expression in HeLa cells and that recombinant ICP22 interacts with the CDK9 subunit of recombinant P-TEFb. ICP22 also interacts with pol II in vitro. Residues 193 to 256 of ICP22 are sufficient for interaction with CDK9 and inhibition of pol II CTD Ser2 phosphorylation but do not interact with pol II. These results indicate that discrete regions of ICP22 interact with either CDK9 or pol II and that ICP22 interacts directly with CDK9 to inhibit expression of host cell genes.

Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue‐specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3‐Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3‐Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two‐step mechanism for UTX‐mediated demethylation at muscle‐specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.