RNA Polymerase I Research Articles

Frequent TRPS1 expression in synovial sarcoma is associated with SS18-SSX fusion oncoprotein activity

Synovial sarcoma is an aggressive translocation-associated soft tissue tumor driven by an SS18-SSX fusion oncoprotein. TRPS1 is a recently identified marker for breast carcinoma, but less is known about its expression in other tumor types. We encountered a case of synovial sarcoma showing strong and diffuse expression of TRPS1. To better characterize this observation, we examined the immunohistochemical expression of TRPS1 in 165 cases of synovial sarcoma, including 70 primary, 21 recurrent, and 74 metastatic tumors, using tissue microarrays. TRPS1 expression was observed in 86% of cases. Among the positive cases, TRPS1 labeled >50% of tumor cells in 57% of cases, and staining intensity was strong or moderate in 68%. Metastatic tumors more frequently demonstrated strong and diffuse TRPS1 expression compared to primary tumors. To understand the mechanism of TRPS1 expression, we interrogated publicly available gene expression and ChIP-seq datasets and found that TRPS1 transcript levels are increased in synovial sarcoma compared to other soft tissue sarcomas. Data from ChIP-seq experiments showed enrichment of SS18-SSX protein at the TRPS1 locus and co-localization with RNA pol II. The TRPS1 locus is also enriched in several histone modifications associated with active transcription. In functional knockdown data, repression of SS18::SSX in synovial sarcoma cell lines is associated with reduced TRPS1 transcript levels, further supporting a model whereby TRPS1 expression is mediated, at least in part, by the SS18-SSX fusion oncoprotein. Knowledge of TRPS1 expression in synovial sarcoma may help avoid potential diagnostic pitfalls in the immunohistochemical workup of tumors.

Abstract B012: Visualization of EWSR1’s colocalization with phosphorylated RNA-Polymerase II reveals its concentration at a subset of active regions of transcription in ewing sarcoma cells

Abstract EWSR1, a member of the FET protein family, contains low complexity and nucleic acid binding domains and functions in transcription regulation and RNA metabolism. Recent biochemical and EWSR1-depletion studies demonstrated that EWSR1 regulates critical phosphorylation events that control basal transcription. In Ewing sarcoma (EWS) cells, the interaction of EWSR1 and the fusion oncoprotein EWS-FLI1 results in EWSR1 no longer functioning as an effective regulator of transcription, which following DNA damage, enhances R-loop formation. To further elucidate EWSR1’s function in EWS and its contribution to EWS-FLI1’s deregulation of gene expression, we have generated reporter systems to visualize and quantify its endogenous expression within EWS cells. We used CRISPR-Cas9 and sgRNAs targeting sequences at the 5’ end of the first exon of EWSR1 to insert a fluorescent mNeonGreen (mNG) reporter gene into the EWSR1 loci of A673 or TC-32 EWS cell lines. We employed sequencing and RNAi-based analysis to identify and validate successfully modified clones. All modified clones harbored insertions into the unrearranged EWSR1 allele. We then employed super-resolution confocal microscopy to assess EWSR1’s localization in EWS cells. Analysis of modified EWS-cells showed mNG-EWSR1 forms puncta, restricted to the nucleoplasm, consistent with a nuclear protein in an active state. A subset of puncta exhibits a high density (HD) mNG-EWSR1 signal, defined by fluorescence at least twice the background signal. Our results show minimal colocalization of mNG-EWSR1 (total or HD) and a marker of chromatin accessibility, H3K27Ac. A small percentage (~5%) of total Ser5-phosphorylated RNA-pol II (pS5-RP-II), a marker of transcription initiation, colocalizes with total mNG-EWSR1, but critically the HD mNG-EWSR1 puncta all colocalize with pS5-RP-II. About 20% of total Ser2-phosphorylated RNA-pol II (pS2-RP-II), a marker of transcription elongation colocalizes with total mNG-EWSR1. As observed for pS5-RP-II, 100% of HD-mNG-EWSR1 puncta colocalize with pS2-RP-II. Finally, when we examined nuclear speckle structures (SC35/SRSF2 >200 µm), we observed a 20% overlap in their signals. We have seen comparable results using the mNG-EWSR1 expressing A673 cells. These findings demonstrate image-based quantification of endogenous EWSR1’s colocalization with RNA-polymerase II within EWS cells. Overall, EWSR1 colocalizes with about 20% of RNA-pol II in a state consistent with active transcription, and over 90% of high-density EWSR1 colocalizes with phosphorylated RNA pol II. Ongoing studies will assess changes in the distribution of EWSR1’s interactions with different proteins following inhibition of RNA-pol II phosphorylation, alterations in EWS-FLI1 expression, or the disruption of low complexity domain interactions. We anticipate findings from these studies will offer critical insights into the functional interactions that EWSR1 contributes to regulating gene expression in EWS cells. Citation Format: Natasha J. Caplen, Soumya Sundara Rajan, Vernon Ebegboni, Tamara L. Jones, Michael J. Kruhlak, Jan Wisniewski, Patricio Pichling, Katelyn R. Ludwig, Javed Khan, Raj Chari. Visualization of EWSR1’s colocalization with phosphorylated RNA-Polymerase II reveals its concentration at a subset of active regions of transcription in ewing sarcoma cells [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B012.

Localization of Epigenetic Markers in Leishmania Chromatin.

Eukaryotes use histone variants and post-translation modifications (PTMs), as well as DNA base modifications, to regulate DNA replication/repair, chromosome condensation, and gene expression. Despite the unusual organization of their protein-coding genes into large polycistronic transcription units (PTUs), trypanosomatid parasites also employ a “histone code” to control these processes, but the details of this epigenetic code are poorly understood. Here, we present the results of experiments designed to elucidate the distribution of histone variants and PTMs over the chromatin landscape of Leishmania tarentolae. These experiments show that two histone variants (H2A.Z and H2B.V) and three histone H3 PTMs (H3K4me3, H3K16ac, and H3K76me3) are enriched at transcription start sites (TSSs); while a histone variant (H3.V) and the trypanosomatid-specific hyper-modified DNA base J are located at transcription termination sites (TTSs). Reduced nucleosome density was observed at all TTSs and TSSs for RNA genes transcribed by RNA polymerases I (RNAPI) or RNAPIII; as well as (to a lesser extent) at TSSs for the PTUs transcribed by RNAPII. Several PTMs (H3K4me3, H3K16ac H3K20me2 and H3K36me3) and base J were enriched at centromeres, while H3K50ac was specifically associated with the periphery of these centromeric sequences. These findings significantly expand our knowledge of the epigenetic markers associated with transcription, DNA replication and/or chromosome segregation in these early diverging eukaryotes and will hopefully lay the groundwork for future studies to elucidate how they control these fundamental processes.

The Potential Mechanism of HDAC1-Catalyzed Histone Crotonylation of Caspase-1 in Nonsmall Cell Lung Cancer.

Nonsmall cell lung cancer (NSCLC) is a predominant subtype of lung cancer and accounts for over 80% of all lung cancer cases. The resistance to pemetrexed (PEM) is frequently occurred and severely affects the NSCLC therapy. Proteomic analysis of histones indicated that the histone deacetylase 1 (HDAC1) complex could hydrolyze lysine crotonylation on histone3 (H3K18cr), affecting epigenetic regulation in cancers. However, the effect of HDAC1-mediated H3K18cr on the PEM resistance of NSCLC is still unclear. Here, we aimed to explore the function of HDAC1-mediated H3K18cr in NSCLC PEM resistance. The expression of HDAC1 was upregulated in clinical NSCLC tissues and cell lines and correlated with the poor prognosis of NSCLC samples. We constructed the PEM-resistant NSCLC cell lines, and the depletion of HDAC1 remarkably reduced the viability of the cells. The proliferation of PEM-resistant NSCLC cells was decreased by HDAC1 knockdown, and the IC50 of PEM was repressed by the silencing of HDAC1 in the cells. Mechanically, we identified the enrichment of HDAC1 on the promoter of caspase-1 in PEM-resistant NSCLC cells. The depletion of HDAC1 inhibited the enrichment of histone H3K18cr and RNA polymerase II (RNA pol II) on the caspase-1 promoter in the cells. The expression of caspase-1 was suppressed by HDAC1 knockdown. The knockdown of HDAC1 reduced proliferation of PEM-resistant NSCLC cells, in which caspase-1 or GSDMD depletion reversed the effect. Clinically, the HDAC1 expression was negatively associated with caspase-1 and GSDMD in clinical NSCLC tissues, while caspase-1 and GSDMD expression was positively correlated in the samples. Therefore, we concluded that HDAC1-catalyzed histone crotonylation of caspase-1 modulates PEM sensitivity of NSCLC by targeting GSDMD.

HDAC1 and PRC2 mediate combinatorial control in SPI1/PU.1-dependent gene repression in murine erythroleukaemia.

Although originally described as transcriptional activator, SPI1/PU.1, a major player in haematopoiesis whose alterations are associated with haematological malignancies, has the ability to repress transcription. Here, we investigated the mechanisms underlying gene repression in the erythroid lineage, in which SPI1 exerts an oncogenic function by blocking differentiation. We show that SPI1 represses genes by binding active enhancers that are located in intergenic or gene body regions. HDAC1 acts as a cooperative mediator of SPI1-induced transcriptional repression by deacetylating SPI1-bound enhancers in a subset of genes, including those involved in erythroid differentiation. Enhancer deacetylation impacts on promoter acetylation, chromatin accessibility and RNA pol II occupancy. In addition to the activities of HDAC1, polycomb repressive complex 2 (PRC2) reinforces gene repression by depositing H3K27me3 at promoter sequences when SPI1 is located at enhancer sequences. Moreover, our study identified a synergistic relationship between PRC2 and HDAC1 complexes in mediating the transcriptional repression activity of SPI1, ultimately inducing synergistic adverse effects on leukaemic cell survival. Our results highlight the importance of the mechanism underlying transcriptional repression in leukemic cells, involving complex functional connections between SPI1 and the epigenetic regulators PRC2 and HDAC1.

Decreased DNA Damage and Improved p53 Specificity of RITA Analogs.

Reactivation of p53 tumor-suppressor function by small molecules is an attractive strategy to defeat cancer. A potent p53-reactivating molecule RITA, which triggers p53-dependent apoptosis in human tumor cells in vitro and in vivo, exhibits p53-independent cytotoxicity due to modifications by detoxification enzyme Sulfotransferase 1A1 (SULT1A1), producing a reactive carbocation. Several synthetic modifications to RITA's heterocyclic scaffold lead to higher energy barriers for carbocation formation. In this study, we addressed the question whether RITA analogs NSC777196 and NSC782846 can induce p53-dependent apoptosis without SULT1A1-dependent DNA damage. We found that RITA analog NSC782846, but not NSC777196, induced p53-regulated genes, targeted oncogene addiction, and killed cancer cells upon p53 reactivation, but without induction of DNA damage and inhibition RNA pol II. Our results might demonstrate a method for designing more specific and potent RITA analogs to accelerate translation of p53-targeting compounds from laboratory bench to clinic.

Investigation of the Presence of <em>Xylella fastidiosa</em>: A Quarantine Pathogen in Potential Host Plants in Sri Lanka

Xylella fastidiosa, a gram-negative, xylem-inhabiting devastating plant pathogenic bacterium is reported in around 600 host plant species. Xylella fastidiosa is a regulated-quarantine pathogen in the European Union where the confirmation of pathogen-free certification is required in international trade. At present, Taiwan is the only country in Asia with a long-term presence of this bacterium. Hence there may be a risk of introduction into Sri Lanka through international trade. Therefore, an investigation was conducted from December 2020 to March 2021 to find out the presence of X. fastidiosa in potential hosts in Sri Lanka. A total number of 54 composite samples were obtained from 23 potential host plant species declared in the regulation (EU) 2020/1201 of 14 August 2020 including ornamental plant species from nine districts where most of the export nurseries are scattered. These were tested for the presence of X. fastidiosa by PCR using specific primers targeting the conserved genomic region of the RNA polymerase sigma factor of the rpoD gene of 733 bp. The positive DNA was sourced from the French collection of Plant Associated Bacteria, CIRM-CFBP for comparison. All local samples were found to be free from X. fastidiosa. These preliminary investigation results are considered as an encouraging indication, considering that X. fastidiosa was not found in Sri Lanka, at least in the surveyed areas. However, frequent studies are needed to be conducted to monitor the pathogen invasion.

Genome Analysis Coupled With Transcriptomics Reveals the Reduced Fitness of a Hot Spring Cyanobacterium Mastigocladus laminosus UU774 Under Exogenous Nitrogen Supplement.

The present study focuses on the stress response of a filamentous, AT-rich, heterocystous cyanobacterium Mastigocladus laminosus UU774, isolated from a hot spring, Taptapani, located in the eastern part of India. The genome of UU774 contains an indispensable fragment, scaffold_38, of unknown origin that is implicated during severe nitrogen and nutrition stress. Prolonged exposure to nitrogen compounds during starvation has profound adverse effects on UU774, leading to loss of mobility, loss of ability to fight pathogens, reduced cell division, decreased nitrogen-fixing ability, reduced ability to form biofilms, reduced photosynthetic and light-sensing ability, and reduced production of secreted effectors and chromosomal toxin genes, among others. Among genes showing extreme downregulation when grown in a medium supplemented with nitrogen with the fold change > 5 are transcriptional regulator gene WalR, carbonic anhydrases, RNA Polymerase Sigma F factor, fimbrial protein, and twitching mobility protein. The reduced expression of key enzymes involved in the uptake of phosphate and enzymes protecting oxygen-sensitive nitrogenases is significant during the presence of nitrogen. UU774 is presumed to withstand heat by overexpressing peptidases that may be degrading abnormally folded proteins produced during heat. The absence of a key gene responsible for heterocyst pattern formation, patS, and an aberrant hetN without a functional motif probably lead to the formation of a chaotic heterocyst pattern in UU774. We suggest that UU774 has diverged from Fischerella sp. PCC 9339, another hot spring species isolated in the United States.

Rpb4/7, a key element of RNA pol II to coordinate mRNA synthesis in the nucleus with cytoplasmic functions in Saccharomyces cerevisiae

Rpb4/7, a key element of RNA pol II to coordinate mRNA synthesis in the nucleus with cytoplasmic functions in Saccharomyces cerevisiae

A genomic meta-analysis of clinical variables and their association with intrinsic molecular subsets in systemic sclerosis.

Four intrinsic molecular subsets (inflammatory, fibroproliferative, limited, normal-like) have previously been identified in SSc and are characterized by unique gene expression signatures and pathways. The intrinsic subsets have been linked to improvement with specific therapies. Here, we investigated associations between baseline demographics and intrinsic molecular subsets in a meta-analysis of published datasets. Publicly available gene expression data from skin biopsies of 311 SSc patients measured by DNA microarray were classified into the intrinsic molecular subsets. RNA-sequencing data from 84 participants from the ASSET trial were used as a validation cohort. Baseline clinical demographics and intrinsic molecular subsets were tested for statistically significant associations. Males were more likely to be classified in the fibroproliferative subset (P = 0.0046). SSc patients who identified as African American/Black were 2.5 times more likely to be classified as fibroproliferative compared with White/Caucasian patients (P = 0.0378). ASSET participants sera positive for anti-RNA pol I and RNA pol III autoantibodies were enriched in the inflammatory subset (P = 5.8 × 10-5, P = 9.3 × 10-5, respectively), while anti-Scl-70 was enriched in the fibroproliferative subset. Mean modified Rodnan Skin Score (mRSS) was statistically higher in the inflammatory and fibroproliferative subsets compared with normal-like (P = 0.0027). The average disease duration for inflammatory subset was less than fibroproliferative and normal-like intrinsic subsets (P = 8.8 × 10-4). We identified multiple statistically significant differences in baseline demographics between the intrinsic subsets that may represent underlying features of disease pathogenesis (e.g. chronological stages of fibrosis) and have implications for treatments that are more likely to work in certain SSc populations.

First Report of Bacterial Soft Rot Caused by Pantoea agglomerans on Chinese yam (Dioscorea opposita) in China.

Chinese yam (Dioscorea opposita Thunb.), which belongs to the family of Dioscorea, is widely naturalized throughout China, due to its high economic and medicinal value. Since 2019, water-soaked lesions were frequently observed in the underground tubers of Chinese yam located in Xinyang City, Henan Province. To identify the causal agent, ten pieces of tissue from the underground tubers with disease symptoms were collected. Those infected tissues (5×5 mm) were crushed in 500 μL sterilized water after surface sterilization and streaked onto Luria-Bertani agar plates. Pale-yellowish, rod-shaped, slimy single bacterial colonies with smooth margin were observed after 24 hours of incubation, and three bacterial colonies (named CY-1, CY-2 and CY-3) were randomly selected for further biochemical and molecular characterization. These bacteria were gram-negative with the cell length of 1.0 to 3.0 μm, width of 0.5 to 1.0 μm, and with peritrichous flagella. Subsequently, the bacteria were biochemically analyzed through BIOLOG (Hayward, CA) and identified as Pantoea agglomerans with 99% probability. Furthermore, the phylogenetic analysis results based on 16S rDNA, DNA gyrase subunit B (gyrB), and RNA polymerase sigma factor (rpoD) showed these three isolates were most closely related to P. agglomerans. The sequence of 16S rDNA, gyrB and rpoD of each strain was submitted to GenBank with the accession numbers MZ541065 MZ541066 and MZ541067 for 16S rDNA; MZ669846, MZ669847 and MZ669848 for gyrB; MZ669849, MZ669850 and MZ669851 for ropD. Pathogenicity test was performed to complete Koch's postulates. Tubers of Chinese yam were wounded by sterile needle and inoculated with 500 μL 108 CFU/mL bacterial suspension. Sterilized water was used as a control. Five pots were inoculated for each isolate. Water-soaked lesions appeared after five days incubation at 25°C in a biochemical incubator and no lesions were observed on the control. Bacteria re-isolated from the lesions were similar in phenotypic and molecular characteristics to the original isolates. In brief, based on colony morphology, biochemical tests, characteristic sequence analysis, and pathogenicity verification, the pathogen responsible for the soft rot of Chinese yam in Henan Province was identified as P. agglomerans. In China, P. agglomerans has been reported to associate with bacterial soft rot on Chinese cabbage (Guo et al., 2020). To our knowledge, this work is the first report of bacterial rot caused by P. agglomerans on Chinese yam.

Abstract 3950: Targeting ribosome biogenesis is a novel strategy to suppress the growth of pancreatic cancer

Abstract Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States with limited therapeutic approaches. Dysregulation of the ribosome biogenesis process was observed in various cancer including pancreatic cancer. Therefore, strategically targeting ribosome biogenesis could be a novel approach for pancreatic cancer treatment. One such approach is to target ribosome biogenesis is via small molecule inhibitors of RNA Polymerase I as these inhibitors have shown promising anti-cancer activity in pre-clinical and clinical studies. We observed differential constitutive expression of various ribosomal proteins including RPA-194 in human PanCa cells when compared with normal HPDE cells. Therefore, we hypothesized that targeting RNA pol I will inhibit the growth of pancreatic cancer. In this study, we for the first time evaluated the therapeutic effect of a novel RNA polymerase I inhibitor (BMH-21) against PanCa in pre-clinical model systems. BMH-21 significantly inhibited the ribosome biogenesis process in various pancreatic cancer cells (Panc-1, AsPC1, BxPC3, MiaPaCa-2) as determined by inhibition of rRNA synthesis and POL I occupancy on rDNA. BMH-21 treatment of pancreatic cancer cells significantly degraded RPA194 protein as determined by Western blot and confocal microscopy analysis. BMH-21 treatment (0.25-5µM) showed differential IC50 in various pancreatic cancer cells which were correlated with the expression of its target proteins RPA194 and RPA135 expression. In our flow cytometry experiment, we observed a significant G0/G1 cell cycle arrest and increase Annexin V and Annexin V7-AAD positive cells in BMH-21 treated pancreatic cancer cells as compared to the vehicle treatment group suggesting its apoptosis-inducing potential in pancreatic cancer cells. BMH-21 treatment inhibited growth and metastatic phenotypes of various PanCa cells as analyzed by various functional assays (colony formation and atomic force microscopy analyses). We also evaluated the effect of BMH-21 on various kinases, which regulate ribosome biogenesis. Interestingly, we observed BMH-21 significantly (P<0.01) downregulated the phosphorylation of AKT (Ser473) and WNK-1 (Thr60) kinases and Stat3 phosphorylation at Ser727 and upregulates pChk2 kinase in PanCa cells. BMH-21 (2 mg/kg i.p) significantly (P<0.01) inhibited the growth of pancreatic tumor in an orthotopic xenograft mouse model. Excised tumor tissues of BMH-21 treated mice showed decreased expression of various ribosomal proteins including RPA194 and RPA195 along with various cell proliferative markers (ki67 and PCNA) as compared to vehicle-treated group. Taken together, our results demonstrated that targeting the ribosome biogenesis process is a novel therapeutic strategy for the management of pancreatic cancer and BMH-21 could be used as a potential therapeutic agent for the treatment of advanced pancreatic cancer. Citation Format: Carlos Perez, Mudassier Ahmad, Andrew Massey, Asif Shahriar, Emmanuel Anning, Vivek Kashyap, Neeraj Chauhan, Anupam Dhasmana, Manish Tripathi, Subhash Chauhan, Bilal Hafeez. Targeting ribosome biogenesis is a novel strategy to suppress the growth of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3950.

Abstract 5393: An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity

Abstract Background: CDK12 has emerged as an attractive cancer target due to its role in transcription and DNA damage repair regulation. In pre-clinical models, small molecule CDK12 inhibitors have demonstrated promising tumor killing effect, especially in combination with DNA damaging agents. Here we profiled new oral and selective CDK12 inhibitors. Material and methods: ADP-glo࣪ assays: inhibition of recombinant CDK12/CyclinK, CDK2/CyclinE, CDK7/CyclinH/MAT1 and CDK9/CyclinT2 was measured by assessing ADP converted from ATP via luminescent signal at Km and 20x Km ATP. Kinome screen: 1 µM of Compound A was tested in duplicate against 370 kinases at 10 µM ATP in the radiometric HotSpot™ assay. Cellular assays: p-Ser2, p-Ser5 and total RNA pol II signals were assessed 4hrs after compound treatment while ɣH2AX and BRCA1 signals were measured after 24-48hr compound treatment via Immunofluorescence (IF) assays. Cell proliferation was determined using cell titer glo after 48-72hrs compound treatment. Apoptosis was measured by annexin V/PI staining and flow cytometry analysis after 48-72hrs of treatment. Cell cycle profile was evaluated with Click-iT-EdU and FxCycle violet stain and flow cytometry analysis following 24-48hrs of treatment. Mouse xenograft: balb/c mice were implanted subcutaneously with H1048 or MDA-MB-468 cells and randomized for treatment with test drug or vehicle when tumors reached 150-200mm3. Mice were dosed BID through oral administration for 4 weeks. Combination effect of Compound A and lurbinectedin was tested in vivo. Lurbinectedin and Compound A were dosed QW and BID respectively for 28 days in H1048 CDX model. Results: A series of CDK12 inhibitors were designed and profiled in biochemical and cellular assays. A representative member of the class, Compound A, exhibited selectivity over CDK2, CDK7, and CDK9 of 46-, 27-, and 9-fold, respectively. Compound A inhibited proliferation in a panel of cell lines with EC50 in the low nanomolar range. Compound A treatment led to dose dependent apoptosis in multiple cancer cell lines and induced G2/M arrest in cancer cell lines. In vitro, combination treatment with Compound A and lurbinectedin lead to increased DNA damage accumulation, decreased homologous recombination repair, and overall enhanced antiproliferation. Dose dependent tumor growth inhibition in SCLC and TNBC CDX models was observed with Compound A treatment. Enhanced and durable antitumor effect was observed with lurbinectedin and Compound A combination compared to single agent treatment in vivo. Conclusions: We designed and profiled orally available, CDK12 selective inhibitors with potent activity as single agent in vitro and in vivo in multiple cancer models. Compound A demonstrated enhanced antitumor effect in vitro and in vivo when combined with DNA damaging agents. These data support the rationale for advancing one or more members of this class toward clinical development. Citation Format: Shan hu, David Moebius, Wojciech Dworakowski, Elliott Cooper, Derek LaPlaca, Sydney Alnemy, Phone Perera, Jason Marineau, Claudio Chuaqui, John P. Carulli, Eric Olson. An oral and selective CDK12 inhibitor demonstrates robust anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5393.

MAF1, a repressor of RNA polymerase III-dependent transcription, regulates bone mass.

MAF1, a key repressor of RNA polymerase (pol) III-mediated transcription, has been shown to promote mesoderm formation in vitro. Here, we show that MAF1 plays a critical role in regulating osteoblast differentiation and bone mass. Global deletion of MAF1 (Maf1-/- mice) produced a high bone mass phenotype. However, osteoblasts isolated from Maf1-/- mice showed reduced osteoblastogenesis ex vivo. Therefore, we determined the phenotype of mice overexpressing MAF1 in cells from the mesenchymal lineage (Prx1-Cre;LSL-MAF1 mice). These mice showed increased bone mass. Ex vivo, cells from these mice showed enhanced osteoblastogenesis concordant with their high bone mass phenotype. Thus, the high bone mass phenotype in Maf1-/- mice is likely due to confounding effects from the global absence of MAF1. MAF1 overexpression promoted osteoblast differentiation of ST2 cells while MAF1 downregulation inhibited differentiation, indicating MAF1 enhances osteoblast formation. However, other perturbations used to repress RNA pol III transcription, inhibited osteoblast differentiation. However, decreasing RNA pol III transcription through these perturbations enhanced adipogenesis in ST2 cells. RNA-seq analyzed the basis for these opposing actions on osteoblast differentiation. The different modalities used to perturb RNA pol III transcription resulted in distinct gene expression changes, indicating that this transcription process is highly sensitive and triggers diverse gene expression programs and phenotypic outcomes. Specifically, MAF1 induced genes known to promote osteoblast differentiation. Furthermore, genes that are induced during osteoblast differentiation displayed codon bias. Together, these results reveal a novel role for MAF1 and RNA pol III-mediated transcription in osteoblast fate determination, differentiation, and bone mass regulation.

Nuclear oligo hashing improves differential analysis of single-cell RNA-seq

Single-cell RNA sequencing (scRNA-seq) offers a high-resolution molecular view into complex tissues, but suffers from high levels of technical noise which frustrates efforts to compare the gene expression programs of different cell types. “Spike-in” RNA standards help control for technical variation in scRNA-seq, but using them with recently developed, ultra-scalable scRNA-seq methods based on combinatorial indexing is not feasible. Here, we describe a simple and cost-effective method for normalizing transcript counts and subtracting technical variability that improves differential expression analysis in scRNA-seq. The method affixes a ladder of synthetic single-stranded DNA oligos to each cell that appears in its RNA-seq library. With improved normalization we explore chemical perturbations with broad or highly specific effects on gene regulation, including RNA pol II elongation, histone deacetylation, and activation of the glucocorticoid receptor. Our methods reveal that inhibiting histone deacetylation prevents cells from executing their canonical program of changes following glucocorticoid stimulation.

First Report of Bacterial Leaf Spot of Coriander Caused by Pseudomonas syringae pv. coriandricola in China.

Coriander (Coriandrum sativum L.) or Chinese parsley is a culinary herb with multiple medicinal effects that are widely used in cooking and traditional medicine. From September to November 2019, symptoms were observed in 2-month-old coriander plants from coriander fields in Lanzhou and Wenzhou, China. The disease developed rapidly under cold and wet climatic conditions, and the infection rate was almost 80% in open coriander fields. Typical symptoms on leaves included small, water-soaked blotches and irregular brown spots surrounding haloes; as the disease progressed, the spots coalesced into necrotic areas. Symptomatic leaf tissue was surface sterilized, macerated in sterile distilled water, and cultured on nutrient agar plates at 28 °C for 48 h (Koike and Bull, 2006). After incubation, six bacterial colonies, which were individually isolated from collected samples from two different areas, were selected for further study. Colonies on NA plate were small, round, raised, white to cream-colored, and had smooth margins. All bacterial isolates were gram-negative, rod-shaped and nonfluorescent on King's B medium. The bacteria were positive for levan production, Tween 80 hydrolysis, and tobacco hypersensitivity but negative for oxidase, potato slice rot test, arginine dihydrolase, ice nucleation activity, indole production and H2S production. The suspension of representative isolate for inoculating of plants was obtained from single colony on King's B medium for 2-3 days at 28 °C. DNA was extracted from bacterial suspensions of YS2003200102 cultured in 20 ml of King's B medium broth at 28 °C for 1 day. Extraction was performed with a TIANamp Bacterial DNA Kit (TIANGEN, China) according to the manufacturer's recommendations. The pathogen was confirmed by amplification and sequencing of the glyceraldehyde-3-phosphate dehydrogenase A (gapA) gene, the citrate synthase (gltA) gene, the DNA gyrase B (gyrB) gene and the RNA polymerase sigma factor 70 (rpoD) gene using gapA-For/gapA-Rev, gltA-For/gltA-Rev, gyrB-For/gryB-Rev, rpoD-For/rpoD-Rev primers, respectively (Popović et al., 2019). The sequences of the PCR products were deposited in GenBank with accession numbers MZ681931 (gapA), MZ681932 (gltA), MZ681933 (gyrB), and MZ681934 (rpoD). Phylogenetic analysis of multiple genes (Xu and Miller, 2013) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolates and ten published sequences of P. syringae pv. coriandricola were clustered into one clade with a 100% confidence level. To confirm the pathogenicity of isolate YS2003200102, 2-month-old healthy coriander plants were inoculated by spraying the leaves with a bacterial suspension (108 CFU ml-1) at 28 °C incubation temperature and 70% relative humidity condition, and sterile distilled water was applied as a negative control treatment (Cazorla et al. 2005). Three replicates were conducted for every isolate, and each replicate included 6 coriander plants. After twelve days, only the inoculated leaves with bacterial suspension showed bacterial leaf spot resembling those observed on naturally infected coriander leaves. Cultures re-isolated from symptomatic leaves showed the same morphological characteristics and molecular traits as those initially isolated from infected leaves in the field. This bacterium was previously reported causing leaf spot of coriander in India and Spain (Gupta et al. 2013; Cazorla et al. 2005). To our knowledge, this is the first report of P. syringae pv. coriandricola causing leaf spot disease on coriander in China. Studies are needed on strategies to manage P. syringae pv. coriandricola in crops, because its prevalence may cause yield loss on coriander in China.

KAT6B May Be Applied as a Potential Therapeutic Target for Glioma.

Glioma is a prevalent malignancy among brain tumors with high modality and low prognosis. Ferroptosis has been identified to play a crucial role in the progression and treatment of cancers. KAT6B, as a histone acetyltransferase, is involved in multiple cancer development. However, the function of KAT6B in glioma is still elusive. Here, we aimed to evaluate the effect of KAT6B on ferroptosis in glioma cells and explored the potential mechanisms. We observed that the expression of KAT6B was enhanced in clinical glioma samples. The viability of glioma cells was repressed by erastin and the overexpression of KAT6B rescued the phenotype in the cells. Meanwhile, the apoptosis of glioma cells was induced by the treatment of erastin, while the overexpression of KAT6B blocked the effect in the cells. The levels of lipid ROS and iron were promoted by the treatment of erastin and the overexpression of KAT6B could reverse the effect in the cells. Mechanically, we identified that the expression of STAT3 was repressed by the KAT6B knockdown in glioma cells. The KAT6B was able to enrich on the promoter of STAT3 in glioma cells. Meanwhile, ChIP assay showed that the knockdown of KAT6B inhibited the enrichment of histone H3 lysine 23 acetylation (H3K23ac) and RNA polymerase II (RNA pol II) on STAT3 promoter in the cells. Depletion of STAT3 reversed KAT6B-regulated viability, apoptosis, and ferroptosis of glioma cells. Thus, we concluded that KAT6B contributes to glioma progression by repressing ferroptosis via epigenetically inducing STAT3.

RNA polymerase I (Pol I) lobe-binding subunit Rpa12.2 promotes RNA cleavage and proofreading

Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides.

A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana

BackgroundCRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS).ResultsIn this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger.ConclusionsThe tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

Functional characterization of the human Cdk10/Cyclin Q complex.

Cyclin-dependent kinases (CDKs) are key players in cell cycle regulation and transcription. The CDK-family member Cdk10 is important for neural development and can act as a tumour suppressor, but the underlying molecular mechanisms are largely unknown. Here, we provide an in-depth analysis of Cdk10 substrate specificity and function. Using recombinant Cdk10/CycQ protein complexes, we characterize RNA pol II CTD, c-MYC and RB1 as in vitro protein substrates. Using an analogue-sensitive mutant kinase, we identify 89 different Cdk10 phosphosites in HEK cells originating from 66 different proteins. Among these, proteins involved in cell cycle, translation, stress response, growth signalling, as well as rRNA, and mRNA transcriptional regulation, are found. Of a set of pan-selective CDK- and Cdk9-specific inhibitors tested, all inhibited Cdk10/CycQ at least five times weaker than their proposed target kinases. We also identify Cdk10 as an in vitro substrate of Cdk1 and Cdk5 at multiple sites, allowing for a potential cross-talk between these CDKs. With this functional characterization, Cdk10 adopts a hybrid position in both cell cycle and transcriptional regulation.