title: Evaluation of genome-wide chromatin library of Stat5 binding sites in human breast cancer.
abstract: BACKGROUND
  There is considerable interest in identifying target genes and chromatin binding sites for transcription factors in a genome-wide manner. Such information may become useful in diagnosis and treatment of disease, drug target identification, and for prognostication. In cancer diagnosis, patterns of transcription factor binding to specific regulatory chromatin elements are expected to complement and enhance current diagnostic predictions of tumor behavior based on protein and mRNA analyses. Signal transducer and activator of transcription-5 (Stat5) is a cytokine-activated transcription factor implicated in growth and progression of many malignancies, including hematopoietic, prostate, and breast cancer. We have explored immunoaffinity purification of Stat5-bound chromatin from breast cancer cells to identify Stat5 target sites in an unbiased, genome-wide manner.
  
  
  RESULTS
  In this report, we evaluate the efficacy of a Stat5-bound chromatin library to identify valid Stat5 chromatin binding sites within the oncogenome of T-47D human breast cancer cells. A general problem with cloning of immunocaptured, transcription factor-bound chromatin fragments is contamination with non-specific chromatin. However, using an optimized strategy, five out of ten randomly selected clones could be experimentally verified to bind Stat5 both in vitro and in vivo as tested by electrophoretic mobility shift assay and chromatin immunoprecipitation, respectively. While there was no binding to fragments lacking a Stat5 consensus binding sequence, presence of a Stat5 binding sequence did not assure binding.
  
  
  CONCLUSIONS
  A chromatin library coupled with experimental validation may productively identify novel in vivo Stat5 chromatin binding sites in cancer, including abnormal regulatory sites in tumor-specific neochromatin.
authors.affiliation: Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, USA. ruih@georgetown.edu
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: LeBaron
authors.forename: Jianwu
authors.initials: J
authors.lastname: Xie
authors.forename: Hallgeir
authors.initials: H
authors.lastname: Rui
mesh_terms: D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D016384:Consensus Sequence / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D015723:Gene Library; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D015203:Reproducibility of Results; D020218:Response Elements / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000378:metabolism*
chemical_list: D002843:Chromatin; D050799:STAT5 Transcription Factor