Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activation
Creators: name: Pasini, Luigi <1978>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: BIO/18 Genetica

description: Transcription is controlled by promoter-selective transcriptional factors (TFs),
which bind to cis-regulatory enhancers elements, termed hormone response
elements (HREs), in a specific subset of genes. Regulation by these factors
involves either the recruitment of coactivators or corepressors and direct
interaction with the basal transcriptional machinery (1). Hormone-activated
nuclear receptors (NRs) are well characterized transcriptional factors (2) that
bind to the promoters of their target genes and recruit primary and secondary
coactivator proteins which possess many enzymatic activities required for
gene expression (1,3,4).
In the present study, using single-cell high-resolution fluorescent microscopy
and high throughput microscopy (HTM) coupled to computational imaging
analysis, we investigated transcriptional regulation controlled by the estrogen
receptor alpha (ERalpha), in terms of large scale chromatin remodeling and
interaction with the associated coactivator SRC-3 (Steroid Receptor
Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a
steroid-dependent transcriptional factor (16) that belongs to the NRs
superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2),
regulates transcription of distinct target genes involved in development,
puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the
nucleus and undergoes a rapid (within minutes) intranuclear redistribution
following the addition of either agonist or antagonist (17,18,19).
We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated
reporter gene array (PRL-array) containing multicopy hormone
response-binding elements for ERalpha that are derived from the physiological
enhancer/promoter region of the prolactin gene. Following GFP-ER
transfection of PRL-HeLa cells, we were able to observe in situ ligand
dependent (i) recruitment to the array of the receptor and associated
coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout
of the reporter gene. Addition of E2 causes a visible opening
(decondensation) of the PRL-array, colocalization of RNA Polymerase II, and
transcriptional readout of the reporter gene, detected by mRNA FISH. On the
contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI),
a dramatic condensation of the PRL-array was observed, displacement of
RNA Polymerase II, and complete decreasing in the transcriptional FISH
signal.
All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid
Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160
family member and a known oncogenic protein (4,34). SRC-3 is regulated by a
variety of posttranslational modifications, including methylation,
phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These
events have been shown to be important for its interaction with other
coactivator proteins and NRs and for its oncogenic potential (37,39). A number
of extracellular signaling molecules, like steroid hormones, growth factors and
cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by
a wide range of kinases, including extracellular-regulated kinase 1 and 2
(ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs)
(41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein,
whose cellular localization is regulated by phosphorylation and interaction with
ERalpha. Using a combination of high throughput and fluorescence microscopy,
we show that both chemical inhibition (with U0126) and siRNA downregulation
of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in
SRC-3 localization, whereas stimulation by EGF signaling enhances its
nuclear localization by inducing phosphorylation at T24, S857, and S860, known
partecipants in the regulation of SRC-3 activity (39). Accordingly, the
cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further
supports these results. In the presence of ERalpha, U0126 also dramatically
reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the
nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell
lysates; localization of SRC-3 at the ER-targeted prolactin promoter array
(PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also
function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of
estrogen receptor.
While a wealth of studies have revealed the molecular functions of NRs and
coregulators, there is a paucity of data on how these functions are
spatiotemporally organized in the cellular context. Technically and
conceptually, our findings have a new impact upon evaluating gene
transcriptional control and mechanisms of action of gene regulators.