Transcription Apparatus Research Articles

Identification of target genes regulated by encystation-induced transcription factor Myb2 using knockout mutagenesis in Giardia lamblia

BackgroundEncystation is one of the two processes comprising the life cycle of Giardia lamblia, a protozoan pathogen with tetraploid genome. Giardia lamblia Myb2 (GlMyb2) is a distinct encystation-induced transcription factor whose binding sites are found in the promoter regions of many encystation-induced genes, including its own.MethodsTwo sequential CRISPR/Cas9 experiments were performed to remove four glmyb2 alleles. The expression level of G. lamblia cyst wall protein 1 (GlCWP1), a well-known target gene of GlMyb2, was measured via western blotting and immunofluorescence assays. Chromatin immunoprecipitation experiments using anti-GlMyb2 antibodies were performed on the encysting G. lamblia cells. Quantitative real-time PCR was performed to confirm an expression of candidate GlMyb2-regulated genes by comparing the transcript level for each target candidate in wild-type and knockout mutant Giardia. The promoter region of glcwp1 was analyzed via deletion and point mutagenesis of the putative GlMyb2 binding sites in luciferase reporters.ResultsCharacterization of the null glmyb2 mutant indicated loss of functions related to encystation, i.e. cyst formation, and expression of GlCWP1. The addition of the wild-type glmyb2 gene to the null mutant restored the defects in encystation. Chromatin immunoprecipitation experiments revealed dozens of target genes. Nineteen genes were confirmed as GlMyb2 regulons, which include the glmyb2 gene, six for cyst wall proteins, five for signal transduction, two for transporter, two for metabolic enzymes, and three with unknown functions. Detailed analysis on the promoter region of glcwp1 defined three GlMyb2 binding sites important in its encystation-induced expression.ConclusionsOur data confirm that GlMyb2 acts as a transcription activator especially during encystation by comparing the glmyb2 knockout mutant with the wild type. Further investigation using glmyb2 null mutant will provide knowledge regarding transcriptional apparatus required for the encystation process of G. lamblia.Graphical

The role of PAP4/FSD3 and PAP9/FSD2 in heat stress responses of chloroplast genes

Chloroplasts’ mechanisms of adaptation to elevated temperatures are largely determined by the gene expression of the plastid transcription apparatus. Gene disruption of iron-containing superoxide dismutase PAP4/FSD3 and PAP9/FSD2, which are parts of the DNA–RNA polymerase complex of plastids, contributed to a decrease in resistance to oxidative stress caused by the prolonged action of elevated temperatures (5 days, 30 °C). Under heat stress conditions, pap4/fsd3 and pap9/fsd2 mutants showed a decline in chlorophyll content and photosynthesis level, as measured by photosynthetic parameters, and a different amplitude of HSP gene response to heat stress. The expression of nuclear- and plastid-encoded photosynthesis genes and corresponding proteins was strongly inhibited in the mutants as compared with wild-type plants and was further suppressed or displayed no additional changes at 30 °C. NEP-dependent plastid genes, as well as NEP genes RPOTp and RPOTmp, were also downregulated in the mutants by high temperature or remained stable, unlike in wild-type seedlings where these genes were strongly upregulated. The results obtained correspond to the concept of the complex effect of various forms of reactive oxygen species under all types of stresses, including heat stress, and confirm the hypothesis of a new regulatory function in plastid transcription acquired by enzymatic proteins during evolution.

Abstract 6264: Mechanisms of resistance to doxorubicin in HOS-MNNG cell line

Abstract The use of poly-chemotherapy combined with surgical resection has good efficiency and increased the survival rates of osteosarcoma (OS) patients to 70%, however these rates have not evolved since the 1970’s. On the other hand, survival drops to 30% among patients with metastases and/or bad response to treatment. This suggests that metastasis and resistance to treatment are key obstacles to tackle in order to reduce mortality. In this context this study has the aim to identify mechanisms that could participate in the emergence of resistance to doxorubicin, one of the most often used chemotherapeutic molecules to treat OS. For this purpose, HOS-MNNG cells were exposed to increasing concentrations of doxorubicin over a long period of time (over 6 months). Transcriptional and super-enhancer (SE) profiles of highly resistant (IC50 greater by 250 times) cells obtained as such were then compared to naive untreated cells. We hypothesized identification of target genes of resistant-specific super-enhancers, which are regulatory regions that mobilize a large proportion of the cell’s transcriptional apparatus and epigenetic regulators, would allow to unveil key genes in the emergence of resistance. We observed the presence of highly efficient super-enhancers within loci located in close proximity to genes encoding for ATP-Binding Cassette (ABC) transporters only within the high dose doxorubicin resistant cells. The associated genes were among the most highly up-regulated genes in the resistant cells and western blot analysis confirms higher concentration of encoded proteins compared to naive cells. The use of one BETi namely JQ1 - that inhibits recruitment of transcriptional co-factors on regulatory DNA regions such as enhancers and SEs - sensitizes resistant cells to doxorubicin. This suggests that the identified SEs in close proximity could play key role in the emergence of resistance. In order to track emergence of the mechanisms of resistance and to appreciate the transcriptional and epigenetic heterogeneity among resistant cells, we performed single cell 10x multiome (ATAC+RNA) on naive, intermediate and high dose doxorubicin resistant cells. While the high expression of genes encoding for the ABC transporters revealed in bulk analysis seemed ubiquitous among all the clusters of high-dose resistant cells, intermediate-dose resistant cells demonstrate no or very low expression for these genes. This could suggest that the first form of resistance passes through other mechanisms. In conclusion ABC transporters could be highly involved in the emergence of resistance to high-doses of doxorubicin in OS. A heterogeneous feature could be expected in patients as resistance to intermediate doses do not seem to depend on this mechanism. Finally, the use of BETi could be suggested in patients with bad response to treatment. This project is supported by Région Pays de la Loire, ANR, la Ligue contre le cancer and SFCE. Citation Format: Robel A. Tesfaye, Mélanie Lavaud, Anaïs Postec, Céline Charrier, Rose-Anne Thépault, Franck Verrecchia, Marc Baud'huin, François Lamoureux, Steven Georges, Benjamin Ory. Mechanisms of resistance to doxorubicin in HOS-MNNG cell line [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 6264.

The Translational Regulation in mTOR Pathway.

The mechanistic/mammalian target of rapamycin (mTOR) plays a master role in cell proliferation and growth in response to insulin, amino acids, energy levels, and oxygen. mTOR can coordinate upstream signals with downstream effectors, including transcriptional and translational apparatuses to regulate fundamental cellular processes such as energy utilization, protein synthesis, autophagy, cell growth, and proliferation. Of the above, protein synthesis is highly energy-consuming; thus, mRNA translation is under the tight and immediate control of mTOR signaling. The translational regulation driven by mTOR signaling mainly relies on eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP), ribosomal protein S6 kinase (S6K), and its downstream players, which are significant in rapid cellular response to environmental change. mTOR signaling not only controls the general mRNA translation, but preferential mRNA translation as well. This means that mTOR signaling shows the stronger selectivity to particular target mRNAs. Some evidence has supported the contribution of 4E-BP and La-related proteins 1 (LARP1) to such translational regulation. In this review, we summarize the mTOR pathway and mainly focus on mTOR-mediated mRNA translational regulation. We introduce the major components of mTOR signaling and their functions in translational control in a general or particular manner, and describe how the specificity of regulation is coordinated. Furthermore, we summarize recent research progress and propose additional ideas for reference. Because the mTOR pathway is on the center of cell growth and metabolism, comprehensively understanding this pathway will contribute to the therapy of related diseases, including cancers, type 2 diabetes, obesity, and neurodegeneration.

Structural insights into nuclear transcription by eukaryotic DNA-dependent RNA polymerases.

The eukaryotic transcription apparatus synthesizes a staggering diversity of RNA molecules. The labour of nuclear gene transcription is, therefore, divided among multiple DNA-dependent RNA polymerases. RNA polymerase I (Pol I) transcribes ribosomal RNA, Pol II synthesizes messenger RNAs and various non-coding RNAs (including long non-coding RNAs, microRNAs and small nuclear RNAs) and Pol III produces transfer RNAs and other short RNA molecules. Pol I, Pol II and Pol III are large, multisubunit protein complexes that associate with a multitude of additional factors to synthesize transcripts that largely differ in size, structure and abundance. The three transcription machineries share common characteristics, but differ widely in various aspects, such as numbers of RNA polymerase subunits, regulatory elements and accessory factors, which allows them to specialize in transcribing their specific RNAs. Common to the three RNA polymerases is that the transcription process consists of three major steps: transcription initiation, transcript elongation and transcription termination. In this Review, we outline the common principles and differences between the Pol I, Pol II and Pol III transcription machineries and discuss key structural and functional insights obtained into the three stages of their transcription processes.

The Codependent Expression of the Essential Mammalian RNA Polymerase I PAF49/PAF53 Heterodimer

Ribosome biogenesis (RB) is a complex process that plays a central role in maintaining cellular homeostasis and supporting cell growth. Therefore, dysregulation of RB contributes to pathologies such as cancer, ribosomopathies, neurodegenerative diseases, and aging. Our lab studies transcription of ribosomal RNA by RNA polymerase I (Pol I), the rate‐limiting step in ribosome biogenesis. Many studies have demonstrated that inhibiting Pol I transcription in cancer cells causes cell death while untransformed cells arrest, thereby making the Pol I apparatus a target for chemotherapeutic agents. This study focuses on two mammalian polymerase associated factors of the Pol I transcription apparatus, PAF49 and PAF53. The PAFs form a heterodimeric complex and are required for rDNA transcription and cell proliferation. The goal of this study is to determine the role(s) of PAF49 and PAF53 in rDNA transcription and understand how these two proteins are regulated.To rapidly knock down (KD) either PAF49 or 53 in HEK293 cells, a novel system that utilizes CRISPR/Cas 9 and an auxin inducible degron was used. Currently, we are using this system to determine if KD of PAF49 effects the expression of PAF53 and vice versa. Our data demonstrates that KD of PAF49 causes the rapid depletion of PAF53 protein levels while KD of PAF53 has no effect on PAF49. Additionally, we have found that the dimerization domain of PAF49 is not sufficient to stabilize PAF53. Rescue of PAF53 protein levels requires PAF49 to dimerize and bind to the polymerase. Dimerization to PAF53 and binding to the polymerase is also necessary to support cell proliferation and rDNA transcription. Further, KD of PAF49 or PAF53 has no effect on the protein levels of the core subunits of Pol I. These findings are significant because they aid in further understanding the process of rDNA transcription by Pol I. Additionally, they will contribute to the discovery of novel drug targets that could be utilized in effective cancer treatments. For example, disrupting the interaction between PAF49 and Pol I should inhibit Pol I transcription and cause cell death in cancer cells.

Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock.

Heat-shock transcription factor 1 (HSF1) orchestrates the fast and vast cellular response to heat shock through increased expression of heat-shock proteins. However, how HSF1 rapidly and reversibly regulates transcriptional reprogramming remains poorly defined. Here by combining super-resolution imaging, in vitro reconstitution and high-throughput sequencing, we reveal that HSF1 forms small nuclear condensates via liquid-liquid phase separation at heat-shock-protein gene loci and enriches multiple transcription apparatuses through co-phase separation to promote the transcription of target genes. Furthermore, the phase-separation capability of HSF1 is fine-tuned through phosphorylation at specific sites within the regulatory domain. Last, we discovered that HSP70 disperses HSF1 condensates to attenuate transcription following the cessation of heat shock and further prevents the gel-like phase transition of HSF1 under extended heat-shock stress. Our work reveals an inducible and reversible phase-separation feedback mechanism for dynamic regulation of HSF1 activity to drive the transcriptional response and maintain protein homeostasis during acute stress.

CTCF-mediated chromatin looping provides a topological framework for the formation of phase-separated transcriptional condensates.

CTCF is crucial to the organization of mammalian genomes into loop structures. According to recent studies, the transcription apparatus is compartmentalized and concentrated at super-enhancers to form phase-separated condensates and drive the expression of cell-identity genes. However, it remains unclear whether and how transcriptional condensates are coupled to higher-order chromatin organization. Here, we show that CTCF is essential for RNA polymerase II (Pol II)-mediated chromatin interactions, which occur as hyperconnected spatial clusters at super-enhancers. We also demonstrate that CTCF clustering, unlike Pol II clustering, is independent of liquid-liquid phase-separation and resistant to perturbation of transcription. Interestingly, clusters of Pol II, BRD4, and MED1 were found to dissolve upon CTCF depletion, but were reinstated upon restoration of CTCF, suggesting a potent instructive function for CTCF in the formation of transcriptional condensates. Overall, we provide evidence suggesting that CTCF-mediated chromatin looping acts as an architectural prerequisite for the assembly of phase-separated transcriptional condensates.

The Reasonable Effectiveness of Randomness in Scalable and Integrative Gene Regulatory Network Inference and Beyond

Gene regulation is orchestrated by a vast number of molecules, including transcription factors and co-factors, chromatin regulators, as well as epigenetic mechanisms, and it has been shown that transcriptional misregulation, e.g., caused by mutations in regulatory sequences, is responsible for a plethora of diseases, including cancer, developmental or neurological disorders. As a consequence, decoding the architecture of gene regulatory networks has become one of the most important tasks in modern (computational) biology. However, to advance our understanding of the mechanisms involved in the transcriptional apparatus, we need scalable approaches that can deal with the increasing number of large-scale, high-resolution, biological datasets. In particular, such approaches need to be capable of efficiently integrating and exploiting the biological and technological heterogeneity of such datasets in order to best infer the underlying, highly dynamic regulatory networks, often in the absence of sufficient ground truth data for model training or testing. With respect to scalability, randomized approaches have proven to be a promising alternative to deterministic methods in computational biology. As an example, one of the top performing algorithms in a community challenge on gene regulatory network inference from transcriptomic data is based on a random forest regression model. In this concise survey, we aim to highlight how randomized methods may serve as a highly valuable tool, in particular, with increasing amounts of large-scale, biological experiments and datasets being collected. Given the complexity and interdisciplinary nature of the gene regulatory network inference problem, we hope our survey maybe helpful to both computational and biological scientists. It is our aim to provide a starting point for a dialogue about the concepts, benefits, and caveats of the toolbox of randomized methods, since unravelling the intricate web of highly dynamic, regulatory events will be one fundamental step in understanding the mechanisms of life and eventually developing efficient therapies to treat and cure diseases.

KSHV transactivator-derived small peptide traps coactivators to attenuate MYC and inhibits leukemia and lymphoma cell growth

In herpesvirus replicating cells, host cell gene transcription is frequently down-regulated because important transcriptional apparatuses are appropriated by viral transcription factors. Here, we show a small peptide derived from the Kaposi’s sarcoma-associated herpesvirus transactivator (K-Rta) sequence, which attenuates cellular MYC expression, reduces cell proliferation, and selectively kills cancer cell lines in both tissue culture and a xenograft tumor mouse model. Mechanistically, the peptide functions as a decoy to block the recruitment of coactivator complexes consisting of Nuclear receptor coactivator 2 (NCOA2), p300, and SWI/SNF proteins to the MYC promoter in primary effusion lymphoma cells. Thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM seq) with target-transcriptional analyses further confirm that the viral peptide directly attenuates MYC and MYC-target gene expression. This study thus provides a unique tool to control MYC activation, which may be used as a therapeutic payload to treat MYC-dependent diseases such as cancers and autoimmune diseases.

Methylation of Host Genes Associated with Coronavirus Infection from Birth to 26 Years.

DNA methylation (DNAm) patterns over time at 1146 CpGs on coronavirus-related genes were assessed to understand whether the varying differences in susceptibility, symptoms, and the outcomes of the SARS-CoV-2 infection in children and young adults could be explained through epigenetic alterations in a host cell’s transcriptional apparatus to coronaviruses. DNAm data from the Isle of Wight birth cohort (IOWBC) at birth, 10, 18, and 26 years of age were included. Linear mixed models with repeated measurements stratified by sex were used to examine temporal patterns, and cluster analysis was performed to identify CpGs following similar patterns. CpGs on autosomes and sex chromosomes were analyzed separately. The association of identified CpGs and expression of their genes were evaluated. Pathway enrichment analyses of the genes was conducted at FDR = 0.05. DNAm at 635 of the 1146 CpGs on autosomes showed statistically significant time effects (FDR = 0.05). The 635 CpGs were classified into five clusters with each representing a unique temporal pattern of DNAm. Of the 29 CpGs on sex chromosomes, DNAm at seven CpGs in males and eight CpGs in females showed time effects (FDR = 0.05). Sex-specific and non-specific associations of DNAm with gene expression were found at 24 and 93 CpGs, respectively. Genes which mapped the 643 CpGs represent 460 biological processes. We suggest that the observed variability in DNAm with advancing age may partially explain differing susceptibility, disease severity, and mortality of coronavirus infections among different age groups.

Comparative Analysis of Transcriptional Regulation Patterns: Understanding the Gene Expression Profile in Nucleocytoviricota.

The nucleocytoplasmic large DNA viruses (NCLDV) possess unique characteristics that have drawn the attention of the scientific community, and they are now classified in the phylum Nucleocytoviricota. They are characterized by sharing many genes and have their own transcriptional apparatus, which provides certain independence from their host’s machinery. Thus, the presence of a robust transcriptional apparatus has raised much discussion about the evolutionary aspects of these viruses and their genomes. Understanding the transcriptional process in NCLDV would provide information regarding their evolutionary history and a better comprehension of the biology of these viruses and their interaction with hosts. In this work, we reviewed NCLDV transcription and performed a comparative functional analysis of the groups of genes expressed at different times of infection of representatives of six different viral families of giant viruses. With this analysis, it was possible to observe a temporal profile of their gene expression and set of genes activated in specific phases throughout the multiplication cycle as a common characteristic of this group. Due to the lack of information regarding the transcriptional regulation process of this group of pathogens, we sought to provide information that contributes to and opens up the field for transcriptional studies of other viruses belonging to Nucleocytoviricota.

Widespread remodeling of the m6A RNA-modification landscape by a viral regulator of RNA processing and export

N6-methyladenosine (m6A) is the most abundant internal messenger RNA (mRNA) modification, contributing to the processing, stability, and function of methylated RNAs. Methylation occurs in the nucleus during pre-mRNA synthesis and requires a core methyltransferase complex consisting of METTL3, METTL14, and WTAP. During herpes simplex virus (HSV-1) infection, cellular gene expression is profoundly suppressed, allowing the virus to monopolize the host transcription and translation apparatus and antagonize antiviral responses. The extent to which HSV-1 uses or manipulates the m6A pathway is not known. Here, we show that, in primary fibroblasts, HSV-1 orchestrates a striking redistribution of the nuclear m6A machinery that progresses through the infection cycle. METTL3 and METTL14 are dispersed into the cytoplasm, whereas WTAP remains nuclear. Other regulatory subunits of the methyltransferase complex, along with the nuclear m6A-modified RNA binding protein YTHDC1 and nuclear demethylase ALKBH5, are similarly redistributed. These changes require ICP27, a viral regulator of host mRNA processing that mediates the nucleocytoplasmic export of viral late mRNAs. Viral gene expression is initially reduced by small interfering RNA (siRNA)-mediated inactivation of the m6A methyltransferase but becomes less impacted as the infection advances. Redistribution of the nuclear m6A machinery is accompanied by a wide-scale reduction in the installation of m6A and other RNA modifications on both host and viral mRNAs. These results reveal a far-reaching mechanism by which HSV-1 subverts host gene expression to favor viral replication.

Glial-Specific Deletion of Med12 Results in Rapid Hearing Loss via Degradation of the Stria Vascularis.

Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult CNS results in region-specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiologic analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis.SIGNIFICANCE STATEMENT Mutations in Mediator protein complex subunit 12 (Med12) are associated with X-linked intellectual disability syndromes and hearing loss. Using temporal-conditional genetic approaches in CNS glia, we found that loss of Med12 results in severe hearing loss in adult animals through rapid degeneration of the stria vascularis. Our study describes the first animal model that recapitulates hearing loss identified in Med12-related disorders and provides a new system in which to examine the underlying cellular and molecular mechanisms of Med12 function in the adult nervous system.

RNA Polymerase III, Ageing and Longevity.

Transcription in eukaryotic cells is performed by three RNA polymerases. RNA polymerase I synthesises most rRNAs, whilst RNA polymerase II transcribes all mRNAs and many non-coding RNAs. The largest of the three polymerases is RNA polymerase III (Pol III) which transcribes a variety of short non-coding RNAs including tRNAs and the 5S rRNA, in addition to other small RNAs such as snRNAs, snoRNAs, SINEs, 7SL RNA, Y RNA, and U6 spilceosomal RNA. Pol III-mediated transcription is highly dynamic and regulated in response to changes in cell growth, cell proliferation and stress. Pol III-generated transcripts are involved in a wide variety of cellular processes, including translation, genome and transcriptome regulation and RNA processing, with Pol III dys-regulation implicated in diseases including leukodystrophy, Alzheimer’s, Fragile X-syndrome and various cancers. More recently, Pol III was identified as an evolutionarily conserved determinant of organismal lifespan acting downstream of mTORC1. Pol III inhibition extends lifespan in yeast, worms and flies, and in worms and flies acts from the intestine and intestinal stem cells respectively to achieve this. Intriguingly, Pol III activation achieved through impairment of its master repressor, Maf1, has also been shown to promote longevity in model organisms, including mice. In this review we introduce the Pol III transcription apparatus and review the current understanding of RNA Pol III’s role in ageing and lifespan in different model organisms. We then discuss the potential of Pol III as a therapeutic target to improve age-related health in humans.

Hierarchy of transcription factor network in Escherichia coli K-12: H-NS-mediated silencing and Anti-silencing by global regulators.

Transcriptional regulation for genome expression determines growth and adaptation of single-cell bacteria that are directly exposed to environment. The transcriptional apparatus in Escherichia coli K-12 is composed of RNA polymerase core enzyme and two groups of its regulatory proteins, seven species of promoter-recognition subunit sigma and about 300 species of transcription factors. The identification of regulatory targets for all these regulatory proteins is critical toward understanding the genome regulation as a whole. For this purpose, we performed a systematic search in vitro of the whole set of binding sites for each factor by gSELEX system. This review summarizes the accumulated knowledge of regulatory targets for more than 150 TFs from E. coli K-12. Overall TFs could be classified into four families: nucleoid-associated bifunctional TFs; global regulators; local regulators; and single-target regulators, in which the regulatory functions remain uncharacterized for the nucleoid-associated TFs. Here we overview the regulatory targets of two nucleoid-associated TFs, H-NS and its paralog StpA, both together playing the silencing role of a set of non-essential genes. Participation of LeuO and other global regulators have been indicated for the anti-silencing. Finally, we propose the hierarchy of TF network as a key framework of the bacterial genome regulation.

Abstract 1141: CDK9 inhibition is selective for transcriptionally addicted tumors harboring MYC genomic amplifications

Abstract Transcriptional deregulation is a hallmark of many cancers, including a subset that are “transcriptionally addicted” and depend on high levels of transcription for oncogenic program genes. Although these tumors are thought to be highly susceptible to targeting of the transcriptional apparatus, their molecular definition remains challenging. To better define molecular sensitivity to transcriptional inhibition, we profiled pan-cancer sensitivity to KB-0742 — a potent, selective, and orally bioavailable small molecule inhibitor of the transcription elongation cofactor CDK9. Multiplexed sensitivity profiling across ~1,000 adherent and suspension immortalized cell lines using the Broad PRISM platform revealed lineage and molecular determinants of sensitivity. Consistent with prior reports, MYC genomic amplification emerged as a key driver of CDK9 inhibitor sensitivity, and this was especially pronounced in non-small cell lung cancer. Sensitivity to CDK9 inhibition was further observed in MYC amplified/over-expressed ex vivo primary patient tumor cell cultures and patient-derived xenografts. Analysis of the temporal kinetics of CDK9 inhibition revealed a rapid collapse of oncogenic transcription programs comprised largely of short half-life transcripts including key oncogenes such as MYC and MCL1. In CDK9 sensitive ex vivo and in vivo models, suppression of oncogenic transcription for >8 hours was followed by apoptosis. In ex vivo models, CDK9 inhibitor sensitivity was observed for both treatment naïve and heavily pretreated patient samples. For in vivo models, CDK9 inhibition on an intermittent dosing schedule achieved sustained target coverage, as evidenced by both direct readouts of CDK9 activity and corresponding transcriptional response, and ultimately resulted in sustained tumor growth inhibition in multiple solid tumor types. These data suggest that MYC genomic amplification may serve as an important feature defining sensitivity to CDK9 inhibition in patients with advanced solid tumors. Citation Format: Melinda A. Day, Nikolaus D. Obholzer, Akanksha Pandey, Tom Chen, Tong Liang, Rosa A. Villagomez, Mulini Pingili, Jost V. Koren, David B. Freeman, Holly M. Nguyen, Jennifer L. Conor, Eva Corey, Matthew G. Reese, Andrew Boghossian, Brienne Engel, Melissa M. Ronan, Jennifer A. Roth, Joesph P. Vacca, Peter B. Rahl, Marius S. Pop, Benjamin W. Trotter, Charles Y. Lin, Jorge C. DiMartino, Pavan Kumar, Douglas C. Saffran. CDK9 inhibition is selective for transcriptionally addicted tumors harboring MYC genomic amplifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1141.

Abstract 1146: Targeted degradation of the enhancer lysine acetyltransferases CBP and p300

Abstract The chromatin regulators CREB-binding protein (CBP) and p300 are factors critical for establishing and activating enhancer-mediated transcription. These large proteins are made up of at least ten distinct functional domains and are known to interact with over 400 other proteins including DNA-binding transcription factors, other chromatin regulators, and components of the general transcription apparatus. In addition to mediating protein-protein interactions on chromatin, p300/CBP exert enzymatic activity through a lysine acetyltransferase (KAT) domain. p300/CBP can dynamically acetylate ~21,000 lysines on over 5,000 proteins including more than three dozen known enhancer-associated regulators. In cancer, p300/CBP have been implicated as both oncogenes and tumor suppressors in a large number of malignant cell types and active drug development projects by a number of groups have focused on the discovery of potent and selective compounds. Small molecules that target the p300/CBP bromodomain and KAT domains have provided a unique toolset for chromatin and chemical biologists to probe various p300/CBP functions at enhancers. However, it is clear that simultaneous inhibition of multiple functional domains, or even complete protein depletion, is required to ablate the enhancer activity mediated by p300/CBP. To pursue this goal further and to provide the research community with additional tools to probe the function of this important class of chromatin regulators, we have designed and characterized the first heterobifunctional chemical degrader of p300/CBP, dCBP-1. Bifunctional, or chimeric, degraders that leverage the activity of E3 ligases such as cereblon (CRBN) through chemical-induced proximity to proteins of interest have transformed the capabilities for chemical degradation. Aided by in silico modeling of a ternary complex of ligand-bound CRBN with the KAT and bromodomains of p300/CBP, we were able to efficiently synthesize a highly potent and selective degrader, dCBP-1. Using dCBP-1 as a tool, we profile the consequences of complete p300/CBP loss in cellular models of multiple myeloma, a tumor type uniquely addicted to the activity of both p300 and CBP. Proteomics profiling reveals selective effects on p300 and CBP, with loss of oncogenic MYC expression. Epigenomic profiles of gene expression and chromatin structures with dCBP-1 treatment reveals augmented effects of oncogene expression compared to inhibitors alone or in combination, with complete ablation of histone lysine acetylation and chromatin accessibility at enhancers, including the immunoglobulin locus enhancer that can drive oncogenic MYC expression in ~20% of multiple myeloma. Taken together, dCBP-1 is the first rapid potent and selective chemical degrader of p300/CBP and enables pharmacologic disruption of p300/CBP-regulated enhancers. Citation Format: Samuel Ojeda, Raghu Vannam, Jan Sayilgan, Barbara Karakyriakou, Eileen Hu, Johannes Kreuzer, Robert Morris, Xcanda Ixchel Herrera Lopez, Sumit Rai, Wilhelm Haas, Michael Lawrence, Christopher J. Ott. Targeted degradation of the enhancer lysine acetyltransferases CBP and p300 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1146.

The Plastid-Encoded RNA Polymerase-Associated Protein PAP9 Is a Superoxide Dismutase With Unusual Structural Features.

In Angiosperms, the plastid-encoded RNA polymerase (PEP) is a multimeric enzyme, essential for the proper expression of the plastid genome during chloroplast biogenesis. It is especially required for the light initiated expression of photosynthesis genes and the subsequent build-up of the photosynthetic apparatus. The PEP complex is composed of a prokaryotic-type core of four plastid-encoded subunits and 12 nuclear-encoded PEP-associated proteins (PAPs). Among them, there are two iron superoxide dismutases, FSD2/PAP9 and FSD3/PAP4. Superoxide dismutases usually are soluble enzymes not bound into larger protein complexes. To investigate this unusual feature, we characterized PAP9 using molecular genetics, fluorescence microscopy, mass spectrometry, X-ray diffraction, and solution-state NMR. Despite the presence of a predicted nuclear localization signal within the sequence of the predicted chloroplast transit peptide, PAP9 was mainly observed within plastids. Mass spectrometry experiments with the recombinant Arabidopsis PAP9 suggested that monomers and dimers of PAP9 could be associated to the PEP complex. In crystals, PAP9 occurred as a dimeric enzyme that displayed a similar fold to that of the FeSODs or manganese SOD (MnSODs). A zinc ion, instead of the expected iron, was found to be penta-coordinated with a trigonal-bipyramidal geometry in the catalytic center of the recombinant protein. The metal coordination involves a water molecule and highly conserved residues in FeSODs. Solution-state NMR and DOSY experiments revealed an unfolded C-terminal 34 amino-acid stretch in the stand-alone protein and few internal residues interacting with the rest of the protein. We hypothesize that this C-terminal extension had appeared during evolution as a distinct feature of the FSD2/PAP9 targeting it to the PEP complex. Close vicinity to the transcriptional apparatus may allow for the protection against the strongly oxidizing aerial environment during plant conquering of terrestrial habitats.

Structure of the bacteriophage PhiKZ non-virion RNA polymerase.

Bacteriophage ΦKZ (PhiKZ) is the archetype of a family of massive bacterial viruses. It is considered to have therapeutic potential as its host, Pseudomonas aeruginosa, is an opportunistic, intrinsically antibiotic resistant, pathogen that kills tens of thousands worldwide each year. ΦKZ is an incredibly interesting virus, expressing many systems that the host already possesses. On infection, it forms a ‘nucleus’, erecting a barrier around its genome to exclude host endonucleases and CRISPR-Cas systems. ΦKZ infection is independent of the host transcriptional apparatus. It expresses two different multi-subunit RNA polymerases (RNAPs): the virion RNAP (vRNAP) is injected with the viral DNA during infection to transcribe early genes, including those encoding the non-virion RNAP (nvRNAP), which transcribes all further genes. ΦKZ nvRNAP is formed by four polypeptides thought to represent homologues of the eubacterial β/β′ subunits, and a fifth with unclear homology, but essential for transcription. We have resolved the structure of ΦKZ nvRNAP to better than 3.0 Å, shedding light on its assembly, homology, and the biological role of the fifth subunit: it is an embedded, integral member of the complex, the position, structural homology and biochemical role of which imply that it has evolved from an ancestral homologue to σ-factor.