Evaluation of genome-wide chromatin library of Stat5 binding sites in human breast cancer

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.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.Transcription factors function uniquely at the interface of the genome and the proteome. A significant portion of transcription factors serve not only as executors of gene transcription programs, but also as biochemical sensors of extracellular stimuli. For instance, members of the nuclear receptor family are directly activated by lipophilic extracellular ligands, and transcription factors of the Smad and Stat families are activated by phosphorylation in response to cytokine stimulation of cell surface receptors. Chromatin-bound transcription factors that act both as sensors of extracellular cues and as transcriptional effectors carry exceptional instructive value about the biological state of individual cells. Their high biological information value makes such factors particularly attractive for use as markers to predict disease activity and outcome, as well as predictive markers of disease-responsiveness to drugs.Based on related and broader rationale, the second phase of the human genome project, ENCODE (ENCyclopedia Of DNA Elements), has been initiated wi