The ability to elicit transcription factor binding and transcriptional activation of specific genes by addition of a small molecular weight hormone to cell culture medium makes steroid hormone-regulated transcription an ideal system to study transcriptional activation. Steroid hormone receptors are hormone-regulated transcription factors belonging to the nuclear receptor family. Binding to their cognate hormone causes steroid hormone receptors to bind to and activate or repress transcription of specific target genes. Transcriptional activation by the DNA-bound receptor is accomplished by recruitment of a large number of coregulator proteins which remodel chromatin conformation and promote the assembly and/or activation of a transcription complex on the target gene promoter1–5
. Chromatin immunoprecipitation (ChIP) studies on selected target genes of estrogen receptor (ER) α (e.g. the TFF1
gene) during the first 60 min of hormone treatment revealed a hormone-initiated sequence of transient steady state occupancy of the promoter and associated ER binding sites by ERα and many coregulator proteins and histone modifications, culminated by enhanced occupancy by RNA polymerase II4–6
. Among the earliest coregulator occupants is the ATP-dependent chromatin remodeling complex SWI/SNF containing ATPase subunit BRG1, followed closely in time by a succession of histone modifying enzymes, including the histone acetyltransferase TIP60. Subsequent target gene occupants include Steroid Receptor Coactivator proteins (SRC-1, SRC-2, and SRC-3), Mediator complex, and other coregulators.
TIP60 belongs to the MYST (MOZ, YBF2, SAS2, and TIP60) family of histone acetyltransferases, which participate in diverse cellular processes, such as transcriptional regulation, DNA damage repair and apoptosis7–10
. Recombinant TIP60 acetylates core histones H2A, H3 and H4 in vitro11,12
; in cells TIP60 is found in a stable multi-protein complex which can acetylate nucleosomes9,12
and several non-histone proteins including the transcription factors p53 (ref. 13–15
) and MYC16
. TIP60 is also known as a nuclear receptor coactivator. It binds to the ligand binding domain of the androgen receptor and enhances hormone-dependent activation of transiently transfected reporter genes by several steroid hormone receptors, including ERα17
. However, little is known about the mechanism by which TIP60 is recruited to endogenous target genes of steroid receptors upon hormonal activation or how TIP60 contributes to chromatin remodeling and transcriptional activation.
To further elucidate the mechanisms of chromatin remodeling and transcription complex assembly and activation, we set out to define the carefully coordinated sequence of physical and functional interactions among the participating coregulators during the early stages of these processes. In this study we demonstrate that TIP60 is required for efficient estrogen-induced expression of a subset of the target genes of ERα. We then use the combination of RNA interference and ChIP to define a cascade of chromatin remodeling events and protein-protein interactions occurring on target gene promoters during the first hour of estrogen treatment, including chromatin remodeling by SWI/SNF ATPase BRG1, subsequent methylation of histone H3 at lysine 4, and the ability of TIP60 to interact with ERα and the enhancer element-specific histone mark, monomethylation of histone H3 at Lys4.