Ser2 Phosphorylation Research Articles

Contributions to nucleosome dynamics in chromatin from interactive propagation of phosphorylation/acetylation and inducible histone lysine basicities.

The effect of phosphorylation on the basicities of amines in histone H3 peptides and their acetylation kinetics is probed with a mild chemical acetylating agent. Phosphorylation of Ser-10 lowers the rate of chemical acetylation of Lys-9, Lys-14, and Lys-18 by methyl acetyl phosphate in that order consistent with a higher pKa of these Lys residues induced by phosphorylation; basicities increase up to 3 pKa units as a function of distance from Ser-10 phosphate. Enzymic acetylation of Lys residues with high pKa values in nucleosomes is also expected to be enhanced by phosphorylation, consistent with the known mechanism involving binding of protonated amines to N-acetyltransferases; fetal hemoglobin has a related linkage of increased basicity at a specific site, its acetylation, and a resulting decrease in subunit interaction strength. In the absence of a phosphate on Ser-10, the amines of Lys-9, Lys-14, and Lys-18 have lowered pKa values. Chemical acetylation of glycine and glycinamide have analogous kinetic profiles to the histone peptides but the phosphate inductive effect in histone H3 is more potent since the linkage between phosphorylation and acetylation is propagated with a range extending 9-10 amino acids in either direction from the phosphorylation site enhancing protonation of amino groups. We conclude that lysine amine basicities in histone tails are not static but inducible and variable due to a dynamic and immediate interaction between phosphorylation/acetylation that may contribute to inactive heterochromatin by compaction through such Ser phosphate-Lys amine electrostatic interactions and their relaxation by acetylation in euchromatin.

Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer

Event Abstract Back to Event Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer Yanxin Wang1* 1 National University of Singapore, Singapore Background Human BAD is a pro-apoptotic Bcl-2 family member, whose apoptotic functions can be inactivated through phosphorylation of specific residues including Ser99. Clinically, BAD phosphorylation has been reported to indicate poor survival and cisplatin resistance in ovarian cancer patients. Methods NPB, a novel small molecule which specifically inhibits BAD Ser99 phosphorylation, has been developed in our laboratory. Cell function assays performed include Alamar Blue cell viability assay, caspase3/7 assay, PI-Annexin V apoptosis assay, and 3D growth in Matrigel. The CI values are calculated using Chou-Talalay method. The cancer stem cell-like cell population was examined by ALDEFLOUR and sphere formation assays. Results The level of BAD Ser99 phosphorylation is negatively correlated with cisplatin sensitivity in a panel of ovarian cancer cell lines and it is increased upon acute cisplatin treatment in ovarian cancer cells. The inhibition of BAD Ser99 phosphorylation by NPB alone increased apoptosis, decreased anchorage-independent growth of both parental and cisplatin resistant ovarian cancer cells, with the combination of NPB and cisplatin showing a synergistic effect. An upstream BAD kinase, AKT has also been reported to mediate cisplatin resistance. Correspondingly, the combination of NPB and AKT inhibitor AZD5363 exhibited strong synergistic effects in both parental and cisplatin resistant cell lines. Both the phosphorylation of BAD and its upstream kinase AKT was increased in this CSC-like population. NPB treatment alone was observed to decrease the CSC-like population, while the combination of AZD5363 and NPB produced a synergistic decrease in the CSC-like population. Conclusion NPB, as a novel inhibitor of BAD Ser99 phosphorylation, can potentially be used in combination with cisplatin as a therapy for naïve ovarian cancer patients to increase cisplatin sensitivity. Furthermore, the combination of NPB and AKT inhibitor AZD5363 is a potential therapeutic approach in the treatment of cisplatin resistant ovarian cancer. Keywords: Bad phosphorylation, cisplatin resistance, ovarian cancer, NPB, Akt inhibitor Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Speed Presentation Topic: Cancer Citation: Wang Y (2019). Efficacy of inhibition of BAD Ser99 phosphorylation by a novel small molecule in cisplatin resistant ovarian cancer. Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00013 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 07 Nov 2018; Published Online: 17 Jan 2019. * Correspondence: Ms. Yanxin Wang, National University of Singapore, Singapore, Singapore, wangyanxin98@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yanxin Wang Google Yanxin Wang Google Scholar Yanxin Wang PubMed Yanxin Wang Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Mitogen- and stress-activated protein kinase 1 is required for gonadotropin-releasing hormone–mediated activation of gonadotropin α-subunit expression

Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific β-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.

YAP repression of the WNT3 gene controls hESC differentiation along the cardiac mesoderm lineage

Activin/SMAD signaling in human embryonic stem cells (hESCs) ensures NANOG expression and stem cell pluripotency. In the presence of Wnt ligand, the Activin/SMAD transcription network switches to cooperate with Wnt/β-catenin and induce mesendodermal (ME) differentiation genes. We show here that the Hippo effector YAP binds to the WNT3 gene enhancer and prevents the gene from being induced by Activin in proliferating hESCs. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) data show that YAP impairs SMAD recruitment and the accumulation of P-TEFb-associated RNA polymerase II (RNAPII) C-terminal domain (CTD)-Ser7 phosphorylation at the WNT3 gene. CRISPR/CAS9 knockout of YAP in hESCs enables Activin to induce Wnt3 expression and stabilize β-catenin, which then synergizes with Activin-induced SMADs to activate a subset of ME genes that is required to form cardiac mesoderm. Interestingly, exposure of YAP-/- hESCs to Activin induces cardiac mesoderm markers (BAF60c and HAND1) without activating Wnt-dependent cardiac inhibitor genes (CDX2 and MSX1). Moreover, canonical Wnt target genes are up-regulated only modestly, if at all, under these conditions. Consequently, YAP-null hESCs exposed to Activin differentiate precisely into beating cardiomyocytes without further treatment. We conclude that YAP maintains hESC pluripotency by preventing WNT3 expression in response to Activin, thereby blocking a direct route to embryonic cardiac mesoderm formation.

Differential Mechanisms Underlying Antidepressant Responses of Ketamine and Imipramine.

Ketamine, a noncompetitive NMDA receptor antagonist, exhibits rapid antidepressant actions, but the underlying mechanism remains obscure. AMPA receptor and cAMP response element-binding protein (CREB) are involved in the antidepressant actions of Ketamine and imipramine, a traditional tricyclic antidepressant. However, ketamine exerts its therapeutic actions much faster than imipramine. Understanding the discrepancy of antidepressant efficiency between ketamine and the traditional antidepressant is important for elucidating the mechanism underlying ketamine's fast-acting antidepressant responses as well as designing new rapid antidepressants. Here we show that the enhancement of the phosphorylation of CREB Ser133 and expression of CREB and glutamate receptor 1 (GluR1) are necessary for both ketamine's and imipramine's antidepressant actions, but the enhancements at early stage may account for the faster onset of ketamine's antidepressant action than imipramine. Notably, ketamine but not imipramine enhances CREBregulated transcription coactivator-1 (CRTC1) expression and induces potentiation of excitatory synaptic transmission at Schaffer collateral CA1 synapses, which indicates critical targets for unveiling ketamine's rapid antidepressant actions. Our study suggests that differential regulation of CRTC1 expression may contribute to the discrepancy of antidepressant efficacy between ketamine and imipramine, which may lead to a better understanding of ketamine's fast antidepressant responses.

MiR-206 inhibits the growth of hepatocellular carcinoma cells via targeting CDK9.

miR‐206 plays an important role in regulating the growth of multiple cancer cells. Cyclin‐dependent kinase 9 (CDK9) stimulates the production of abundant prosurvival proteins, leading to impaired apoptosis of cancer cells. However, it is unknown whether CDK9 is involved in the miR‐206‐mediated growth suppression of hepatocellular carcinoma (HCC) cells. In this study, we found that the expression level of miR‐206 was significantly lower in HCC cell lines than that in normal hepatic cell line (L02). Meanwhile, CDK9 was upregulated in HCC cell lines. Moreover, miR‐206 downregulated CDK9 in HCC cells via directly binding to its mRNA 3′ UTR, which resulted in a decrease of RNA PolII Ser2 phosphorylation and Mcl‐1 level. Additionally, miR‐206 suppressed the cell proliferation, and induced cell cycle arrest and apoptosis. Similarly, silence or inhibition of CDK9 also repressed the cell proliferation, and induced cell cycle arrest and apoptosis. Taken together, the results demonstrated that miR‐206 inhibited the growth of HCC cells through targeting CDK9, suggesting that the miR‐206‐CDK9 pathway may be a novel target for the treatment of HCC.

Phosphorylation of Suppressor of Hairless impedes its DNA-binding activity

Notch signalling activity governs cellular differentiation in higher metazoa, where Notch signals are transduced by the transcription factor CSL, called Suppressor of Hairless [Su(H)] in Drosophila. Su(H) operates as molecular switch on Notch target genes: within activator complexes, including intracellular Notch, or within repressor complexes, including the antagonist Hairless. Mass spectrometry identified phosphorylation on Serine 269 in Su(H), potentially serving as a point of cross-regulation by other signalling pathways. To address the biological significance, we generated phospho-deficient [Su(H)S269A] and phospho-mimetic [Su(H)S269D] variants: the latter displayed reduced transcriptional activity despite unaltered protein interactions with co-activators and -repressors. Based on the Su(H) structure, Ser269 phosphorylation may interfere with DNA-binding, which we confirmed by electro-mobility shift assay and isothermal titration calorimetry. Overexpression of Su(H)S269D during fly development demonstrated reduced transcriptional regulatory activity, similar to the previously reported DNA-binding defective mutant Su(H)R266H. As both are able to bind Hairless and Notch proteins, Su(H)S269D and Su(H)R266H provoked dominant negative effects upon overexpression. Our data imply that Ser269 phosphorylation impacts Notch signalling activity by inhibiting DNA-binding of Su(H), potentially affecting both activation and repression. Ser269 is highly conserved in vertebrate CSL homologues, opening the possibility of a general and novel mechanism of modulating Notch signalling activity.

Costunolide induces micronuclei formation, chromosomal aberrations, cytostasis, and mitochondrial-mediated apoptosis in Chinese hamster ovary cells.

Costunolide (CE) is a sesquiterpene lactone well-known for its antihepatotoxic, antiulcer, and anticancer activities. The present study focused on the evaluation of the cytogenetic toxicity and cellular death-inducing potential of CE in CHO cells, an epithelial cell line derived from normal ovary cells of Chinese hamster. The cytotoxic effect denoting MTT assay has shown an IC50 value of 7.56μM CE, where 50% proliferation inhibition occurs. The oxidative stress caused by CE was confirmed based on GSH depletion induced cell death, conspicuously absent in N-acetylcysteine (GSH precursor) pretreated cells. The evaluation of genotoxic effects of CE using cytokinesis block micronucleus assay and chromosomal aberration test has shown prominent induction of binucleated micronucleated cells and aberrant metaphases bearing chromatid and chromosomal breaks, indicating CE's clastogenic and aneugenic potential. The apoptotic death in CE treated cells was confirmed by an increase in the number of cells in subG1 phase, exhibiting chromatin condensation and membranous phosphatidylserine translocation. The apoptosis induction follows mitochondrial mediation, evident from an increase in the BAX/Bcl-2 ratio, caspase-3/7 activity, and mitochondrial membrane permeability. CE also induces cytostasis in addition to apoptosis, substantiated by the reduced cytokinetic (replicative indices) and mitotic (mitotic indices and histone H3 Ser-10 phosphorylation) activities. Overall, the cellular GSH depletion and potential genotoxic effects by CE led the CHO cells to commit apoptosis and lowered cell division. The observed sensitivity of CHO cells doubts unintended adverse effects of CE on normal healthy cells, suggesting higher essentiality of further studies in order to establish its safety efficacy in therapeutic explorations.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virus-induced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Abstract LB-317: Identification of a novel preclinical candidate for CDK7 inhibition

Abstract Cyclin-dependent kinase 7 (CDK7) is an important constituent of the cellular transcriptional machinery, where it phosphorylates the C-terminal domain (CTD) of RNAP polymerase II (RNAPII). Because many tumor types are critically dependent on transcription for maintenance of their oncogenic state, pharmacological modulation of CDK7 kinase activity is considered as an approach to treat cancer. Multiple series of covalent CDK7 inhibitors were identified by iterative medicinal chemistry efforts and SAR based approach. These compounds were optimized towards attaining good physicochemical properties, high potency, good selectivity and desirable pharmacokinetic profile to achieve anti-tumor activity. We have now identified a pre-clinical candidate AU-BGB-002 which is highly potent in inhibiting CDK7 in biochemical as well as cellular assays while fully efficiently engaging the target. In a panel of kinases, AU-BGB-002 shows selectivity for CDK7. A panel of cell lines derived from a diverse set of indications are sensitive to AU-BGB-002. AU-BGB-002 exhibits excellent drug-like characteristics including solubility, permeability, metabolic stability and good oral bioavailability. When tested in a xenograft model, AU-BGB-002 treatment resulted in dose dependent tumor growth inhibition in AML xenograft model with tumor stasis at a dose of 10 mg/kg. Potent inhibiton of tumor growth was accompanied by complete target engagement and suppression of pS5RNAPII RNAPolII Ser5 phosphorylation in a parallel PK-PD study. Efficacy studies in additional xenograft models, advanced DMPK and toxicity studies are ongoing for this compound. In summary, we have identified a novel and selective CDK7 covalent inhibitor candidate with desirable drug-like properties that shows excellent efficacy in an AML xenograft model. Findings presented here support further development of AU-BGB-002 for the treatment of cancer. Citation Format: Leena K. Satyam, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Aravind Basavaraju, Lakshmi Narayana Kaza, Manoj Kumar Pothuganti, Shilpa Nayak, Nandish C, Amith A, Ravindra MV, Dabbeeru Madhu Babu, Nagaraju A, Suraj Tgore, Thomas Antony, Chetan Pandit, Murali Ramachandra, Shekar Chelur, Girish Daginakatte, Susanta Samajdar. Identification of a novel preclinical candidate for CDK7 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-317. doi:10.1158/1538-7445.AM2017-LB-317

Abstract 1057: Co-treatment with fenretinide and safingol induced p38 MAPK and/or FOXO3a activated pro-survival autophagy in glioblastoma multiforme cells

Abstract Cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased reactive oxygen species and/or de novo synthesized D-erythro-dihydroceramides. Co-treatment with L-threo-sphinganine (safingol, S), synergistically increased fenretinide cytotoxicity in cell lines of many cancer types coincident with increase of safingol-derived, L-threo-dihydroceramides. Previously, we showed that 4-HPR+S induced ER stress, unfolded protein response (UPR), pro-survival autophagy, and mixed non-apoptotic and apoptotic cell death in glioblastoma multiforme (GBM) cells in vitro. However, the mechanisms regulating such activity remained unclear, including the role of ceramide-activated signal transducers, such as ASK1. We now report that 4-HPR+S activated stress kinase ASK1 via phosphorylation at Thr845 (+1-6 hrs), an event known to induce p38 MAPK phosphorylation. Furthermore, 4-HPR+S induced an increase in the nuclear localization of transcription factor FOXO3a coincident with decreased Akt signaling and increased phosphorylated levels of JNK1/2, p38 MAPK, ERK1/2, and autophagy regulator, AMPK (+1-6 hrs). 4-HPR+S caused a time-dependent (+6-24 hrs) increase of GRP78 protein levels, a key regulator of ER stress response and UPR, and increased levels of pro-apoptotic transcription factor, CHOP. GRP78 increase was p38 MAPK-dependent, as p38 MAPK inhibitor, SB203580, greatly reduced GRP78 increase. Interestingly, 4-HPR+S induced cytotoxicity was unchanged in the presence of an inhibitor of ERK 1/2 (PD98059) but significantly decreased when cells were concurrently treated with p38 MAPK inhibitor, SB203580, or JNK1/2 inhibitor, SP600125 (+24-72 hrs, p ≤ 0.05). Significantly, co-treatment with dorsomorphin, an AMPK inhibitor, increased and accelerated 4-HPR+S cytotoxicity (+24-72 hrs, p ≤ 0.05). Moreover, 4-HPR+S treatment induced FOXO3a Ser7 phosphorylation (+1-6 hrs) and a corresponding decrease in levels of nuclear E3-Ub protein ligase, Skp2. The decrease of SKp2 protein was coincident with increased levels of CARM1 protein, a known transcriptional activator of autophagy-related genes. Activation of ASK1, JNK1/2, p38 MAPK and ERK1/2 by 4-HPR+S occurred earlier, and to a greater extent, than single agent 4-HPR and S exposures at all time points (+1-24 hrs). Together, these results support that 4-HPR+S induced both pro-survival autophagy and cell death at least partly through activation of FOXO3a and ASK1-dependent activation of p38 MAPK, possibly in response to the known progressive increase of de novo synthesized D-erythro and L-threo dihydroceramides. A Phase I trial of intravenous 4-HPR+S in adult solid tumors (ClinicalTrials.gov Identifier:NCT0155307) is currently open in the Texas State-supported, South Plains Oncology Consortium (SPONC.org). Citation Format: Nikhil Vad, Dong Wang, Charlie Linch, Barry J. Maurer. Co-treatment with fenretinide and safingol induced p38 MAPK and/or FOXO3a activated pro-survival autophagy in glioblastoma multiforme cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2017-1057

Ser-261 phospho-regulation is involved in pS256 and pS269-mediated aquaporin-2 apical translocation

Vasopressin catalyzes aquaporin-2 phosphorylation at several serine sites in the C-terminal region. Compared with Ser-256 and Ser-269 phosphorylation, the role of Ser-261 phospho-regulation on vasopressin-regulated AQP2 apical translocation is largely unknown. In addition, recent discovery of transcytotic apical delivery of AQP2 made the concept of its intracellular trafficking even more complicated. In this study, we evaluated how intact phospho-AQP2 signals fit with the transcytosis trafficking model in Madin-Darby canine kidney cells. PS256 and pS269 signals were intracellularly detectable in wild-type AQP2 at the beginning of forskolin stimulation (1 min). These phospho-signals were detectable in basolateral membranes even after 10 min of stimulation. AQP2 stably inserted in the apical membrane increased pS269 and decreased pS261 signals. In an NDI-causing mutant P262L-AQP2, in which Ser-261 phospho-regulation is impaired, the pS256 and pS269 signals were detectable in the basolateral membranes with increased pS261 signals after forskolin stimulation. These results suggest that Ser-261 phospho-regulation is involved in pS256- and pS269-mediated AQP2 apical translocation.

RNA Pol II Dynamics Modulate Co-transcriptional Chromatin Modification, CTD Phosphorylation, and Transcriptional Direction

Eukaryotic genes are marked by conserved post-translational modifications on the RNA pol II C-terminal domain (CTD) and the chromatin template. How the 5'-3' profiles of these marks are established is poorly understood. Using pol II mutants in human cells, we found that slow transcription repositioned specific co-transcriptionally deposited chromatin modifications; histone H3 lysine 36 trimethyl (H3K36me3) shifted within genes toward 5' ends, and histone H3 lysine 4 dimethyl (H3K4me2) extended farther upstream of start sites. Slow transcription also evoked a hyperphosphorylation of CTD Ser2 residues at 5' ends of genes that is conserved in yeast. We propose a "dwell time in the target zone" model to explain the effects of transcriptional dynamics on the establishment of co-transcriptionally deposited protein modifications. Promoter-proximal Ser2 phosphorylation is associated with a longer pol II dwell time at start sites and reduced transcriptional polarity because of strongly enhanced divergent antisense transcription at promoters. These results demonstrate that pol II dynamics help govern the decision between sense and divergent antisense transcription.

Rapid Genome-wide Recruitment of RNA Polymerase II Drives Transcription, Splicing, and Translation Events during T Cell Responses

Activation of immune cells results in rapid functional changes, but how such fast changes are accomplished remains enigmatic. By combining time courses of 4sU-seq, RNA-seq, ribosome profiling (RP), and RNA polymerase II (RNA Pol II) ChIP-seq during Tcell activation, we illustrate genome-widetemporal dynamics for ∼10,000 genes. This approach reveals not only immediate-early and posttranscriptionally regulated genes but also coupled changes in transcription and translation for >90% of genes. Recruitment, rather than release of pausedRNA Pol II, primarily mediates transcriptional changes. This coincides with a genome-wide temporary slowdown in cotranscriptional splicing, even for polyadenylated mRNAs that are localized at the chromatin. Subsequent splicing optimization correlates with increasing Ser-2 phosphorylation of the RNA Pol II carboxy-terminal domain (CTD) and activation of the positive transcription elongation factor (pTEFb). Thus, rapid de novo recruitment of RNA Pol II dictates the course of events during Tcell activation, particularly transcription, splicing, and consequently translation.

Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation

The BET proteins are major transcriptional regulators and have emerged as new drug targets, but their functional distinction has remained elusive. In this study, we report that the BET family members Brd2 and Brd4 exert distinct genomic functions at genes whose transcription they co-regulate during mouse T helper 17 (Th17) cell differentiation. Brd2 is associated with the chromatin insulator CTCF and the cohesin complex to support cis-regulatory enhancer assembly for gene transcriptional activation. In this context, Brd2 binds the transcription factor Stat3 in an acetylation-sensitive manner and facilitates Stat3 recruitment to active enhancers occupied with transcription factors Irf4 and Batf. In parallel, Brd4 temporally controls RNA polymerase II (Pol II) processivity during transcription elongation through cyclin T1 and Cdk9 recruitment and Pol II Ser2 phosphorylation. Collectively, our study uncovers both separate and interdependent Brd2 and Brd4 functions in potentiating the genetic program required for Th17 cell development and adaptive immunity.

Oxidative Stress Delays Prometaphase/Metaphase of the First Cleavage in Mouse Zygotes via the MAD2L1-Mediated Spindle Assembly Checkpoint.

In zygotes, DNA damage delays the first cleavage to enable repair. Our previous study found that 0.03 mM hydrogen peroxide (H2O2) was the minimum concentration required for induction of oxidative DNA damage in mouse zygotes and that this represented the most similar situation to the clinical phenomenon. In this study, we quantified the cleavage rates of cells in blastocysts at different developmental stages, followed by immunofluorescence to detect activation of γ-H2A histone family member X (a marker of DNA damage) in zygotes to confirm that oxidative DNA damage was induced in H2O2-treated zygotes. Monitoring H3S10P (phosphorylation of Ser10 on histone H3; a prometaphase/metaphase marker) levels at different hour postinsemination revealed that treatment of zygotes with 0.03 mM H2O2 resulted in a prometaphase/metaphase delay. Furthermore, immunofluorescence staining for mitotic arrest deficient 2-like 1 and the protein kinase TTK, components of the spindle assembly checkpoint (SAC), suggested that this delay possibly involved SAC activation. These studies of the relationships between oxidative stress and SAC can promote the success rate of in vitro fertilization.

Transient mechanical strain promotes the maturation of invadopodia and enhances cancer cell invasion in vitro.

Cancer cell invasion is influenced by various biomechanical forces found within the microenvironment. We have previously found that invasion is enhanced in fibrosarcoma cells when transient mechanical stimulation is applied within an in vitro mechano-invasion assay. This enhancement of invasion is dependent on cofilin (CFL1), a known regulator of invadopodia maturation. Invadopodia are actin-rich structures present in invasive cancer cells that are enzymatically active and degrade the surrounding extracellular matrix to facilitate invasion. In this study, we examine changes in gene expression in response to tugging on matrix fibers. Interestingly, we find that integrin β3 expression is downregulated and leads to an increase in cofilin activity, as evidenced by a reduction in its Ser3 phosphorylation levels. As a result, invadopodia lengthen and have increased enzymatic activity, indicating that transient mechanical stimulation promotes the maturation of invadopodia leading to increased levels of cell invasion. Our results are unique in defining an invasive mechanism specific to the invasive process of cancer cells that is triggered by tugging forces in the microenvironment, as opposed to rigidity, compression or stretch forces.

SGN-CD48A: a Novel Humanized Anti-CD48 Antibody-Drug Conjugate for the Treatment of Multiple Myeloma

Multiple myeloma (MM) is an incurable hematologic malignancy of transformed plasma cells. New targeted biological therapeutics are needed to increase the stringency and durability of remissions. In this study we describe SGN-CD48A, a potent CD48-targeting antibody-drug conjugate (ADC) utilizing a novel glucuronide-monomethylauristatin E (MMAE) linker, under development for the treatment of MM. CD48, or SLAMF2 (Signaling Lymphocyte Activation Molecule family member 2), is a GPI-anchored membrane protein in the SLAM family of immunoreceptors. CD48 is expressed on B and T lymphocytes, natural killer (NK) cells, and other immune cell types where it functions to modulate immune cell activation, proliferation, and differentiation. CD48 is also a tumor antigen broadly expressed in MM. We observed CD48 expression on the surface of malignant plasma cells in 90% (90/100) of human multiple myeloma patient samples examined by flow cytometry. Monoclonal antibodies (mAbs) specific for human CD48 were evaluated and a lead antibody was selected based on binding characteristics and cytotoxic activity against myeloma cells as an auristatin ADC. SGN-CD48A is a humanized anti-CD48 mAb to which eight molecules of MMAE, a potent microtubule disrupting cytotoxic drug, have been conjugated via a β-glucuronidase-cleavable linker. This novel glucuronide-MMAE drug-linker incorporates a PEG side chain and self-stabilizing maleimide to achieve homogenous drug-to-antibody ratio (DAR) 8 conjugates with decreased plasma clearance and increased preclinical antitumor activity. Following binding of CD48 at the myeloma cell surface, SGN-CD48A internalizes and traffics to lysosomal vesicles. Intracellular MMAE drug released from SGN-CD48A in myeloma cells induced cell cycle arrest at G2/M phase, phospho-histone H3 (Ser-10) phosphorylation, and caspase 3/7 dependent apoptotic cell death. SGN-CD48A demonstrated potent cytotoxic activity (EC50 values 1.0 - 11 ng/mL) against a panel of human MM cell lines, with CD48 expression levels of 135,000 - 480,000 receptors per cell. In contrast, SGN-CD48A had negligible cytotoxic activity against normal resting human B, NK, and T lymphocytes. We evaluated the in vivo antitumor activity of SGN-CD48A in disseminated MM cell line mouse xenograft models. In the NCI-H929 and EJM xenograft models, a single intraperitoneal dose of 0.3 mg/kg SGN-CD48A produced durable complete remissions in 8/8 and 6/8 mice, respectively. Similarly, in the U-266 xenograft model, a single dose of 1.0 mg/kg SGN-CD48A produced durable complete remissions in 7/8 mice. Neither unconjugated mAb nor a non-binding control MMAE ADC were active in these MM xenograft models, demonstrating that targeted delivery of MMAE drug through CD48 binding is required for activity. In summary, CD48 is a highly expressed new multiple myeloma target and the novel glucuronide-MMAE ADC SGN-CD48A shows potent antitumor activity against cell line models of MM. DisclosuresLewis:Seattle Genetics, Inc.: Employment, Equity Ownership. Olson:Seattle Genetics, Inc.: Employment, Equity Ownership. Gordon:Seattle Genetics, Inc.: Employment, Equity Ownership. Sandall:Seattle Genetics, Inc.: Employment, Equity Ownership. Quick:Seattle Genetics, Inc.: Employment, Equity Ownership. Finn:Seattle Genetics, Inc.: Employment, Equity Ownership. Westendorf:Seattle Genetics, Inc.: Employment, Equity Ownership. Linares:Seattle Genetics, Inc.: Employment, Equity Ownership. Leiske:Seattle Genetics, Inc.: Employment, Equity Ownership. Nesterova:Seattle Genetics, Inc.: Employment, Equity Ownership. Law:Seattle Genetics, Inc.: Employment, Equity Ownership.

C-terminal domain (CTD) phosphatase links Rho GTPase signaling to Pol II CTD phosphorylation in Arabidopsis and yeast

Rho GTPases, including the Rho, Cdc42, Rac, and ROP subfamilies, act as pivotal signaling switches in various growth and developmental processes. Compared with the well-defined role of cytoskeletal organization in Rho signaling, much less is known regarding transcriptional regulation. In a mutant screen for phenotypic enhancers of transgenic Arabidopsis plants expressing a constitutively active form of ROP2 (designated CA1-1), we identified RNA polymerase II (Pol II) C-terminal domain (CTD) phosphatase-like 1 (CPL1) as a transcriptional regulator of ROP2 signaling. We show that ROP2 activation inhibits CPL1 activity by promoting its degradation, leading to an increase in CTD Ser5 and Ser2 phosphorylation. We also observed similar modulation of CTD phosphorylation by yeast Cdc42 GTPase and enhanced degradation of the yeast CTD phosphatase Fcp1 by activated ROP2 signaling. Taken together, our results suggest that modulation of the Pol II CTD code by Rho GTPase signaling represents an evolutionarily conserved mechanism in both unicellular and multicellular eukaryotes.

Aquaporin-2 Ser-261 phosphorylation is regulated in combination with Ser-256 and Ser-269 phosphorylation

Aquaporin-2 (AQP2) is a water channel in collecting duct principal cells in the kidney. Vasopressin catalyzes AQP2 phosphorylation at several serine sites in its C-terminus: Ser-256, Ser-261, and Ser-269. Upon stimulation by vasopressin, Ser-269 phosphorylation increases and Ser-261 phosphorylation decreases. Ser-256 phosphorylation is relatively constant. However, whether these types of phospho-regulation occur independently in distinct AQP2 populations or sequentially in the same AQP2 population is unclear. Especially, the manner of vasopressin-mediated Ser-261 phospho-regulation has been in controversy. In this study, we established phospho-specific AQP2 immunoprecipitation assays and investigated how pS256-positive AQP2 and pS269-positive AQP2 are catalyzed by forskolin or vasopressin, focusing on their Ser-261 phosphorylation status in polarized Madin-Darby canine kidney (MDCK) cells and in mice. In forskolin-treated MDCK cells, Ser-269 phosphorylation preceded Ser-261 dephosphorylation and Ser-256 phosphorylation was constant. In both MDCK cells and mouse kidney, phospho-specific immunoprecipitation revealed that the regulated Ser-269 phosphorylation occurred in the pS256-positive AQP2 population. Importantly, basal-state Ser-261 phosphorylation and its regulated dephosphorylation occurred in the pS256- and pS269-positive AQP2 population. These results provide the direct evidence that the Ser-261 dephosphorylation is involved in the pS256- and pS269-related AQP2 regulation.