Progress 02/12/09 to 12/31/12
Outputs
Progress Report Objectives (from AD-416): As described in USDA NRI-funded grant, we will: 1. Identify MDV Meq and c-Jun binding sites in the chicken genome. 2. Identify differentially expressed genes following MDV-reactivation in MSB-1 cells. 3. Determine if differentially expressed genes identified in objective 2 also show differentially expression between MD tumors and CD4+ T cells. 4. Identify differentially expressed genes as a function of genetic resistance status or the presence of Meq. Approach (from AD-416): 1. Utilize chromatin immunoprecipitation (ChIP) using Meq or c-Jun antibodies and determine the DNA sequences via Solexa paired-end reads. 2. Isolate RNA from MSB-1 cells at time 0 and following MDV reactivation, and run on Affymetrix Chicken microarrays. This project is directly linked to project 3635-31320-009-01R titled "Positional Candidate Genes for Resistance to Marek's Disease by Screening for Marek's Disease Virus Meq-Regulated Genes." Marek�s disease (MD) is an economically significant disease in chickens caused by the highly oncogenic Marek�s disease virus (MDV). Meq, a bZIP transcription factor discovered in the 1990s, is critically involved in viral oncogenicity. Only a few of its host target genes have been described, thus, impeding our understanding of MDV-induced tumorigenesis. Over the course of the project, using ChIP-seq and microarray analysis, a high confidence list of Meq-binding sites in the chicken genome and a global transcriptome of Meq-responsive genes was generated. Meq binding sites were found to be enriched in the promoter regions of up-regulated genes, but not in those of down-regulated genes. ChIP-seq was also performed for c-Jun, a known heterodimeric partner of Meq. Close location of binding sites of Meq and c-Jun was noted, suggesting cooperativity between these two factors in modulating transcription. Pathway analysis indicated that Meq transcriptionally regulates many genes that are part of several signaling pathways which include the ERK/MAPK, Jak-STAT, and ErbB pathways that are critical for oncogenesis and/or include signaling mediators involved in apoptosis. Meq activates oncogenic signaling cascades by transcriptionally activating major kinases in the ERK/MAPK pathway and simultaneously repressing phosphatases, as verified using inhibitors of MEK and ERK1/2 in a cell proliferation assay. This study provides significant insights into the mechanistic basis of Meq-dependent cell transformation.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): As described in USDA NRI-funded grant, we will: 1. Identify MDV Meq and c-Jun binding sites in the chicken genome. 2. Identify differentially expressed genes following MDV-reactivation in MSB-1 cells. 3. Determine if differentially expressed genes identified in objective 2 also show differentially expression between MD tumors and CD4+ T cells. 4. Identify differentially expressed genes as a function of genetic resistance status or the presence of Meq. Approach (from AD-416): 1. Utilize chromatin immunoprecipitation (ChIP) using Meq or c-Jun antibodies and determine the DNA sequences via Solexa paired-end reads. 2. Isolate RNA from MSB-1 cells at time 0 and following MDV reactivation, and run on Affymetrix Chicken microarrays. This project is directly linked to project 3635-31320-008-24R titled �Positional Candidate Genes for Resistance to Marek�s Disease by Screening for Marek�s disease Virus Meq-Regulated Genes." Genetic resistance to Marek�s disease (MD) is characterized by the lack of tumors or nerve enlargements following exposure to Marek�s disease virus (MDV), a highly-oncogenic alphaherpesvirus. MDV Meq is a transcription factor and the likely MDV oncogene suggesting that one pathway for resistance is the inability of Meq to regulate the transcription of specific genes in individuals resistant to MD, thereby, failing to initiate transformation. Therefore, it is of interest to define DNA-binding sites and the genes that are directly regulated by Meq. Previous work with chromatin immunoprecipitation (ChIP) has shown that Meq binds to specific sites on the MDV genome that are dependent on whether Meq is a homodimer or a heterodimer with c-Jun, another transcription factor and a key regulator for many biological pathways including those involved with cancer. Also, different forms of Meq are expressed and, in general, the full-length form is highly expressed during viral latency and in MD tumors while the Meq-vIL8 variant, which shows no transactivation ability, is expressed at low levels during lytic replication. Recent advancements in ultra-high throughput sequencing and bioinformatics analyses can identify and define the sequences in the chicken genome bound by Meq alone or in combination with c-Jun, the preferential dimerization partner for Meq. The identification of DNA-binding sites combined with DNA chip analyses that profile genes regulated by Meq may reveal positional candidate genes that confer genetic resistance to MD. Furthermore, identification of Meq binding sites may help to explain some of the allele-specific expression (ASE) identified in a related project. Thus far, using chicken cells that do or do not express Meq, ChIP analysis has identified 22,000+ genomic regions that bind Meq. Examining these peaks, there are approximately 3, 000 and 2,000 genes within 2 kb of the Meq and c-Jun binding sites, respectively, with about 600 of the peaks in common. Binding of Meq and c- Jun are highly enriched for the promoter regions, especially 500-800 bases upstream of the transcriptional start site. Motif analysis for the Meq-binding sites have confirmed existing motifs (AP-1 and MERE II) as well as identified new ones such as those recognized by CREB, GCN4, Myc, and HAC1. In parallel, the same set of cells was processed on DNA microarrays to reveal 1,829 (FDR=0.01) differentially expressed genes with 1,575 being down-regulated in the presence of Meq, which agrees with the previous conclusion that Meq-Meq homodimers repressed gene expression. Integrating the ChIP seq results that detect chromosomal regions bound by a transcription factor with microarray analysis that examines gene expression differences has revealed 354 and 93 genes that are down- or up- regulated by the directly binding of Meq, respectively. Pathway analysis has suggested genes and molecular mechanisms for MDV-induced transformation.
Impacts
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) As described in USDA NRI-funded grant, we will: 1. Identify MDV Meq and c-Jun binding sites in the chicken genome. 2. Identify differentially expressed genes following MDV-reactivation in MSB-1 cells. 3. Determine if differentially expressed genes identified in objective 2 also show differentially expression between MD tumors and CD4+ T cells. 4. Identify differentially expressed genes as a function of genetic resistance status or the presence of Meq. Approach (from AD-416) 1. Utilize chromatin immunoprecipitation (ChIP) using Meq or c-Jun antibodies and determine the DNA sequences via Solexa paired-end reads. 2. Isolate RNA from MSB-1 cells at time 0 and following MDV reactivation, and run on Affymetrix Chicken microarrays. This project is directly linked to project 3635-31320-008-24R titled �Positional Candidate Genes for Resistance to Marek�s Disease by Screening for Marek�s disease Virus Meq-Regulated Genes." Genetic resistance to Marek�s disease (MD) is characterized by the lack of tumors or nerve enlargements following exposure to Marek�s disease virus (MDV), a highly-oncogenic alphaherpesvirus. MDV Meq is a transcription factor and the likely MDV oncogene suggesting that one pathway for resistance is the inability of Meq to regulate the transcription of specific genes in individuals resistant to MD, thereby, failing to initiate transformation. Therefore, it is of interest to define DNA-binding sites and the genes that are directly regulated by Meq. Previous work with chromatin immunoprecipitation (ChIP) has shown that Meq binds to specific sites on the MDV genome that are dependent on whether Meq is a homodimer or a heterodimer with c-Jun, another transcription factor and a key regulator for many biological pathways including those involved with cancer. Also, different forms of Meq are expressed and, in general, the full-length form is highly expressed during viral latency and in MD tumors while the Meq-vIL8 variant, which shows no transactivation ability, is expressed at low levels during lytic replication. Recent advancements in ultra-high throughput sequencing and bioinformatics analyses can identify and define the sequences in the chicken genome bound by Meq alone or in combination with c-Jun, the preferential dimerization partner for Meq. The identification of DNA-binding sites combined with DNA chip analyses that profile genes regulated by Meq may reveal positional candidate genes that confer genetic resistance to MD. Furthermore, identification of Meq binding sites may help to explain some of the allele-specific expression (ASE) identified in a related project. Thus far, using chicken cells that do or do not express Meq, ChIP analysis has identified 22,000+ genomic regions that bind Meq. Examining these peaks, there are approximately 3, 000 and 2,000 genes within 2 kb of the Meq and c-Jun binding sites, respectively, with about 600 of the peaks in common. Motif analysis for the Meq-binding sites have confirmed existing motifs as well as identified a number of new ones. In parallel, the same set of cells was processed on DNA microarrays to reveal differentially expressed genes. Integrating the ChIP seq results that detect chromosomal regions bound by a transcription factor with microarray analysis that examines gene expression differences has revealed 351 genes. Pathway analysis has suggested genes and molecular mechanisms for MDV-induced transformation. This project is monitored by weekly meetings between the collaborating parties.
Impacts
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) As described in USDA NRI-funded grant, we will: 1. Identify MDV Meq and c-Jun binding sites in the chicken genome. 2. Identify differentially expressed genes following MDV-reactivation in MSB-1 cells. 3. Determine if differentially expressed genes identified in objective 2 also show differentially expression between MD tumors and CD4+ T cells. 4. Identify differentially expressed genes as a function of genetic resistance status or the presence of Meq. Approach (from AD-416) 1. Utilize chromatin immunoprecipitation (ChIP) using Meq or c-Jun antibodies and determine the DNA sequences via Solexa paired-end reads. 2. Isolate RNA from MSB-1 cells at time 0 and following MDV reactivation, and run on Affymetrix Chicken microarrays. This project is directly linked to project 3635-31320-008-24R titled �Positional Candidate Genes for Resistance to Marek�s Disease by Screening for Marek�s disease Virus Meq-Regulated Genes." Marek�s disease, a T cell lymphoma of chickens caused by a pathogenic virus, costs the poultry industry approximately $1 billion worldwide annually in losses and control measures. Thus, understanding how the Marek�s disease virus leads to disease is of both scientific and commercial interest. Meq is the putative cancer-causing viral protein and leads to aberrant expression of chicken proteins. Using molecular biology techniques and high-throughput sequencing, an ARS scientist in East Lansing identified all the targets candidate genes in the chicken genome that Meq might alter. This information can help efforts to develop more effective vaccines or improve genetic resistance, both of which would reduce costs and improve animal welfare. This project is monitored by e-mail, telephone calls, and almost weekly face-to-face meeting between the two parties.
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