RNA polymerase II trapped on a molecular treadmill: Structural basis of persistent transcriptional arrest by a minor groove DNA binder

Elongating RNA polymerase II (Pol II) can be paused or arrested by a variety of obstacles. These obstacles include DNA lesions, DNA-binding proteins, and small molecules. Hairpin pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA in a sequence-specific manner and induce strong transcriptional arrest. Remarkably, this Py-Im–induced Pol II transcriptional arrest is persistent and cannot be rescued by transcription factor TFIIS. In contrast, TFIIS can effectively rescue the transcriptional arrest induced by a nucleosome barrier. The structural basis of Py-Im–induced transcriptional arrest and why TFIIS cannot rescue this arrest remain elusive. Here we determined the X-ray crystal structures of four distinct Pol II elongation complexes (Pol II ECs) in complex with hairpin Py-Im polyamides as well as of the hairpin Py-Im polyamides–dsDNA complex. We observed that the Py-Im oligomer directly interacts with RNA Pol II residues, introduces compression of the downstream DNA duplex, prevents Pol II forward translocation, and induces Pol II backtracking. These results, together with biochemical studies, provide structural insight into the molecular mechanism by which Py-Im blocks transcription. Our structural study reveals why TFIIS fails to promote Pol II bypass of Py-Im–induced transcriptional arrest.

Structural basis of Ty3 retrotransposon integration at RNA Polymerase III-transcribed genes

Retrotransposons are endogenous elements that have the ability to mobilise their DNA between different locations in the host genome. The Ty3 retrotransposon integrates with an exquisite specificity in a narrow window upstream of RNA Polymerase (Pol) III-transcribed genes, representing a paradigm for harmless targeted integration. Here we present the cryo-EM reconstruction at 4.0 Å of an active Ty3 strand transfer complex bound to TFIIIB transcription factor and a tRNA gene. The structure unravels the molecular mechanisms underlying Ty3 targeting specificity at Pol III-transcribed genes and sheds light into the architecture of retrotransposon machinery during integration. Ty3 intasome contacts a region of TBP, a subunit of TFIIIB, which is blocked by NC2 transcription regulator in RNA Pol II-transcribed genes. A newly-identified chromodomain on Ty3 integrase interacts with TFIIIB and the tRNA gene, defining with extreme precision the integration site position.

TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation

The conserved HIV-1 LTR cis elements RBE1/3 bind the factor RBF2, consisting of USF1/2 and TFII-I, and are essential for reactivation of HIV-1 by T cell signaling. We determined that TFII-I recruits the tripartite motif protein TRIM24 to the LTR, and this interaction is required for efficient reactivation of HIV-1 expression in response to T cell signaling, similar to the effect of TFII-I depletion. Knockout of TRIM24 did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9 to the LTR. These results demonstrate that TFII-I promotes transcriptional elongation in response to T cell activation through recruitment of the co-factor TRIM24, which is necessary for efficient recruitment of the elongation factor P-TEFb.

Delineating CDK9 Regulated Molecular Events for the Development of Rationally Derived Multiple Myeloma Treatment Strategies

Background: Despite major advances in multiple myeloma (MM) therapy over the last 2 decades, most patients relapse. The identification of novel targets and the development of derived treatment approaches are therefore urgently needed. Aberrant expression of various cyclin-dependent kinases (CDKs) in solid and hematologic malignancies including MM, results in the loss of proliferative control and enhanced survival. The serine-threonine kinase CDK9, a subunit of pTEFb, in particular, is a major transcriptional regulator of numerous oncogenes. Past studies have suggested CDK9 as a potential therapeutic target in MM. However, CDK9-regulated molecular events in MM are only partly understood. By delineating CDK9-dependent pathophysiologic effects, the present study proposes rationally derived anti-CDK9-containing novel MM treatment strategies to improve patient outcome. Methods: Following expression profiling, CRISPR loss-of-function screens and correlation analyses in MM cell line and patient cells, the regulatory impact of CDK9 on downstream target genes was outlined using genomic as well as pharmacological approaches in 2D/3D MM models of the tumor microenvironment. Functionally, CDK9-regulated molecular effects as well as anti-MM activity of anti-CDK9-containing rationally derived treatment combinations were determined by gene arrays, qPCR, flow cytometry, and western blot, proliferation and survival analyses. Results: Strongly suggested by a significant induction of CDK9 mRNA expression levels progressing from normal plasma cells to cells from patients with MGUS, SMM and MM; siRNA and CRISP loss-of-function screens across various MM cell lines verified their dependency on CDK9. Correlative expression levels indicated a functional role of CDK9 (but not for CDK2 and CDK7) on Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, but not other genes (e.g. Bcl-2) in the CCLE as well as CoMMpass and GSE5900/GSE2658 MM patient datasets. Indeed, siRNA-mediated CDK9 silencing decreased protein levels of Mcl-1, cMyc, Mdm2, RNA Pol II, and IRF4, and consequently tumor cell survival. Similarly, the novel, selective CDK9-directed proteolysis-targeting chimera Thal-sns-032 induced a reduction of mRNA/ protein levels of Mcl-1, cMyc, RNA Pol II, but not of other potential targets (e.g. Bcl-2) in a dose- and time-dependent manner. Moreover, Thal-sns-032 reduced Mdm2 and thereby increased p53 protein levels. Consequently, Thal-sns-032 inhibited tumor cell proliferation and survival both in tumor cell- and tumor cell:BMSC co- cultures. Rationally, derived combination strategies of Thal-sns-032 for example with venetoclax, but also other investigational and established MM therapies induced synergistic anti-MM effects within the tumor microenvironment. Conclusion: In summary, by delineating CDK9-regulated molecular events in MM, our studies strongly support the therapeutic role of targeted CDK9-therapy and rationally derive MM combination treatment strategies. Disclosures Vallet: Pfizer: Honoraria; MSD: Honoraria; Roche Pharmaceuticals: Consultancy. Podar: Celgene: Consultancy, Honoraria; Amgen Inc.: Consultancy, Honoraria; Janssen Pharmaceuticals: Consultancy, Honoraria; Roche Pharmaceuticals: Research Funding.

EKLF/Klf1 Regulates Erythroid Transcription By Its Pioneering Activity and Subsequent Control of RNA Pol II Pause-Release

EKLF/Klf1 is a master transcriptional activator of critical genes that regulate both erythroid fate specification and terminal erythroid maturation. EKLF binds to DNA using three Zn-fingers at its C-terminus while the N-terminus constitutes a transcription activation domain (TAD) that interacts with various transcription co-factors including the protein acetylase CBP. An autosomal semi-dominant mutation at a single residue (E339D) in the mouse EKLF Zn-finger leads to Neonatal anemia (Nan). A mutation at the same residue in human EKLF (E325K) causes Congenital Dyserythropoietic Anemia type IV (CDA IV). Nan/Nan mice show lethality at embryonic day E10-11, in contrast to EKLF-/- homozygotes that survive until E15. Nan/+ heterozygotes survive to adulthood but are severely anemic, unlike EKLF+/- heterozygotes that display no aberrant phenotypes. The Nan-EKLF protein has an altered DNA binding specificity leading to a vastly altered transcriptome by two mechanisms. First, Nan-EKLF binding causes ectopic gene expression that significantly contributes to the severe anemia in Nan/+. Second, a subset of EKLF targets is downregulated in heterozygous Nan/+ mutants despite the presence of one copy of wild type EKLF, exacerbating the anemia. Thus, uncovering the mechanism by which gene expression is altered in Nan/+ may illuminate how EKLF normally activates transcription of its targets in vivo. To this end, we first examined the global occupancy of RNA Pol II phospho-Ser5 (as a paused mark) and phospho-Ser2 (as an elongation mark) in the mouse E13.5 fetal liver as a source of primary definitive erythroid cells. At promoters of ectopically expressed genes, where only Nan-EKLF (but not WT) binding is expected, we predominantly find increased levels of both paused and elongating RNA Pol II suggesting that Nan-EKLF binding activates transcription at ectopic genes by RNA Pol II recruitment and promoter proximal pausing. Further, we find increased levels of H3K27ac and CBP occupancy at these sites indicating that the mechanism of Pol II recruitment relies on CBP-mediated H3K27 acetylation and increased chromatin accessibility. Overall, this suggests robust pioneering activity of Nan-EKLF likely mediated by the interaction of its TAD with the CBP/p300 acetylase complex. At genes downregulated in Nan/+ we find two major patterns of Pol II occupancy. One is the converse of that seen at ectopic genes wherein there is a concomitant decrease in both Pol II p-Ser5 and p-Ser2 levels, along with lower H3K27ac and CBP levels suggesting EKLF gene activation has been lost at these sites in Nan/+. This includes cell cycle EKLF targets such as E2f2 and Rgcc. The second set of genes have comparable levels of p-Ser5 (paused) Pol II in Nan/+ and WT, but lower levels of p-Ser2 (elongating) Pol II in Nan/+. This suggests that although Pol II is being recruited to the TSS and pauses effectively, the pause-release step leading to effective transcription elongation is impaired. This subset includes important EKLF targets such as Bcl11a, Pax7, Xpo7, and several membrane transporters. As expected, CBP and H3K27ac levels are similar in WT and Nan/+ at these sites. To determine the cause of impaired RNA Pol II pause-release we examined the global occupancies of key transcription elongation factors such as P-TEFb and NELF. We find that levels of NELF, a negative elongation factor, remain unchanged in WT and Nan/+. However, levels of the P-TEFb subunit Cdk9, a positive elongation factor that facilitates release of paused RNA Pol II, is significantly lower at the TSS of these genes in Nan/+. This suggests that in Nan/+, possible reduction or loss of EKLF binding at some EKLF target promoters impairs effective recruitment of positive transcription elongation factors, resulting in a failure to efficiently release paused RNA Pol II. This causes downregulation of these EKLF target genes and contributes to the severe anemic phenotypes of the Nan mouse. We conclude that: EKLF exhibits expression control of its target genes at both the transcriptional initiation and elongation steps in vivo; EKLF can act as a pioneer transcription factor and increase chromatin accessibility through H3K27 acetylation by CBP leading to recruitment and pausing of RNA Pol II; and EKLF recruits the positive transcription elongation complex P-TEFb, enabling the controlled release of paused RNA Pol II at transcription start sites of a select group of its targets. Disclosures No relevant conflicts of interest to declare.

PER2 mediates CREB-dependent light induction of the clock gene Per1

Light affects many physiological processes in mammals such as entrainment of the circadian clock, regulation of mood, and relaxation of blood vessels. At the molecular level, a stimulus such as light initiates a cascade of kinases that phosphorylate CREB at various sites, including serine 133 (S133). This modification leads CREB to recruit the co-factor CRCT1 and the histone acetyltransferase CBP to stimulate the transcription of genes containing a CRE element in their promoters, such as Period 1 (Per1). However, the details of this pathway are poorly understood. Here we provide evidence that PER2 acts as a co-factor of CREB to facilitate the formation of a transactivation complex on the CRE element of the Per1 gene regulatory region in response to light or forskolin. Using in vitro and in vivo approaches, we show that PER2 modulates the interaction between CREB and its co-regulator CRTC1 to support complex formation only after a light or forskolin stimulus. Furthermore, the absence of PER2 abolished the interaction between the histone acetyltransferase CBP and CREB. This process was accompanied by a reduction of histone H3 acetylation and decreased recruitment of RNA Pol II to the Per1 gene. Collectively, our data show that PER2 supports the stimulus-dependent induction of the Per1 gene via modulation of the CREB/CRTC1/CBP complex.

RNA Polymerase II Is a Therapeutic Target to Overcome FLT3 Inhibitor Resistance Mediated By the Bone Marrow Microenvironment

Background: Gilteritinib is a clinically active FLT3 tyrosine kinase inhibitor (TKI) approved for relapsed/refractory FLT3-mutant AML, but nearly all patients treated with gilteritinib and other FLT3 TKIs eventually develop clinical resistance. Activating RAS/MAPK pathway mutations are a predominant non-FLT3 dependent resistance mechanism in patients treated with gilteritinib. AML blasts can also develop FLT3 TKI resistance secondary to paracrine MAPK activation stimulated by FLT3 Ligand, FGF2, or other protective cytokines within the bone marrow microenvironment (BME). To identify potential targets that sensitize AML cells to gilteritinib-induced apoptosis in a model of the BME, we performed a genome-wide CRISPR/Cas9 death screen in MOLM-14 FLT3-ITD+ human AML cells cultured in bone marrow stromal cell conditioned media. We hypothesize that identified genes represent promising combinatorial therapeutic targets that can enhance clinical efficacy of FLT3 TKIs in AML. Methods: To model stroma-mediated TKI resistance, we used the HS5 human bone marrow stromal cell line that secretes multiple cytokines (G-CSF, GM-CSF, FGF2) and supports myeloid progenitor proliferation in co-culture. MOLM-14 CRISPRi cells transduced with CRISPRi-v2 genome-wide sgRNA library were cultured in HS5 conditioned media for 24 hours and then treated with gilteritinib 250 nM. Cells were stained with a fluorogenic caspase 3/7 reagent and then fixed after 24 hours of drug treatment. Caspase-3 positive cells were sorted from the entire drug-treated cell population by FACS and guide RNAs enriched or depleted in this sample as compared to an untreated T0 sample were determined by NGS. Results: We identified several gene-level hits that were enriched in the apoptotic population (FDR <0.2). Among these, we identified multiple transcription factors or regulators of transcriptional activation. The latter included multiple components of RNA pol II machinery (POLR2G, RTF1) and multiple subunits of Mediator (MED12, MED30, MED21, MED11), a complex that regulates RNA pol II activity and has been shown to modulate super-enhancer-associated genes in AML cells. To validate select hits, we transduced MOLM-14 and MV411 CRISPRi cells with a tetracycline-inducible sgRNA expression vector. Using this system, we found that conditional knockdown of MED12, the top scoring Mediator subunit in our screen, significantly sensitized MOLM-14 (FgH1) cells to gilteritinib while modestly augmenting the cytotoxicity of gilteritinib in MV411 (FgH1) cells when compared to a non-targeting sgRNA. Functionally, MED12 associates with MED13, Cyclin C, and CDK8 to form the CDK8 kinase module of Mediator. MED12 knockdown, as expected, led to suppression of STAT1 S7272 and STAT5 S726 phosphorylation, known targets of CDK8. Based on these results, we hypothesized that CDK8 inhibition would augment apoptosis induced by gilteritinib in HS5 conditioned media. Using SEL120, a novel CDK8 inhibitor already in early phase AML clinical trials, we performed an 8 x 8 dose matrix drug synergy analysis of gilteritinib and SEL120 in multiple FLT3-mutant AML cell lines using Bliss independence modeling. We found that SEL120 was synergistic with gilteritinib in inducing apoptosis in MOLM-14 and MV411 cells in HS5 conditioned media (Bliss synergy scores of 2.44 and 11.45 with most synergistic area scores of 10.91 and 26.85, respectively). We also found combinatorial activity against MOLM-14 cells harboring secondary NRAS activating mutations (G12C and Q61K), suggesting the therapeutic combination could potentially overcome cell intrinsic and extrinsic MAPK-activating resistance mechanisms. Lastly, we found that gilteritinib and SEL120 combined to impart greater cytotoxicity than either drug alone in a primary sample (AML #1) from a patient with newly diagnosed AML possessing a FLT3-ITD mutation at high mutant allele ratio. Conclusions: The results and validation of our CRISPRi screen suggest that combined CDK8 and FLT3 inhibition is a novel strategy for augmenting gilteritinib cytotoxicity. Assessment of the activity of the combination in additional primary AML samples and in vivo murine models of AML is planned. Additional candidate targets already described and other Mediator and RNA pol II subunits from our screen are also being further evaluated to precisely define the transcriptional programs that influence FLT3 inhibitor resistance. Disclosures Logan: Pharmacyclics, Astellas, Jazz, Kite, Kadmon, Autolus, Amphivena: Research Funding; Amgen, Pfizer, AbbVie: Consultancy. Gilbert: Denali Therapeutics: Ended employment in the past 24 months, Other: Spouse/Significant Other's Employment; GSK: Consultancy, Research Funding; AstraZeneca: Research Funding; Chroma Medicine: Consultancy, Other: Co-founder. Smith: Revolutions Medicine: Research Funding; AbbVie: Research Funding; Daiichi Sankyo: Consultancy; Amgen: Honoraria; FUJIFILM: Research Funding; Astellas Pharma: Consultancy, Research Funding.

Transcription recycling assays identify PAF1 as a driver for RNA Pol II recycling

RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.

Localization of RNA Pol II CTD (S5) and Transcriptome Analysis of Testis in Diploid and Tetraploid Hybrids of Red Crucian Carp (♀) × Common Carp (♂)

Polyploidy occurs naturally in fish; however, the appearance of these species is an occasional and gradual process, which makes it difficult to trace the changes in phenotypes, genotypes, and regulation of gene expression. The allotetraploid hybrids (4nAT) of red crucian carp (RCC; ♀) × common carp (CC; ♂) generated from interspecies crossing are a good model to investigate the initial changes after allopolyploidization. In the present study, we focused on the changes in the active sites of the testicular transcriptome of the allotetraploid by localization of RNA Pol II CTD YSPTSPS (phospho S5) using immunofluorescence and RNA-seq data via bioinformatic analysis. The results showed that there was no significant difference in signal counts of the RNA Pol II CTD (S5) between the different types of fish at the same stages, including RCC, CC, 2nF1, and 4nAT, which means that the number of transcriptionally active sites on germ cell chromosomes was not affected by the increase in chromosome number. Similarly, RNA-seq analysis indicated that in the levels of chromosomes and 10-kb regions in the genome, there were no significant changes in the highly active sites in RCC, 2nF1, and 4nAT. These findings suggest that at the beginning of tetraploid origin, the active transcriptome site of 4nAT in the testis was conserved in the regions of the genome compared to that in RCC and 2nF1. In conclusion, 4nAT shared a similar gene expression model in the regions of the genome with RCC and 2nF1 with significantly different expression levels.