RNA Polymerase II Research Articles

NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis

BackgroundNotch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis.MethodsSize exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry.ResultsWe identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC.ConclusionsThis study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.DmMSvvPJYVoBWTrbTitSG7Video abstract

The histone chaperone Spt6 is required for normal recruitment of the capping enzyme Abd1 to transcribed regions

The histone chaperone Spt6 is involved in promoting elongation of RNA polymerase II (RNAPII), maintaining chromatin structure, regulating cotranscriptional histone modifications, and controlling mRNA processing. These diverse functions of Spt6 are partly mediated through its interactions with RNAPII and other factors in the transcription elongation complex. In this study, we used mass spectrometry to characterize the differences in RNAPII-interacting factors between wildtype cells and those depleted for Spt6, leading to the identification of proteins that depend on Spt6 for their interaction with RNAPII. The altered association of some of these factors could be attributed to changes in steady-state protein levels. However, Abd1, the mRNA cap methyltransferase, had decreased association with RNAPII after Spt6 depletion despite unchanged Abd1 protein levels, showing a requirement for Spt6 in mediating the Abd1–RNAPII interaction. Genome-wide studies showed that Spt6 is required for maintaining the level of Abd1 over transcribed regions, as well as the level of Spt5, another protein known to recruit Abd1 to chromatin. Abd1 levels were particularly decreased at the 5′ ends of genes after Spt6 depletion, suggesting a greater need for Spt6 in Abd1 recruitment over these regions. Together, our results show that Spt6 is important in regulating the composition of the transcription elongation complex and reveal a previously unknown function for Spt6 in the recruitment of Abd1.

Spt4 facilitates the movement of RNA polymerase II through the+2 nucleosomal barrier.

SummarySpt4 is a transcription elongation factor with homologs in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, and yet the in vivo function of Spt4 is unclear. Here, we assess the precise position of Spt4 during transcription and the consequences of the loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription, and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

Nanomaterial-Facilitated Cyclin-Dependent Kinase 7 Inhibition Suppresses Gallbladder Cancer Progression via Targeting Transcriptional Addiction.

Gallbladder cancer (GBC) is the most aggressive malignancy of the biliary tract cancer, and there is a lack of effective treatment. Here, we developed a nanoparticle platform (8P4 NP) that can deliver THZ1, a cyclin-dependent kinase 7 (CDK7) inhibitor, to treat GBC. Analysis of datasets demonstrated that CDK7 was positively correlated with poor prognosis. CDK7 inhibition suppressed cell proliferation, induced apoptosis, and caused cell cycle block in GBC cells. THZ1 downregulated CDK7-mediated phosphorylation of RNA polymerase II (RNAPII), resulting in a significant downregulation of transcriptional programs, with a preferential repression of oncogenic transcription factors. To improve the tumor targeting efficiency of THZ1, 8P4 NPs were prepared and assembled with THZ1 to form THZ1@8P4 NPs. Compared with free THZ1, THZ1@8P4 NPs showed more advantages in prolonging blood circulation, escaping from lysosomes and increasing cellular uptake. Importantly, THZ1@8P4 NPs demonstrated a more significant inhibition effect on GBC cells than free THZ1 in vitro. In addition, THZ1@8P4 NPs could efficiently deliver THZ1 to tumor sites in a patient-derived xenograft model of early recurrence, leading to tumor regression and transcriptional inhibition with minimal toxicity. In summary, we conclude that THZ1@8P4 NPs provide a potent therapeutic strategy that targets CDK7-mediated transcriptional addiction in GBC.

Transcription-coupled repair and the transcriptional response to UV-Irradiation

Lesions in genes that result in RNA polymerase II (RNAPII) stalling or arrest are particularly toxic as they are a focal point of genome instability and potently block further transcription of the affected gene. Thus, cells have evolved the transcription-coupled nucleotide excision repair (TC-NER) pathway to identify damage-stalled RNAPIIs, so that the lesion can be rapidly repaired and transcription can continue. However, despite the identification of several factors required for TC-NER, how RNAPII is remodelled, modified, removed, or whether this is even necessary for repair remains enigmatic, and theories are intensely contested. Recent studies have further detailed the cellular response to UV-induced ubiquitylation and degradation of RNAPII and its consequences for transcription and repair. These advances make it pertinent to revisit the TC-NER process in general and with specific discussion of the fate of RNAPII stalled at DNA lesions.

FACT is recruited to the +1 nucleosome of transcribed genes and spreads in a Chd1-dependent manner

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the+1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning.

A CpG island promoter drives the CXXC5 gene expression

CXXC5 is a member of the zinc-finger CXXC family that binds to unmethylated CpG dinucleotides. CXXC5 modulates gene expressions resulting in diverse cellular events mediated by distinct signaling pathways. However, the mechanism responsible for CXXC5 expression remains largely unknown. We found here that of the 14 annotated CXXC5 transcripts with distinct 5′ untranslated regions encoding the same protein, transcript variant 2 with the highest expression level among variants represents the main transcript in cell models. The DNA segment in and at the immediate 5′-sequences of the first exon of variant 2 contains a core promoter within which multiple transcription start sites are present. Residing in a region with high G–C nucleotide content and CpG repeats, the core promoter is unmethylated, deficient in nucleosomes, and associated with active RNA polymerase-II. These findings suggest that a CpG island promoter drives CXXC5 expression. Promoter pull-down revealed the association of various transcription factors (TFs) and transcription co-regulatory proteins, as well as proteins involved in histone/chromatin, DNA, and RNA processing with the core promoter. Of the TFs, we verified that ELF1 and MAZ contribute to CXXC5 expression. Moreover, the first exon of variant 2 may contain a G-quadruplex forming region that could modulate CXXC5 expression.

AGR2-Dependent Nuclear Import of RNA Polymerase II Constitutes a Specific Target of Pancreatic Ductal Adenocarcinoma in the Context of Wild-Type p53

Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2-RNAPII complex. We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation invivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents invivo. Of note, this therapeutic effect is efficient across various cancer types. Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Commuting to Work: Nucleolar Long Non-Coding RNA Control Ribosome Biogenesis from Near and Far.

Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.

A Robust Ex-Vivo Erythropoiesis System For Studying Transcriptional Regulation Of Globin Genes In Thalassaemia

Ex-vivo differentiation of hematopoietic stem cells and progenitors to erythroid cells helped tremendously in our understanding of transcriptional and epigenetic regulation of normal erythroid differentiation as large number of nucleated cells can be generated by these protocols. However, to study the disease mechanisms in haematological diseases, there are practical difficulties in obtaining mobilized CD34+ cells from the patients. Alternatively, peripheral blood mononuclear cells (PBMNCs), which have a small fraction of haematopoietic progenitors, have also been used to obtain cultured erythroid cells (cErCs). Though PBMNCs is an alternative source, the current protocols using these cells do not generate sufficient number of cErCs, which is a major hurdle for performing transcriptional and epigenetic studies in erythroid cells from patients with β thalassaemia and sickle cell disease. We established a modified two phase culture protocol using a serum free basal medium with IL-3, IL-6, Flt3, SCF in phase I and Epo, SCF, IL-3 and iron compounds in phase II and could generate up to 108 cells from PBMNCs isolated from 5-10ml blood of patients with homozygous β thalassaemia and those who were compound heterozygous for β thalassaemia and HPFH or δβ thalassaemia. Real time quantitative PCR (qPCR) analysis showed steady increase in the expression of α and β globin genes in different stages of erythropoiesis. As we obtained a large number of nucleated cells using this protocol, we could perform chromatin immunoprecipitation (ChIP) to study the binding of RNA Polymerase II (RNAPII), transcription cofactors, CBP and p300, and histone modifications at the regulatory sequences of β globin cluster. High levels of RNAPII was found at the coding sequences of β globin gene in the cErCs from both normal individuals and patients with splice site mutations suggesting that these mutations do not cause significant reduction in the transcription rate. Both CBP and p300 were found to bind at the HS3 and HS2 regions of locus control region in both normal and β thalassaemia cultures though they were not found at the promoter sequences of actively transcribed genes in the β globin cluster. In the patients compound heterozygous for β thalassaemia and HPFH-3 or (Aγδβ)° deletion/Inversion, we found increased γ globin gene expression compared to the normal controls and ChIP also showed high levels of RNAPII occupancy at the coding sequences of γ globin gene. As it has been shown earlier that alternative splicing is a transcriptional regulatory mechanism for cell specific expression of genes, we performed experiments to identify the alternatively spliced transcripts of β globin gene and their co-ordinated expression in human erythropoiesis. RT-PCRs and sequencing of β globin cDNA using various sets of primers that bind at the upstream, downstream and coding sequences showed 3 aberrantly spliced transcripts formed by the cryptic splice sites in exon1 and 4 novel transcripts by the activation of splice sites at the 5'UTR. Interestingly, 5' splice site mutations in intron 1 did not activate cryptic splice sites in the coding sequences and instead, they activated specific cryptic splice sites upstream b globin gene. Q-PCR with chimeric primers for the exact quantitation of the splice variants showed that the splice site mutations also cause significant reduction (> 90%) in expression of normally spliced transcripts suggesting that nonsense mediated decay is not the major mechanism for the reduced β globin production by these mutations. We found high levels of one of the aberrantly spliced transcripts with 5' splice site in exon 1 (∼20%) in normal individuals. All the splice variants showed steady increase in their expression from early to late stages of erythropoiesis. Our study shows that ex-vivo erythropoiesis system is an invaluable tool for studying the mechanism of globin gene regulation and switching. Identification of specific splice variants in cErCs of normal individuals and in patients with β thalassaemia could define the molecular mechanisms by which splice site mutations cause β-thalassaemia. The presence of abundant splice variants in normal controls suggests that β globin gene is regulated by multiple promoters and other regulatory elements and identification of these sequences will help in understanding the molecular basis of human globin gene regulation. Disclosures:No relevant conflicts of interest to declare.

BANP opens chromatin and activates CpG-island-regulated genes.

The majority of gene transcripts generated by RNA polymeraseII in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.

Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination

SummarySPT6 is a histone chaperone that tightly binds RNA polymerase II (RNAPII) during transcription elongation. However, its primary role in transcription is uncertain. We used targeted protein degradation to rapidly deplete SPT6 in human cells and analyzed defects in RNAPII behavior by a multi-omics approach and mathematical modeling. Our data indicate that SPT6 is a crucial factor for RNAPII processivity and is therefore required for the productive transcription of protein-coding genes. Unexpectedly, SPT6 also has a vital role in RNAPII termination, as acute depletion induced readthrough transcription for thousands of genes. Long-term depletion of SPT6 induced cryptic intragenic transcription, as observed earlier in yeast. However, this phenotype was not observed upon acute SPT6 depletion and therefore can be attributed to accumulated epigenetic perturbations in the prolonged absence of SPT6. In conclusion, targeted degradation of SPT6 allowed the temporal discrimination of its function as an epigenetic safeguard and RNAPII elongation factor.

RNA:m5c/Methyltransferases Mediate Chromatin Organization and 5-Azacytidine Response/Resistance in Leukemia

Background: The roles of RNA modifications and RNA-modifying enzymes (writers and erasers) in the regulation of chromatin structure and drug response/resistance in leukemia cells are not well understood. Most published studies have been focused on RNA N6-adenosine methylation (RNA:m6A) and its writers and erasers (Li, S., and Mason, C.E., 2014, Annu Rev Genomics Hum Genet 15, 127). Comparatively little is known about the functions of RNA cytosine methylation (RNA:m5C) and its writers and erasers (Gilbert, et al., 2016, Science 352, 1408). To date, ten RNA:m5C methyltransferases (RCMTs) have been identified, including NSUN1 to NSUN7, three versions (A, B and C) of NSUN5 and DNMT2, but the functions of RNA:m5C and RCMTs remains largely unknown (Schapira, M. 2016. ACS Chem Biol 11, 575). This study aims to determine the impact of RNA:m5C/RCMTs on chromatin structure and 5-azacytidine (5-AZA) response/resistance in myeloid leukemia cells.Experiments and Results: Our initial experiments demonstrate a clear relationship between drug resistance and the backbone chemical structures of nucleic acid analogues/drugs and distinct cell lineage-specific drug response patterns in our leukemia cell lines. These results led us to study the roles of RNA:m5C and RCMTs in the regulation of cell lineage-specific chromatin structure and differential drug response in leukemia. Our data demonstrate a marked increase in RNA:m5C and RCMTs as well as their associated active chromatin in our 5-AZA resistant leukemia cell lines, compared to the original 5-AZA sensitive leukemia cell lines. Our RNase-coupled co-immunoprecipitation demonstrates that RCMTs interact with different partners to form functional complexes and distinct chromatin structures, which are essential for the differential drug response and the survival of the leukemia cells. Specifically, NSUN3 and DNMT2 directly interact with a conserved RNA-binding protein hnRNPK to form a 5-AZA-sensitive multi-protein complex. hnRNPK preferentially binds methylated RNA and interacts with multiple proteins, including cell lineage-specific transcription factors GATA1 and SPI1/PU.1 and CDK9/P-TEFb, a kinase phosphorylating the serine 2 (S2) in the C-terminal domain (CTD) of RNA polymerase II (RNA-pol-II). In contrast, NSUN1 and NSUN2 are not associated with hnRNPK directly. Instead, NSUN1 directly binds active (phosphorylated) RNA-pol-II to form active chromatin structure that is completely resistant to 5-AZA. Strikingly, 5-AZA-resistant leukemia cells have a massive increase in NSUN1-and BRD4-associated active RNA-pol-II, compared to the original 5-AZA-sensitive leukemia cells. By employing the newly developed technologies, including 5-ethynyl uridine-clicking chemistry and ligation-mediated rolling circle amplification, we demonstrate that RCMTs recruit active RNA-pol-II to nascent (methylated) RNA to form distinct chromatin structures in the 5-AZA-sensitive vs. 5-AZA-resistant leukemia cells. Knockdown of the expression of these RCMTs causes growth inhibition in both the 5-AZA-sensitive and 5-AZA-resistant leukemia cells and also resensitize the 5-AZA-resistant leukemia cells to a low dose of 5-AZA. Based on our data, we propose the working model of RNA:m5C/RCMT-mediated chromatin structures in 5-AZA-sensitive vs. the 5-AZA-resistant leukemia cells (Figure 1). To test the working model, we applied various CDK and BRD4 inhibitors that target the serine phosphorylation in the CTD of RNA-pol-II to our 5-AZA-sensitive and 5-AZA-resistant-resistant leukemia cell lines. Our data show that these inhibitors synergize with 5-AZA and resensitize the 5-AZA-resistant leukemia cells, leading to growth inhibition in the leukemia cells at very low drug concentrations. By applying these technologies to clinical specimens, we demonstrate the existence of RNA:m5C/RCMT-mediated chromatin structures in clinical MDS/AML cells, supporting their importance and clinical relevance of our working model (Figure 1).Conclusion: Our data demonstrate that RCMTs interact with RNA-pol-II complex to form distinct chromatin structures at nascent (methylated) RNA, which regulate drug response/resistance in leukemia cells. Such RNA:m5C/RCMT-mediated drug-responsive chromatin structures may lead to the identification of new therapeutic avenues. [Display omitted] DisclosuresShammo:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Larson:Astellas: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen Inc.: Research Funding.

Loss of ASXL1 in the Bone Marrow Niche Dysregulates Hematopoietic Stem Cell Fates through RNA Polymerase II Complex

Introduction: Somatic or de novo mutations of Additional Sex Combs-like 1 (ASXL1) frequently occur in patients with myeloid malignancies or Bohring-Opitz syndrome, respectively. Conditional deletion of Asxl1 in hematopoietic system (Asxl1Δ/Δ, Abdel-Wahab et al. J Exp Med 2013) or global loss of Asxl1 (Asxl1-/-, Wang et al. Blood 2014) resulted in myeloid malignancies in mice. Interestingly, hematopoietic disease phenotypes are severer in Asxl1-/- mice than that in Asxl1Δ/Δ mice. The difference in disease severity between the two different mouse models led us to hypothesize that ASXL1 plays an important role in the function of marrow niche for normal hematopoiesis and deletion of Asxl1 in the niche contributes to the hematopoietic defect in vivo.Methods: In the current study, we performed a serial hematopoietic phenotypic analyses in OsxCre;Asxl1fl/fl mice, in which Asxl1 is only deleted in the mesenchymal stromal cells (MSCs) and preosteoblasts. Bone marrow (BM) transplantation was performed to identify the biological effects of ASXL1 loss in the niche on hematopoietic stem cell (HSC) functions. Convergent analyses of RNA-seq and ChIP-seq data were performed to survey ASXL1 target genes that are critical for HSC maintenance.Results:OsxCre;Asxl1fl/fl mice had a reduced LT-HSC pool and skewed cell differentiation favoring granulocytic/monocytic lineages in the BM by flow cytometric analyses. Co-culture of MSC with Lin-cKit+ (LK) cells revealed that Asxl1-/- MSCs induced a preferential myeloid differentiation of LK cells compared with WT MSCs. BM transplantation assay further confirmed that deletion of Asxl1 in MSCs/preosteoblasts resulted in a reduced HSC pool and a preferential myeloid lineage increment. Gene set enrichment analysis of RNA-seq data showed that downregulated genes in Asxl1-/- MSCs were enriched in cell cycle, stem cell division/proliferation, and cell surface receptor signaling pathway. Gene ontology analysis revealed that some of the differentially expressed genes were associated with cell cycle, cell division/proliferation, extracellular matrix organization, and cell adhesion. Protein mass spectrometry analysis and immunoprecipitation demonstrated a novel interaction of ASXL1 with multiple core subunits of RNA polymerase II (RNAPII) complex. Gel filtration chromatography assay verified the interaction between ASXL1 and RNAPII. ChIP-seq analysis using ASXL1 and RNAPII antibodies showed a high degree of the overlap peaks between ASXL1 and RNAPII bound genes. Convergent analyses of RNA-seq and ChIP-seq data revealed that loss of Asxl1 deregulated RNAPII transcriptional function and altered the expression of genes critical for HSC maintenance, such as Cxcl1, Cxcl2, and Vcam1 . Importantly, ASXL1 expression decreased markedly in MSCs of patients with chronic myelomonocytic leukemia (CMML-MSCs) compared with that in healthy donors. Consistently, co-culture of CMML-MSCs with cord blood CD34+ cells revealed that CMML-MSCs had a reduced hematopoietic supportive activity and induced a myeloid differentiation bias of CD34+ cells.Conclusion: Our in vivo studies demonstrate that Asxl1 in MSCs/preosteoblasts is required for normal hematopoiesis through regulating RNAPII transcriptional function. This study provides a mechanistic insight into the function of ASXL1 in the niche to maintain normal hematopoiesis; and ASXL1 alteration in, at least, a subset of the niche cells induces myeloid differentiation bias, thus, contributes the progression of myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

The CDK7 Inhibitor THZ1 Alters RNA Polymerase Dynamics at the 5‘ and 3‘ Ends of Genes

The t(8;21) is one of the most frequent chromosomal translocations in acute myeloid leukemia (AML). This translocation encodes a fusion protein containing the DNA binding domain of RUNX1, a master regulator of hematopoiesis, linked to the majority of the myeloid translocation gene on chromosome 8 (MTG8, also known as eight-twenty-one or ETO; gene name RUNX1T1). This chimeric fusion protein binds to the RUNX1 DNA elements to control gene expression by recruiting transcriptional cofactors via the MTG8/ETO moiety. In addition, MTG/ETO family proteins co-purified in a complex that also contained “E proteins” and the cyclin dependent kinase CDK9, a component of the positive transcription elongation factor b (P-TEFb).P-TEFb contains Cyclin T1 and CDK9 and several potential CDK9 inhibitors have shown promise in the clinics for t(8;21) AML, such as flavopiridol (alvocidib). Transcription initiation, promoter-proximal pausing of RNA polymerase II (RNAPII), and transcription elongation are events that are highly regulated for genes involved in proliferation and differentiation such as MYC and BCL2 . CDK7 phosphorylates Ser5 of the C-terminal domain of RNAPII to trigger promoter-proximal pausing of RNA polymerase II, whereas CDK9 phosphorylates Ser2 of RNAPII and the negative elongation factors DSIF and NELF to release the polymerase into the body of the gene.In this study, we used inhibitors of CDK7 and CDK9 to probe for transcriptional vulnerabilities in t(8;21) AML. We used both broad spectrum (flavopiridol, dinacicilib) and a specific CDK9 inhibitor PHA767491 along with THZ1 (CDK7 inhibitor). Since CDK7 is also a CAK (CDK activating kinase) that activates cell-cycle CDKs and mutations in Cyclin D2 are common in t(8;21) AML, we also tested the highly selective CDK4/6 inhibitor palbociclib (PD-0332991). We observed that the t(8;21)-containing AML cell lines SKNO-1 and Kasumi-1, as well as primary AML patient cells, were extremely sensitive to all the CDK7/9 inhibitors, but not palbociclib. We used precision global run-on transcription sequencing (PROseq) to define the mechanism of action of these compounds. This technique creates high resolution genomic maps of the active RNA polymerases to define the effects on RNA polymerase dynamics. CDK9 inhibitors caused wide spread RNA Polymerase II promoter proximal pausing at nearly 80% of the expressed genes, as well as at enhancers where there was a general impairment of production of eRNAs and lncRNAs. Conversely, inhibition of CDK7, a critical component of TFIIH caused a loss of paused RNA polymerase II at promoters, which was associated with an increase of polymerases in the body of genes. While RUNX1 was reported to be regulated by CDK7 in T-ALL, there was no impact on the expression of RUNX1 or the fusion protein in t(8;21)-containing cells. Although enhancers were not affected, super enhancers were modestly affected by THZ1. Interestingly, within a subset of genes that showed increased RNA polymerase accumulation within the gene body, CDK7 inhibition altered 3' end transcription termination dynamics within 1 hr and this effect remained even at 2 hr. A time course of THZ1 treatment at 0, 15', 30', and 60' suggested that CDK7 inhibition altered RNA polymerase dynamics at 5'ends of the genes, and it's these effects that eventually affected transcription termination at the 3' ends of the genes. DisclosuresSavona:Amgen: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Takeda: Research Funding; Karyopharm: Consultancy, Equity Ownership; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Consultancy, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Altered Enhancer Activity Represses CEBPE Expression in MLL -Rearranged Leukemia

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins resulting from chromosomal translocation between MLL and its partner genes. The resultant fusion proteins can confer aberrant epigenetic patterns at gene regulatory elements to disrupt transcriptional program, and ultimately leads to differentiation blockage at early hematopoietic progenitor stage. CCAAT enhancer binding proteins (C/EBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. We found that Cebpe is markedly downregulated in murine hematopoietic stem/progenitor cells (HSPCs) expressing MLL-EEN, which could be associated with differentiation blockage in MLL -rearranged leukemia. However, how CEBPE is epigenetically dysregulated by MLL fusion proteins remains unclear. In murine granulocytes, we demonstrated long-range chromatin interaction between Cebpe promoter and enhancer by chromatin conformation capture (3C) assay, conferring the strong expression of Cebpe . Strikingly, we noticed gain of DNA methylation at Cebpe promoter in MLL-EEN expressing HSPCs, suggesting the disruption of chromatin interaction through aberrant epigenetic alterations resulted in Cebpe repression. Interestingly, CEBPE expression was also found significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). DNA hypermethylation was observed at both promoter and predicated enhancer regions in MLL AML cells, which were absent in non-MLL AML cells. In addition, reduction of H3K27ac enrichment at the predicted enhancer was found in MLL AML cells. Analysis of the public available RNA Polymerase II (RNAPII) ChIP-seq data demonstrated RNAPII binding at both CEBPE promoter and enhancers in non-MLL AML cell lines, but it was absent at the enhancer region in MLL AML cells. These results suggest a common epigenetic mechanism which disrupts the CEBPE enhancer-promoter interaction in MLL -rearranged leukemia. Besides, treatment of MLL AML cell lines with DNA methylation inhibitor 5'-aza-cytidine (5-aza-C) demonstrated induction of CEBPE and myeloid marker CD11b . While all -trans retinoid acid failed to trigger cell differentiation in ML-2 AML cells, co-treatment with 5-aza-C resulted in further induction of CEBPE and CD11b, compared to 5-aza-C treatment per se . Collectively, our work shows that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells. This study provides novel insights on the aberrant epigenetic regulation in MLL -rearranged leukemia and facilitates the development of differentiation therapy for leukemia. DisclosuresNo relevant conflicts of interest to declare.

MTOR Restricts Chromatin Access and Genomic Activity to Maintain Hematopoietic Stem Cell Quiescence and Engraftment

mTOR kinase is an anti-cancer drug target that integrates signals from nutrients, growth factors, and cellular energy status to control cell growth and proliferation, but its biological function in hematopoietic stem cells remains unclear. Numerous studies by using rapamycin or mTOR kinase inhibitors have indicated that mTOR inhibition suppresses chromatin access and gene expression and inhibits cell growth. Consistent with the notion that mTOR targeting suppresses gene activation, hyperactivation of mTOR by deletion of negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and exhaustion of HSCs. To directly examine the function of mTOR and its kinase activity in HSC regulation, we used the conditional Mx-Cre;mTORflox/flox and the Mx-Cre;mTORflox/knockin mice that produce the mTOR-/- and the mTOR kinase-activity deficient D2338A mutant knockin genotype, respectively, upon poly I:C induced Cre recombination. We have determined the phenotypic, genomic and molecular effects of their HSCs. Our previous studies have found that depletion of mTOR drastically affected hematopoiesis, with a marked reduction in total BM cellularity and transient increase in the number of HSCs in bone marrow. Remarkably, mTOR-/- HSCs displayed a loss of quiescence, increased proliferation and a normal survival index (Guo et al., Haematologic a 98:1353). Our current study has found similar phenotypes of the HSCs in increased number and proliferation in the Mx-Cre;mTORflox/knockin mice where the mTOR kinase activity is abolished. Both mTOR-/- and mTORKI/- HSCs are defective in engraftment upon competitive transplant into recipient mice, with a clear molecular signature of drastically reduced downstream effectors p-S6K and p-4E-BP. ATAC-seq reveals that loss of mTOR or mTOR kinase activity results in selectively increased chromatin access and global gene activation, in contrast to the reported effects by mTOR inhibitors such as rapamycin or pp242/AZD2014. ChIP-seq of two active histone markers, H3K27Ac and H3K4Me3, yields consistent results that chromosome of the HSCs are activated upon the loss of mTOR or its kinase activity. In particular, increased H3K27Ac and H3K4Me3 ChIP-seq activities are observed in both the upstream and downstream region of c-myc locus, consistent with an activated c-myc expression. RNA-seq analysis reveals that upon mTOR KO or KI, thousands of genes exhibit differential expression. The up-regulated genes are particularly enriched in functions involved in ribosome biogenesis/protein translation, cell cycle progression, cell fate determination, and survival. Analysis using the STRING functional interaction network to search for the compensatory genes/pathways discovers a number of likely compensatorily upregulated genes, upon mTOR KO or KI, that share a significant number of interacting genes with mTOR, including c-myc, Fos, and Jun. These genes are enriched in pathways such as EGFR-MAPK pathway, PI3K-Akt pathway, Jak-Stat pathway, and NFkB pathway, which may compensate for mTOR loss. RT-PCR and Western blotting further verify that loss of mTOR or its kinase activity causes elevated mRNA and protein expression of selected compensatory genes, including c-myc, Fos, Jun, and Pik3cd. In addition, broad compensatory elevations in ribosome and translation machinery gene expression, activation of RNA pol-II, and activation of several interesting compensatory pathways are seen. The BET inhibitor, JQ1, that inhibits c-myc activity readily suppresses the hyper-proliferative phenotype of mTOR-/- and mTORKI/-HSCs. Thus, contrary to dogma established mostly by transient rapamycin or kinase inhibitor usages, our genetic and genomic studies demonstrate that mTOR, through its kinase activity, plays a restrictive role to maintain chromosomal access and genomic activity of HSCs. Loss of mTOR or its kinase activity unleashes a compensatory chromosomal and gene activation effects that involve master regulators of gene activities such as c-myc and Fos, protein translation and ribosomal proteins, and cell growth signaling pathways. These genomic changes may cause the HSC loss of quiescence and engraftment phenotypes seen upon mTOR loss, and need to be taken into consideration in the development of new generations of mTOR targeted therapy and combinatory therapy.Disclosures: No relevant conflicts of interest to declareASH Members: Yi Zheng DisclosuresNo relevant conflicts of interest to declare.

EZH1/2, SWI/SNF, and MLL2 Dependent Heterochromatin Formation and Abnormal Transcriptome in Hematological Malignancies

The neomorphic alteration of H3K27me3 patterning is a fundamental trait of cancers. We have developed a new clinical strategy; dual inhibition of EZH1 and EZH2 significantly reprogrammed the abnormal epigenome and restored the transcriptome in lymphomas (Blood, 2016; 58th ASH, 2016; NCT02732275).H3K27me3-associated chromatin regulation contributes to almost all human malignancies, yet the underlying mechanisms of gene silencing remain unclear, owing to a lack of data representing the action of EZH1/2 and other chromatin regulators in practical models. To analyze the dynamic processes of chromatin regulation, we treated H3K27me3-accumulated lymphoma cells with EZH2 inhibitors or EZH1/2 dual inhibitors and then profiled several epigenetic factors and histone marks using time-course chromatin immunoprecipitation (ChIP) and genome-wide ChIP assays. EZH2 single inhibition expelled the EZH2-containing polycomb repressive complex 2 (EZH2-PRC2) from methylated chromatin. This simultaneously triggered the acute recruitment of EZH1-PRC2 to a part of the loci, leading to homeostatic regulation of chromatin. In contrast, pharmacological EZH1/2 dual inhibition or EZH1 depletion prior to EZH2 inhibition resulted in the rapid removal of both PRC2 and PRC1 and their enzymatic products H3K27me3 and H2AK119ub. These initial changes were further accompanied by the recruitment of chromatin remolding factors (SWI/SNF, also called BAF complex), such as ARID1A, BRG1 (SMARCA4), and SNF5 (SMARCB1), histone H3K27 acetylation and H3K4 tri-methylation, formation of accessible focal chromatin, RNA PolII recruitment, and stable induction of transcription. We also found that hyperactivation of EZH1/2 by co-overexpression or EZH2 active mutation, as in a part of B-cell lymphoma, facilitated the eviction of the BAF complex and closed chromatin conformation. Thus, the opposing actions of EZH1/2-containing polycomb and SWI/SNF complexes are required for facultative heterochromatin formation.Genetic lesions and transcriptional downregulation of epigenetic factors are frequently observed in general cancers, including hematological malignancies. We reconstructed the loss-of-function (LoF) models of major genetic targets ARID1A (9% in FL, 7% in BL) , SMARCA4 (21% in BL) , KDM6A (found in AML, CML, T-ALL, MM, HL) , and BAP1 and then analyzed them using epigenome/transcriptome profiling. Inactivation of the tumor-suppressive chromatin remodelers caused EZH1- and EZH2-dependent H3K27me3 accumulation and heterochromatin formation, leading to transcriptional silencing. The new EZH1/2 dual inhibitor effectively cancelled epigenetic abnormalities. The cell models lacking a functional BAF complex were sensitive to the EZH1/2 dual inhibitors compared with the EZH2 single inhibitor.MLL2 (KMT2D)-mediated H3K4me3 regulation is frequently disrupted in DLBCL (~30%) and FL (~90%). MLL2 LoF enhanced cell growth in pre-tumorigenic LCLs, which could be cancelled by EZH1/2 dual inhibition. Genome-wide ChIP revealed that MLL2 LoF-dependent H3K4me3 downregulation is closely associated with EZH1- and EZH2-dependent H3K27me3 accumulation. Collectively, the EZH1/2 dual inhibitors may be promising for use in (1) EZH1/2 dual expressing tumors, such as ATL and other T- and B-cell lymphomas; (2) DLBCL/FL with actively mutated EZH2; and (3) additional cancer types (~20% of human malignancies) with mutations in the Trithorax and other epigenetic genes (ARID1A, SMARCA4, SMARCB1, KDM6A, BAP1, and MLL2).The EZH1/2-dependent epigenetic pathway of cancer development is also initiated by viral infection (Curr. Opin. Oncol., 2017). Whole epigenome and transcriptome analyses revealed that infection by lymphotropic oncoviruses, HTLV-1 and EBV, reprogrammed global EZH1/2-H3K27me3 patterns and established unique transcriptomes in malignant progenitors. Low-dose EZH1/2 inhibitor selectively depleted the HTLV-1-infected cell population in asymptomatic carriers (31/35, 88.6%) and EBV-infected immortalized LCLs. Data integration with established lymphoma epigenome suggested that this heritable EZH1/2 dysfunctional state is epigenetically imprinted at the non-malignant, virus-infected step. The identification of this novel epigenetic pathway will lead to precision oncology and new therapeutic strategies in human malignancies, particularly for elimination of the founder cell populations. DisclosuresYamagishi:Daiichi Sankyo Co., Ltd.: Research Funding. Honma:Daiichi Sankyo Co., Ltd.: Employment. Adachi:Daiichi Sankyo Co., Ltd.: Employment. Utsunomiya:HUYA Bioscience International: Honoraria; Roche Diagnostics: Honoraria; Japan Blood Products Organization: Honoraria; Celgene: Honoraria; Astellas Pharma: Honoraria; Pfizer: Honoraria; Nippon Shinyaku: Honoraria; Daiichi Sankyo: Honoraria; Kyowa Hakko Kirin: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Siemens: Honoraria; Novartis Pharma: Honoraria; Chugai Pharma: Honoraria. Tsukasaki:HUYA: Honoraria; Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Mundypharma: Research Funding; Zenyaku Kogyo: Honoraria; Chugai/Roche: Honoraria; Kyowa-Kirin: Honoraria; DaiichiSankyo: Consultancy. Tobinai:AbbVie: Research Funding; HUYA Bioscience: Honoraria; Mundipharma: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Daiichi Sankyo Co., Ltd: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Servier: Research Funding; Takeda: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria. Araki:Daiichi Sankyo: Employment. Uchimaru:Daiichi Sankyo Co., Ltd.: Research Funding.

How Single-Molecule Localization Microscopy Expanded Our Mechanistic Understanding of RNA Polymerase II Transcription.

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.