KSHV LANA upregulates the expression of EGFL7 proteins in BJAB cells
ABSTRACT: The objective of this study was to determine the effects of LANA on the expressions of the cellular genes. BJAB cells were transduced with lentiviral vector expressing LANA or the control vector, total RNA was extracted for the detection of relative expression of cellular genes in LANA expressing cells.
Project description:LANA is essential for tethering the KSHV genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR), and to a few minor binding sites within the latency control region encoding that LANA transcript. We identified 256 LANA binding peaks with p < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Two candidate DNA sequence motifs were identified, and confirmed to bind purified LANA protein, although with weaker affinity compared to viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and TNF regulatory network. Further analysis revealed partial overlap of LANA binding sites with STAT1 binding sites in several interferon (IFN)-g regulated genes. We show that ectopic expression of LANA can down-modulate IFN-g mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9) required for class I antigen presentation. Our data provides a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements. Study of KSHV LANA
Project description:Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently-infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and 2) on viral DNA, and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here we have examined LANA interactions with host chromatin on a genome-wide scale using ChIP-seq, and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LBS1 motif in KSHV DNA. Comparison of the ChIP-seq profile with whole transcriptome (RNA-seq) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. Profiling of KSHV LANA positioning on the host genome and examination of gene expression from promoters bound by KSHV LANA.
Project description:The objective of this study was to determine the effects of LANA on the expressions of the cellular genes. Overall design: BJAB cells were transduced with lentiviral vector expressing LANA or the control vector, total RNA was extracted for the detection of relative expression of cellular genes in LANA expressing cells.
Project description:Gene expression profiling of three PEL cell lines compare to three Burkitt's lymphoma lines to figure out the changed genes under KSHV latent infection. Gene expression profiling of two time points on TIVE cells after infection by KSHV compare to TIVE cell without infection by KSHV to figure out the changed genes on TIVE cell under latent infection of KSHV. Gene expression profiling of four time points after inducing recombinant LANA protein expression when compare to no inducing BJAB/Tet-On/LANA cells to figure out the changed genes under the latency-associate nuclear antigen (LANA) of KSHV expression. Gene expression profiling of three time points after inducing recombinant LANA protein expression when compare to no inducing Jurkat/Tet-On/LANA cell line to figure out the changed genes under the latency-associate nuclear antigen (LANA) of KSHV expression. Gene expression profiling of two time points after inducing recombinant LANA protein expression when compare to no inducing 293/Tet-On/LANA cell line to figure out the changed genes under the latency-associate nuclear antigen (LANA) of KSHV expression.
Project description:Human SLK cells were infected with wildtype (wt) and LANA knockout (KO) Kaposi's sarcoma-associated herpesvirus (KSHV), separately for 3 days. Cellular gene expression changes were identified upon the wild type and LANA KO KSHV virus infection compared to the uninfected SLK cells using the human gene expression microarray U133plus2.0. 2 independent biological replicates from uninfected SLK cells, wild type KSHV infected SLK cells at 72hrs post-infection (hpi) , and LANA KO infected SLK cells at 72 hrs post-infection were collected and RNA was prepared for microarray analysis.
Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to identify genes specifically modulated by p21 we compared with the cell line with overexpression of p27, because p21 and p27 belong to the same gene family and regulated the same genes specially in cell cycle. So, our intention is to identify only genes regulated by p21 and not p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. We chose human primary keratinocytes because they are non-tumorigenic, non-immortalized and epithelial cells, in contrast to human myeloid leukemia K562 cells. Human primary keratinocytes were infected with recombinant adenoviruses expressing the full-length p21 protein. A dramatic increase in p21 in infected keratinocytes was demonstrated by RT-qPCR (as we show in the manuscript). As controls, we also infected the keratinocytes with adenovirus carrying the genes for p27 which overexpression was also confirmed by RT-qPCR (as we show in the manuscript). We prepared RNA 24 h after infection and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27 in cell cycle and mitosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The success of this study is to describe a new role of p21 as a transcriptional co-repressor in some systems.
Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus which establishes latent infection in endothelial and B cells, as well as in primary effusion lymphoma (PEL). During latency, the viral genome exists as a circular DNA minichromosome (episome) and is packaged into chromatin analogous to human chromosomes. Only a small subset of promoters, those which drive latent RNAs, are active in latent episomes. In general, nucleosome depletion (“open chromatin”) is a hallmark of eukaryotic regulatory elements such as promoters and transcriptional enhancers or insulators. We applied formaldehyde-assisted isolation of regulatory elements (FAIRE) followed by next-generation sequencing to identify regulatory elements in the KSHV genome and integrated these data with previously identified locations of histone modifications, RNA polymerase II occupancy, and CTCF binding sites. We found that (i) regions of open chromatin were not restricted to the transcriptionally defined latent loci; (ii) open chromatin was adjacent to regions harboring activating histone modifications, even at transcriptionally inactive loci; and (iii) CTCF binding sites fell within regions of open chromatin with few exceptions, including the constitutive LANA promoter and the vIL6 promoter. FAIRE-identified nucleosome depletion was similar among B and endothelial cell lineages, suggesting a common viral genome architecture in all forms of latency. Ten total samples analyzed by FAIRE-seq from latent KSHV-infected cell lines. Two replicates were performed for BC1, KSHV-BJAB, KSHV-HUVEC, and L1-TIVE cells using the Illumina HiSeq 2000 platform. For BCBL1 cells, 1 FAIRE-seq sample and 1 non-cross-linked control BCBL1 sample was analyzed using the Illumina GAIIx
Project description:miRNA-Sequencing of RUNX1 overexpressing K562 cells were compared to K562 cells transduced with an empty vector control to analyse the differential expression of miRNAs in hematopoiesis. K562 cells were transduced either with an empty lentiviral vector as control or a lentiviral vector overexpressing human RUNX1. The samples were biological triplicates and the results were validated by qRT-PCR using SYBR green.
Project description:2nd generation sequencing was used to compare expression profiles of MBP-specific T cells retrieved from blood, CSF, spinal cord meninges and parenchyma. The overall expression profiles were found to be very similar.However, genes regulated during T cell activation were found to be upregulated in T cells from spinal cord meninges and parenchyma compared to blood and CSF. 2nd generation sequencing of MBP-specific T cells retrieved from blood and CNS compartments during experimental autoimmune encephalomyelitis
Project description:To characterize gene expression changes in AML induced by CNTRL-FGFR1, we used RNA-seq analysis of sorted Mac+Gr1+B220+ AML cells (n = 2) from the SP and BM of leukemic mice, as well as sorted Mac+Gr1+ myeloid cells (n=3) from normal mice. Methods: cDNA libraries were generated using the Illumina TruSeq RNA Sample Preparation kit following the manufacturer’s instructions. The quality of the resulting cDNA libraries were assessed using a Bioanalyzer DNA 1000 chip (Agilent) and quantified by quantitative PCR (Bio-Rad). The cDNA libraries were sequenced using paired end Illumina Hiseq2000 protocols (50 cycles). The Illumina sequencing pipeline version 1.8 was employed for transferring raw images to base calls, generating sequence reads, and de-multiplexing the reads. The generated FASTQ files were imported to CLC Genomics Workbench and aligned to the mouse genome, NCBI37/mm9. The transcript expression levels were determined in terms of number of reads per kilobase per million (RPKM) and compared between sorted normal myeloid (Gr1+Mac1+) cells and sorted leukemic myeloid cells. Results: The result showed a remarkable difference between normal (Gr1+Mac1+) and leukemic (Mac+Gr1+B220+) myeloid cells. Gene Set Enrichment Analysis and leading-edge analysis suggested that Mac+Gr1+B220+ cells have the potential to differentiate into either myeloid or T-cell, but not a B-cell, lineages. Quantitative RT-PCR analysis of lineage-specific genes showed increased expression of Flt3, Gfi1, and Kit related to early myeloid-lineage commitment, while genes related to myeloid differentiation, Irf8, Klf4, Csf1r, Myc, and Spi1 were decreased compared with normal myeloid cells. Quantitative RT-PCR also confirmed that the Cd3e and Lmo2 T-cell specific genes were markedly increased in AML cells. Conclusions: We demonstrate that the CNTRL-FGFR1 fusion kinase induces bi-lineage myeloid and T-lymphoid disease in model mice. Genetic and molecular analyses in these models demonstrated that multiple signaling pathways, specifically related to myeloid and T-lymphoid lineages, were altered in neoplasms, which underscores the importance of developing therapies that target all of these signaling pathways to eradicate the leukemia-initiating-cells RNA-seq analysis of sorted Mac+Gr1+B220+ AML cells (n = 2) from the SP and BM of leukemic mice, as well as sorted Mac+Gr1+ myeloid cells (n=3) from normal mice.