FA 5 Progress Report WV-INBRE - Joan C. Edwards School of ...

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Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 32to adipokines. Given that the AHR is a ligand-activated transcription factor, we hypothesize thatAHR regulation of gene expression determines the maximal level of cancer cell growth that canoccur in response to adipokines. The objectives of this proposal are to define the genome-wide setof cis elements bound by the AHR (the AHR cistrome) in the human MCF-7 breast cancer cell lineto further investigate how the AHR controls cancer growth by regulating gene expression.b) Methods & Results: Chromatin immunoprecipitation coupled with next generation sequencing(ChIP-Seq) was used to define the AHR cistrome in MCF-7 cells. AHR-ChIP-Seq was performedwith chromatin isolated from MCF-7 cells treated with DMSO (vehicle) or the AHR agonist 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). With total chromatin input controls, Model-based Analysis ofChIP-Seq (MACS) was performed to generate peaks of sequence tag enrichment to identifyspecific AHR DNA binding site. Using MACS and peak finding, we identified the binding of the AHRto a region upstream from the CYP1B1 gene known to contain 5 AHR binding GCGTC motifs (thespecific peak region identified by MACS was ~650 to 1100 bp upstream from the CYP1B1transcription start site). In TCDD treated MCF-7 cells, this peak had a false discovery rate (FDR)of less than 1.5 % with an AHR binding motif within 13 bp of the peak summit (area of highestsequence build up, and predicted to the precise AHR binding site). The CYP1B1 promoter spanningpeak in DMSO treated MCF-7 cells had a FDR of less than 4.5% and an AHR binding sequencemotif within 57 bp of the peak summit. Using MACS we also identified the AHR binding to theCYP1A1 gene promoter in regions containing AHR binding motifs with FDRs ranging from less 3%to less than 6 %. Thus, with MACS we have identified AHR binding to known AHR binding motifsthat have been shown to have transcriptional activity. Conventional AHR-ChIP confirmed recruitmentof AHR to CYP1A1 and CYP1B1 gene promoters, providing further validation of our AHR-ChIP-Seqexperiments. With total input as a control, MACS analysis of AHR-ChIP-Seq identified ~134,000peaks in TCDD-treated MCF-7 cells and ~97,700 peaks in vehicle-treatedMCF-7 cells. Currently, we are validating additional AHR gene targets and will use bioinformatics tofurther characterize the biology of AHR binding as it pertains to cancer growth.NGS Project #6Title: Global Analysis of DNase I Hypersensitivity Sites in Treg CellsSubproject investigator: Wei-ping Zeng, PhDIntroduction: Regulatory T (Treg) cells function as active suppressors of immune responses ofconventional T cells and other immune cells. They play important regulatory roles in immuneresponses to infections and cancers. Therefore, understanding the pathogenesis and designingnew therapies of infectious diseases and cancers will benefit from studies of regulation of genesimportant for Treg cell function and frequencies. In the past, many molecules expressed by Tregcells have been identified to mediate their immune suppressive function. In addition, other genesexpressed in Treg cells, including those important for homeostatic maintenance of the Treg cells,have been analyzed with gene expression arrays. However, little is known about the regulation ofthese genes in Treg cells. DNAse I hypersensitive (DHS) sites often contain important ciselements, such as locus control regions, enhancers and sometimes silencers.Methods: To study the regulation of the important genes in Treg cells, we wish to obtain a map ofthe global distribution of the DHS sites in Treg cells. Towards this goal, we are adapting a methodfor DNase-Seq analysis for high throughput on the Illumina next generation sequencer.Result and Discussion: Since the Treg cells are a minor population of CD4 T cells, before applyingthe method to this rare population of cells we determined the best experimental conditions usingtotal CD4 T cells. We have tried different concentrations of DNAse I, temperatures and times ofdigestion of the nuclei. In addition, we made several modifications to improve the yields of DNA atvarious steps. After pulling down the DNA fragments derived from ends generated by DNase IPHS 2590 (Rev. 06/09)Continuation Format Page
Program Director/Principal Investigator (Last, First, Middle): Rankin, Gary O 33digestion, we performed PCR to determine whether such isolated DNA fragments are indeedenriched for the DNase I hypersensitive sites by examining a previously known DHS site at the CD4gene. Preliminary results showed that the sequence of CD4 DHS site is preferentially amplifiedcompared with nearby sequences with no DHS site. This result indicated successful enrichmentof the DHS sequences. In the future, we will attempt to apply the same conditions to study of theTreg cells.PROTECTION AGAINST RESEARCH RISKSY 1. Will human subjects be involved next year?If yes, provide complete the above Targeted/Planned Enrollment Table and the Inclusion EnrollmentReport. Provide the date of IRB approval and enclose with transmittal.05/06/2011 (Cuff Project)Y 2. Will vertebrate animals be used next year?If yes, provide the date of Institutional Animal Care and Use Committee (IACUC) approval and enclosewith transmittal.05/06/2010If no approval date, please explain.Is this IACUC approval date different from the date reported last year?Y 3. Will recombinant DNA experiment(s) be conducted next year?If yes, provide the date of Office of Recombinant DNA Activities (ORDA), NIH approval:EXEMPTN 4. Are there potential hazards to laboratory workers (carcinogens, pathogens, ionizing radiation, etc.)involved in the proposed research for next year? If yes, identify:N5. Will any of the research-risk categories,not involved next year, be involved future years? If yes, identify:PHS 2590 (Rev. 06/09)Continuation Format Page
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