AbstractTo further explore the role of Sp1 and Sp3 in the estrogen regulated TFF1 gene transcription, chromatin immunoprecipitation (ChIP) assay was used to determine the association of estrogen receptor α (ERα), Sp1 and Sp3 with the endogenous trefoil factor 1 (TFF1) gene promoter in MCF‐7 breast cancer cells. ERα and serine 5 phosphorylated RNA polymerase II, the form of RNA polymerase II associated with transcription initiation, were recruited to the TFF1 gene promoter following estrogen addition to MCF‐7 cells cultured under estrogen deplete conditions. Both Sp1 and Sp3 were bound to the TFF1 gene promoter before and after estrogen treatment. Using the re‐ChIP assay, we demonstrate that either Sp1 or Sp3 but not both bind to a TFF1 promoter. The co‐occupancy of ERα and Sp1 on TFF1 promoter remains at similar level with and without estrogen, while that of ERα and Sp3 increased in the presence of estrogen. Further, we observed increased co‐occupancy of Sp3 and serine 5 phosphorylated RNA polymerase II on the TFF1 promoter after estrogen treatment of cells. Taken together, these results provide evidence that Sp3 and ERα are involved in the estrogen induced transcription of the TFF1 gene. J. Cell. Biochem. 105: 365–369, 2008. © 2008 Wiley‐Liss, Inc.