Biology subjects

Journo, G.

Publications and source records attributed to Journo, G..

3 recordsLinked to original sources

bioRxiv2026

KSHV upregulates the long non-coding RNA PURPL via p73 to suppress p53

Long non-coding RNAs (lncRNAs) are a class of regulatory RNAs with lengths exceeding 200 nucleotides. The lncRNA PURPL (p53 upregulated regulator of p53 levels) is a p53-regulated gene. PURPL is an oncogenic lncRNA that represses p53 function; it is upregulated in many tumors and promotes cancer cell proliferation. Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of all forms of Kaposi sarcoma (KS) and is tightly associated with primary effusion lymphoma (PEL). We found upregulation of PURPL in both de novo and chronically KSHV-infected cells. We found that p73 (a p53 homolog) associates with the PURPL promoter and upregulates PURPL expression. Previously, it has been shown that KSHV-encoded latency-associated nuclear antigen (LANA) stabilizes p73. We found that ectopic expression of LANA increased both p73 protein levels and PURPL expression. p73 knockdown abrogates PURPL upregulation following KSHV infection. Antisense oligonucleotides (ASO) targeting PURPL reduced PEL cell proliferation and increased acetylated p53 levels. In contrast to p53, p73 is not repressed by PURPL RNA. In summary, our study reveals how an oncogenic virus can utilize p73 to upregulate the oncogenic lncRNA PURPL as an additional way to repress p53. Moreover, we propose another mechanism by which p73 may promote tumor growth.

cancer biology↗

bioRxiv2021

Unidirectional recruitment relations between proteins can be determined by a CRISPR/dCas9 recruitment assay

Kaposis sarcoma associated herpesvirus (KSHV, HHV-8) is associated with several human malignancies. During latency the viral genomes reside in the nucleus of infected cells as large non-integrated plasmids, known as episomes. All KSHV infected cells express LANA, and LANA is essential for viral latency. LANA binding to the viral episomes is critical for both replication of the viral genomes during latency, and for tethering the viral episomes to the cell chromosomes during cell division. Directional recruitment of protein complexes are critical for proper function of many nuclear processes. To test for recruitment directionality between LANA and cellular proteins we directed LANA via catalytically inactive Cas9 (dCas9) to a repeat sequence to obtain easily detectable dots. Then, recruitment of nuclear proteins to these dots can be evaluated. We found that LANA recruited its known interactors ORC2 and SIN3A. Interestingly, LANA was unable to recruit MeCP2, but MeCP2 recruited LANA. Both LANA and histone deacetylase 1 (HDAC1) interact with the transcriptional-repression domain (TRD) of MeCP2. Similar to LANA, HDAC1 was unable to recruit MeCP2. While heterochromatin protein 1 (HP1) which interacts with the N-terminal of MeCP2, was able to recruit MeCP2. Forcing MeCP2 dimerization via the tandem epitopes of SunTag, allows LANA to recruit MeCP2 in infected cells. We propose that available interacting domains force this recruitment directionality. Knock-down of MeCP2 by shRNA, as well as cells derived from Rett syndrome and express a mutant MeCP2 (T158M), dramatically reduced the ability of LANA to support viral latency. Therefore, this unidirectional recruitment of LANA by MeCP2 identified MeCP2 as a critical factor for KSHV viral maintenance. Significance StatementUsing a CRISPR/Cas9 recruitment assay, we show that some interacting proteins have a unidirectional recruitment property, where only one of the proteins can recruit its partner. We found unidirectional recruitment relations between the methylated DNA binding protein MeCP2 and KSHV encoded LANA. Where MeCP2 recruits LANA, but LANA is unable to recruit MeCP2. We were able to break this unidirectional recruitment by forcing MeCP2 dimerization. We propose that this unidirectional recruitment is the result of available interacting domains. Furthermore, this unidirectional recruitment seems to be critical for viral latency since LANA fails to maintain the viral genomes in MeCP2 mutant cells. Therefore, in this case unidirectional recruitment is a matter of survival or extinction.

microbiology↗

bioRxiv2019

RNA-sequencing analysis of a multistep and hit-and-run cell and animal model of KSHV tumorigenesis reveal the roles of mutations, CpG methylation, and viral-infection footprints in oncogenesis.

Human viral oncogenesis is the consequence of cell transformation mediated by virally encoded oncogenes in combination with host oncogenic alterations. Kaposis sarcoma (KS), caused by the Kaposis sarcoma-associated herpes virus (KSHV), is an AIDS-associated cancer characterized by angiogenesis and spindle-cells proliferation. KSHV-infected KS lesions are composed of latently-infected cells, as well as cells expressing lytic genes that have been implicated in the development of the KS angioproliferative phenotype. The existence of KS lesions with varying levels of KSHV-infected cells suggests also the existence of virus-independent \"hit-and-run\" mechanisms of sarcomagenesis, whereby viral infection irreversibly induce genetic or epigenetic oncogenic alterations in host cells. We have integrated genetic mutations, changes in expression signatures and methylation analysis to dissect genetic and epigenetic signaling pathways in an unbiased manner in the mECK36 mouse model of KSHV tumorigenesis. Pathway analysis of differential expressed genes (DEGs) showed KSHV lytic switch, DNA methylation and Epigenetic as the most regulated pathways during KSHV-dependent in vivo tumorigenesis. Methylation analysis data indicates that during the development of KSHV-infected tumors the most changes were towards hypo-methylation of tissues specific genes and oncogenic signature pathways, on the other hand during viral loss and development of KSHV-negative tumors changes are towards hyper-methylation. Mutational analysis of KSHV-infected cells and tumors revealed a set of mutations, including mutations in three inflammasome-related IFN response genes, that were absent in KSHV-infected cells but present in all KSHV-infected tumors in the same loci pointing to clonal selection \"in vivo\". This result suggests that in the context of in vivo tumorigenesis both these mutations and the virus may determine tumor growth. On the other hand, clustering analysis of mutations driving KSHV-negative tumors reveal a network comprising PDGFRA D842V, Pak1 and Nucleolin mutations implicated in cell proliferation. Our results have uncovered novel specific aspects of the interplay between host oncogenic alterations and virus-induced transcriptional effects as well as the epigenetic changes induced by KSHV infection and tumorigenesis. The existence virally-induced irreversible genetic and epigenetic oncogenic alterations support the possibility for hit-and-run KSHV sarcomagenesis which is consistent with pathological and clinical findings.\n\nAUTHOR SUMMARYWe performed whole genome RNA sequencing and CpG DNA methylation analysis in a mouse bone-marrow endothelial-lineage cells (mEC) transfected with the KSHVBac36 (mECK36 cells), that are able to form KSHV-infected tumors in nude mice, which were thoroughly characterized as KS-like tumors. This unique model allowed us to dissect genetic and epigenetic mechanisms of KSHV dependent and hit-and-run sarcomagenesis. We found that during KSHV in vivo lytic switch and KSHV-dependent tumorigenesis DNA methylation and Epigenetic regulation are among the most host-regulated pathways. CpG DNA methylation analysis during transformation supports the notion that loss of methylation (hypo-methylation) is the major epigenetic change during this process. Sequence analysis of KSHV-positive tumors revealed that KSHV tumorigenesis not only selects for the presence of the virus but also pre-existing host mutations that allow the KSHV oncovirus to express the oncogenic lytic program and creates a permissive environment of inflammation and viral tumorigenesis providing a selective advantage in vivo.

cancer biology↗