Maureen G. Mancini

FRAP reveals that mobility of oestrogen receptor-α is ligand- and proteasome-dependent

Here we report the use of fluorescence recovery after photobleaching (FRAP) to examine the intranuclear dynamics of fluorescent oestrogen receptor-α (ER). After bleaching, unliganded ER exhibits high mobility (recovery t1/2 < 1 s). Agonist (oestradiol; E2) or partial antagonist (4-hydroxytamoxifen) slows ER recovery (t1/2 ∼5–6 s), whereas the pure antagonist (ICI 182,780) and, surprisingly, proteasome inhibitors each immobilize ER to the nuclear matrix. Dual FRAP experiments show that fluorescent ER and SRC-1 exhibit similar dynamics only in the presence of E2. In contrast to reports that several nuclear proteins show uniform dynamics, ER exhibits differential mobility depending upon several factors that are linked to its transcription function.

Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

Estrogen-receptor-α exchange and chromatin dynamics are ligand- and domain-dependent

We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor α (ERα) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERα- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in koff. ERα-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERα DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.

Regulation of SRC-3 Intercompartmental Dynamics by Estrogen Receptor and Phosphorylation

ABSTRACT The steroid receptor coactivator 3 gene (SRC-3) (AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family transcription coactivator and a known oncogene. Despite its importance, the functional regulation of SRC-3 remains poorly understood within a cellular context. Using a novel combination of live-cell, high-throughput, and fluorescent microscopy, we report SRC-3 to be a nucleocytoplasmic shuttling protein whose intracellular mobility, solubility, and cellular localization are regulated by phosphorylation and estrogen receptor α (ERα) interactions. We show that both chemical inhibition and small interfering RNA reduction of the mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by epidermal growth factor signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known participants in the phosphocode that regulates SRC-3 activity. Accordingly, the cytoplasmic localization of a nonphosphorylatable SRC-3 mutant further supported these results. In the presence of ERα, U0126 also dramatically reduces (i) ligand-dependent colocalization of SRC-3 and ERα, (ii) the formation of ER-SRC-3 complexes in cell lysates, and (iii) SRC-3 targeting to a visible, ERα-occupied and -regulated prolactin promoter array. Taken together, these results indicate that phosphorylation coordinates SRC-3 coactivator function by linking the probabilistic formation of transient nuclear receptor-coactivator complexes with its molecular dynamics and cellular compartmentalization. Technically and conceptually, these findings have a new and broad impact upon evaluating mechanisms of action of gene regulators at a cellular system level.

SUBNUCLEAR PARTITIONING AND FUNCTIONAL REGULATION OF THE PIT-1 TRANSCRIPTION FACTOR

Subnuclear compartmentation is postulated to play an important role in many aspects of nuclear metabolism. To directly test an application of this model to transcription factor function, we examined the subnuclear partitioning behavior of Pit‐1, a tissue‐specific, POU‐class transactivator. Biochemical and in situ assays indicate the nuclear pool of Pit‐1 is normally divided between two compartments: the majority being differentially soluble in detergent, and a significant insoluble fraction (∼20%) bound to the nuclear matrix. Examination of Pit‐1 deletion mutants and chimeric fusions reveal the highly conserved 66 amino acid POU‐specific domain contains a necessary and sufficient nuclear matrix targeting signal. The nuclear partitioning behavior of several natural or engineered point mutations of Pit‐1 was also examined. Surprisingly, the inactive point mutants were completely matrix‐bound, irrespective of their ability to bind Pit‐1 specific DNA. These results suggest that dynamic partitioning of Pit‐1 is a component of its normal transactivator function that takes place upon the insoluble nuclear substructure where transcription occurs. J. Cell. Biochem. 72:322–338, 1999.

Defining Estrogenic Mechanisms of Bisphenol A Analogs through High Throughput Microscopy-Based Contextual Assays

Environmental exposures to chemically heterogeneous endocrine-disrupting chemicals (EDCs) mimic or interfere with hormone actions and negatively affect human health. Despite public interest and the prevalence of EDCs in the environment, methods to mechanistically classify these diverse chemicals in a high throughput (HT) manner have not been actively explored. Here, we describe the use of multiparametric, HT microscopy-based platforms to examine how a prototypical EDC, bisphenol A (BPA), and 18 poorly studied BPA analogs (BPXs), affect estrogen receptor (ER). We show that short exposure to BPA and most BPXs induces ERα and/or ERβ loading to DNA changing target gene transcription. Many BPXs exhibit higher affinity for ERβ and act as ERβ antagonists, while they act largely as agonists or mixed agonists and antagonists on ERα. Finally, despite binding to ERs, some BPXs exhibit lower levels of activity. Our comprehensive view of BPXs activities allows their classification and the evaluation of potential harmful effects. The strategy described here used on a large-scale basis likely offers a faster, more cost-effective way to identify safer BPA alternatives.

Activation of Estrogen Receptor-α by E2 or EGF Induces Temporally Distinct Patterns of Large-Scale Chromatin Modification and mRNA Transcription

Estrogen receptor-α (ER) transcription function is regulated in a ligand-dependent (e.g., estradiol, E2) or ligand-independent (e.g., growth factors) manner. Our laboratory seeks to understand these two modes of action. Using a cell line that contains a visible prolactin enhancer/promoter array (PRL-HeLa) regulated by ER, we analyzed ER response to E2 and EGF by quantifying image-based results. Data show differential recruitment of GFP-ER to the array, with the AF1 domain playing a vital role in EGF-mediated responsiveness. Temporal analyses of large-scale chromatin dynamics, and accumulation of array-localized reporter mRNA over 24 hours showed that the EGF response consists of a single pulse of reporter mRNA accumulation concomitant with transient increase in array decondensation. Estradiol induced a novel cyclical pattern of mRNA accumulation with a sustained increase in array decondensation. Collectively, our work shows that there is a stimuli-specific pattern of large-scale chromatin modification and transcript levels by ER.

Differential phosphorylation of perilipin 1A at the initiation of lipolysis revealed by novel monoclonal antibodies and high content analysis.

Lipolysis in adipocytes is regulated by phosphorylation of lipid droplet-associated proteins, including perilipin 1A and hormone-sensitive lipase (HSL). Perilipin 1A is potentially phosphorylated by cAMP(adenosine 3′,5′-cyclic monophosphate)-dependent protein kinase (PKA) on several sites, including conserved C-terminal residues, serine 497 (PKA-site 5) and serine 522 (PKA-site 6). To characterize perilipin 1A phosphorylation, novel monoclonal antibodies were developed, which selectively recognize perilipin 1A phosphorylation at PKA-site 5 and PKA-site 6. Utilizing these novel antibodies, as well as antibodies selectively recognizing HSL phosphorylation at serine 563 or serine 660, we used high content analysis to examine the phosphorylation of perilipin 1A and HSL in adipocytes exposed to lipolytic agents. We found that perilipin PKA-site 5 and HSL-serine 660 were phosphorylated to a similar extent in response to forskolin (FSK) and L-γ-melanocyte stimulating hormone (L-γ-MSH). In contrast, perilipin PKA-site 6 and HSL-serine 563 were phosphorylated more slowly and L-γ-MSH was a stronger agonist for these sites compared to FSK. When a panel of lipolytic agents was tested, including multiple concentrations of isoproterenol, FSK, and L-γ-MSH, the pattern of results was virtually identical for perilipin PKA-site 5 and HSL-serine 660, whereas a distinct pattern was observed for perilipin PKA-site 6 and HSL-serine 563. Notably, perilipin PKA-site 5 and HSL-serine 660 feature two arginine residues upstream from the phospho-acceptor site, which confers high affinity for PKA, whereas perilipin PKA-site 6 and HSL-serine 563 feature only a single arginine. Thus, we suggest perilipin 1A and HSL are differentially phosphorylated in a similar manner at the initiation of lipolysis and arginine residues near the target serines may influence this process.

Inactivating Pit-1 mutations alter subnuclear dynamics suggesting a protein misfolding and nuclear stress response.

Pit‐1, a POU‐class nuclear DNA‐binding transcription factor, specifies three of the parenchymal cell types in anterior pituitary ontogeny. Using fluorescent fusions and live cell imaging, we have compared the dynamic behavior of wild‐type and inactivating Pit‐1 point mutations. Fluorescence recovery after photobleaching (FRAP) and real‐time extraction data indicate that wild‐type Pit‐1 has a dynamic mobility profile, with t1/2s ∼ 5–7 s when expressed from low to high amounts, respectively. Biochemically, Pit‐1 is ∼50% retained according to direct observation during extraction, indicating a dynamic interaction with nuclear structure. An analysis of transiently expressed Pit‐1 carrying two different debilitating mutations reveals that they translocate normally to the nucleus, but exhibit two different levels of mobility, both clearly distinguishable from wild‐type Pit‐1. At low expression levels, the t1/2s of PitW261C and PitA158P are extremely rapid (0.3 and 0.6 s t1/2s, respectively). At higher expression levels, unlike wild‐type Pit‐1, both mutant proteins become immobilized and insoluble, and fractionate completely with the insoluble nuclear matrix. Relative to wild‐type, over expression of mutated Pit‐1 elicits a nuclear stress response indicated by increased levels of heat shock inducible heat shock protein 70 (Hsp70), and reorganization of heat shock factor‐1. The decreased mobility of PitA158P relative to PitW261C at low expression levels correlates with its ability to partially activate when expressed at low levels and its ability to bind cognate DNA. At high expression levels, lower PitA158P activation correlates with its immobilization and insolubility. These data suggest a link between specific rates of intranuclear mobility and Pit‐1 transcription function, perhaps to insure sufficient interactions with chromatin, or in the case of non‐DNA binding Pit‐1, interaction as a repressor (Scully and Rosenfeld [2002]: Science 295:2231–2235). These data imply inactivating mutations can lead to an intranuclear sorting away from transcription related pathways, and at least in part to a misfolded protein pathway. Taken together, caution is suggested when interpreting point (or other) mutational analyses of transactivator function, as new compartmentation, especially in the context of expression levels, may cloud the distinction between defining functional molecular domains and intranuclear processing of misfolded proteins.

High-Content Screening Identifies Src Family Kinases as Potential Regulators of AR-V7 Expression and Androgen-Independent Cell Growth

AR‐V7 is an androgen receptor (AR) splice variant that lacks the ligand‐binding domain and is isolated from prostate cancer cell lines. Increased expression of AR‐V7 is associated with the transition from hormone‐sensitive prostate cancer to more advanced castration‐resistant prostate cancer (CRPC). Due to the loss of the ligand‐binding domain, AR‐V7 is not responsive to traditional AR‐targeted therapies, and the mechanisms that regulate AR‐V7 are still incompletely understood. Therefore, we aimed to explore existing classes of small molecules that may regulate AR‐V7 expression and intracellular localization and their potential therapeutic role in CRPC.