Tiling Experiment Sheds New Light on Estrogen Regulation October 2005 Researchers at Harvard Medical School and Furman University have acquired new insights into estrogen regulation. Estrogen plays essential roles in reproduction, endometrial cancer, and breast cancer--which is the second leading cause of cancer deaths in women worldwide. With the completion of the human genome, the expressed regions of protein-coding genes for estrogen have been identified, but a great deal remains to be learned about estrogen's cis-regulatory elements. Cis-regulatory elements physically link to target genes to identify the genes for regulating factors. Scientists hope this additional information will be useful in developing better understanding of estrogen-related gene expression and also new cancer therapies. A number of potential ER target genes in human breast cancer and a few cis-elements targeted directly by ER have been identified. Previous studies have focused on promoter regions and promoter-rich sequences. As reported in the July 2005 issue of Cell, Dr. Jason Carroll and his colleagues at Harvard Medical School and Furman University have identified a number of distal estrogen receptor (ER) binding sites involved with transcriptional regulation. Additionally, the team found that the Forkhead protein FoxA1 is required at these sites for ER activity. This study used the powerful combination of chromatin immunoprecipitation (ChIP) and tiled oligonucleotide arrays to study protein-DNA interactions across two human chromosomes. The team's goal was to identify all regulatory regions playing a role in ER-mediated transcription. Chromatin immunoprecipitation (ChIP) was used in combination with Affymetrix' GeneChip® Chromosome 21/22 Tiling Array Set which has one million probe pairs at 35 bp density, covering the entire non-repetitive sequences of chromosomes 21 and 22. The Chromosome 21/22 Tiling Array Set is currently available to qualified laboratories through the Affymetrix Early Access Program.* Using the Chromosome 21/22 Tiling Array Set, the researchers identified fifty-seven ER binding sites containing 32 ER binding clusters, including some proximal to genes previously identified as estrogen targets. There were also ER binding sites discovered within 200 kb of genes not previously implicated as estrogen targets. Further experimentation demonstrated estrogen-dependent interaction of distal ER binding sites and promoters, confirming the distal sites' function as transcriptional enhancers. There were two sequence motifs found by an unbiased search within the 57 ER binding sites. One was a consensus 15-base sequence identical to the canonical ERE, and the other was a consensus Forkhead factor binding site. The researchers found that a Forkhead protein (FoxA1) binding was necessary for expression of estrogen gene targets. In fact, knockdown of the Forkhead protein FoxA1 blocked the expression of ER with chromatin and estrogen-induced gene expression, thus showing that FoxA1 mediates estrogen response in breast cancer cells. By using the ChIP and GeneChip tiling arrays, Dr. Carroll and his team were able to identify chromatin regions, both proximal and distal to promoters, which are involved in mediating ER transcriptional activity. Finding the Forkhead binding motifs and identifying the functional role of FoxA1 in estrogen signaling demonstrates the power of this approach for identifying critical regulatory regions within previously unexplored sequences of the human genome. *The Early Access Program offers developing technologies to qualified laboratories for independent research. To inquire about enrollment in the Early Access Program, please contact your local Affymetrix sales representative.