Abstract Background. Herceptin, which targets the protein HER2, has poor efficacy as a monotherapy. To better understand the role of HER2, we compared isogenic HER2 expressing and non-expressing cells for the distribution for RNA polymerase II (POL II) on promoters and RNA expression. Methods. Chromatin immunoprecipitation was performed with anti-RNA polymerase II (POL II) in high HER2-expressing and non-expressing MCF7 cells as well as two breast cancer cell lines with naturally amplified HER2; MDA453 and BT474. Binding of POL II was assessed on Agilent promoter arrays. Gene activation in the same cells was assessed by expression analysis using U133plus2 arrays. Results. De novo expression of HER2 in MCF7 cells causes POLII to interact strongly with 606 genes not bound in empty vector control cells (p < 0.05). 30% of these genes exhibited a significant increase in transcription (p < 0.05; Fold-change > 1.4). POL II also interacts 1079 genes that are common between HER2-expressing and control cells. Conversely, 678 genes were bound more tightly by POL II in the control (p < 0.05). 20% of these genes exhibited a significant decrease in transcription. An additional 825 genes, bind POLII “loosely” in MCF7HER2 cells but not in MCF7 cells. 30% of these genes were significantly differentially regulated relative to control. The transcription of 68 loosely bound genes was tested by qPCR for all three cell lines. When compared with the array data, the correlation coefficients were 0.74 - 0.90;(p < 0.01). In both HER2 and non-expressing cells, loosely bound POL II sites are largely associated with 3′ positions of the Agilent probes within the transcribed region, whereas strong binding sites were largely localized to + 250 bp of the transcription start sites (TSSs). The transcriptome of MCF7 was compared to MCF7HER2 and the two HER2-expressing cancer cell lines. 633 genes were differentially expressed between the control and the three lines. Conclusions. The expression of HER2 is associated with a marked alteration in the number of genes that interact with POL II, involving hundreds of genes. The resulting gene activity depends upon the binding strength of POL II. Strong localized binding in one part of the promoter is often associated with less transcription, and diffused looser binding is often associated with higher transcription, whereas a lack of POL II binding is usually associated with no transcription. This is consistent with a mass action model in which transcription is maximal for POL II sites with an optimal koff rate and declines with either an increase (stalled) or decrease (not interacting) in binding. 42%, of the HER2-dependent gene interactions and transcription changes occur in both of the human cancer cell lines with naturally amplified HER2. These observations reveal a much expanded role of HER2 in gene regulation and indicate a greatly extended role in gene activation in HER2-positive breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3001. doi:1538-7445.AM2012-3001