Biology subjects

Gottwein, E.

Publications and source records attributed to Gottwein, E..

5 recordsLinked to original sources

bioRxiv2025

KSHV-encoded vIRF3 Cooperates with Cellular IRF4 to Drive Super-Enhancer Activity through Complex DNA Elements

The Kaposis sarcoma-associated herpesvirus (KSHV) oncoprotein vIRF3 is essential for the survival of primary effusion lymphoma (PEL) cells. vIRF3 cooperates with cellular IRF4 to activate super-enhancers (SEs) driving oncogenes including MYC and IRF4 itself. However, the vIRF3/IRF4-responsive DNA sequences underlying this cooperation are unknown. Investigating the IRF4-SE, we mapped its vIRF3/IRF4-responsiveness to a complex [~]83 bp region, which retained cooperative activation by vIRF3 and IRF4 and was activated by vIRF3 but not IRF4 alone. vIRF3-mediated activation depended on an AP1 site, while the cooperation of vIRF3 with IRF4 required the DNA binding ability of IRF4 and IRF-related motifs that do not participate in canonical AP1-IRF (AICE) composite sites. These motifs are necessary but insufficient to confer responsiveness outside their native sequence context, suggesting that vIRF3/IRF4-mediated IRF4-SE activation requires an extended composite element. DNA pulldowns confirmed the importance of the identified motifs for association of vIRF3 and IRF4 with the IRF4-SE. A PEL MYC-SE similarly depended on an extended responsive element containing a critical AP1 site, within a functional AICE motif. Together, our results show that vIRF3 activates oncogenic SEs by co-opting complex genetic elements that may accommodate previously unknown IRF4 binding configurations, improving our understanding of vIRF3 and IRF4-dependent oncogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/669493v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@8eb737org.highwire.dtl.DTLVardef@19ea21eorg.highwire.dtl.DTLVardef@1a5909dorg.highwire.dtl.DTLVardef@b947c0_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Cytotoxicity of Activator Expression in CRISPR-based Transcriptional Activation Systems

CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of published and new CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and the p300 acetyltransferase core domain. We show that the expression of potent transcriptional activators in lentiviral producer cells leads to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection bottlenecks that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.

molecular biology↗

bioRxiv2022

Cellular FLIP Protects KSHV-Transformed Lymphoma Cells from TRAIL-Independent TRAIL Receptor 1-Mediated Cell Death

Kaposis sarcoma-associated herpesvirus (KSHV) causes primary effusion lymphoma (PEL). PEL cell lines require expression of the cellular FLICE inhibitory protein (cFLIP) for survival, although KSHV encodes a viral homolog of this protein (vFLIP). Cellular and viral FLIP proteins have several functions, including, most importantly, the inhibition of pro-apoptotic caspase 8 and modulation of NF-{kappa}B signaling. To investigate the essential role of cFLIP and its potential redundancy with vFLIP in PEL cells, we first performed rescue experiments with human or viral FLIP proteins known to affect FLIP target pathways differently. The long and short isoforms of cFLIP and molluscum contagiosum virus MC159L, which are all strong caspase 8 inhibitors, efficiently rescued the loss of endogenous cFLIP activity in PEL cells. KSHV vFLIP was unable to fully rescue the loss of endogenous cFLIP and is therefore functionally distinct. Next, we employed genome-wide CRISPR/Cas9 synthetic rescue screens to identify loss of function perturbations that can compensate for cFLIP knockout. Results from these screens and our validation experiments implicate the canonical cFLIP target caspase 8 and TRAIL receptor 1 (TRAIL-R1 or TNFRSF10A) in promoting constitutive death signaling in PEL cells. However, this process was independent of TRAIL receptor 2 or TRAIL, the latter of which is not detectable in PEL cell cultures. The requirement for cFLIP is also overcome by inactivation of the ER/Golgi resident chondroitin sulfate proteoglycan synthesis and UFMylation pathways, Jagunal homolog 1 (JAGN1) or CXCR4. UFMylation and JAGN1, but not chondroitin sulfate proteoglycan synthesis or CXCR4, contribute to TRAIL-R1 expression. In sum, our work shows that cFLIP is required in PEL cells to inhibit ligand-independent TRAIL-R1 cell death signaling downstream of a complex set of ER/Golgi-associated processes that have not previously been implicated in cFLIP or TRAIL-R1 function.

microbiology↗

bioRxiv2019

The oncogenic Kaposi sarcoma-associated herpesvirus encodes a mimic of the tumor suppressive miR-15/16 miRNA family

Many tumor viruses encode oncogenes of cellular origin. Here, we report the first instance of an oncoviral mimic of a cellular tumor suppressor. The Kaposis sarcoma-associated herpesvirus (KSHV) miRNA miR-K6-5p shares sequence similarity to the tumor suppressive cellular miR-15/16 miRNA family. We show that miR-K6-5p inhibits cell cycle progression, a hallmark function of miR-16. miR-K6-5p regulates conserved miR-15/16 targets, including many cell cycle regulators. Inhibition of miR-K6-5p in KSHV-transformed B cells conferred a significant growth advantage. Altogether, our data show that KSHV encodes a functional mimic of the miR-15/16 miRNA family. While it is exceedingly well established that oncogenic viruses encode oncogenes of cellular origin, this is the first example of an oncogenic virus that encodes a viral mimic of a cellular tumor suppressor. Encoding a tumor suppressive miRNA could help KSHV balance viral oncogene expression and thereby avoid severe pathogenesis in the healthy host.

microbiology↗

bioRxiv2019

Genome-wide CRISPR Screens Reveal Genetic Mediators of Cereblon Modulator Toxicity in Primary Effusion Lymphoma

Genome-wide CRISPR/Cas9 screens represent a powerful approach to study mechanisms of drug action and resistance. Cereblon modulating agents (CMs) have recently emerged as candidates for therapeutic intervention in primary effusion lymphoma (PEL), a highly aggressive cancer caused by Kaposis sarcoma-associated herpesvirus. CMs bind to cereblon (CRBN), the substrate receptor of the cullin-RING type E3 ubiquitin ligase CRL4CRBN, and thereby trigger the acquisition and proteasomal degradation of neosubstrates. Downstream mechanisms of CM toxicity are incompletely understood, however. To identify novel CM effectors and mechanisms of CM resistance, we performed positive selection CRISPR screens using three CMs with increasing toxicity in PEL: lenalidomide (LEN), pomalidomide (POM), and CC-122. Results identified several novel modulators of the activity of CRL4CRBN. The number of genes whose inactivation confers resistance decreases with increasing CM efficacy. Only inactivation of CRBN conferred complete resistance to CC-122. Inactivation of the E2 ubiquitin conjugating enzyme UBE2G1 also conferred robust resistance against LEN and POM. Inactivation of additional genes, including the Nedd8-specific protease SENP8, conferred resistance to only LEN. SENP8 inactivation indirectly increased levels of unneddylated CUL4A/B, which limits CRL4CRBN activity in a dominant negative manner. Accordingly, sensitivity of SENP8-inactivated cells to LEN is restored by overexpression of CRBN. In sum, our screens identify several novel players in CRL4CRBN function and define pathways to CM resistance in PEL. These results provide rationale for increasing CM efficacy upon patient relapse from a less efficient CM. Identified genes could finally be developed as biomarkers to predict CM efficacy in PEL and other cancers.\n\nKey PointsO_LIGenome-wide CRISPR/Cas9 screens identify novel mediators of resistance to lenalidomide, pomalidomide and CC-122 in PEL cells.\nC_LIO_LIUBE2G1 and SENP8 are modulators of CRL4CRBN and their inactivation drives resistance to CMs in PEL-derived cell lines.\nC_LI

cancer biology↗