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Published in final edited form as: Essays Biochem. 2021 Dec 17;65(6):857–866. doi: 10.1042/EBC20210019

Steroid receptor-coregulator transcriptional complexes: new insights from CryoEM

Ping Yi 1, Xinzhe Yu 2, Zhao Wang 1,2, Bert W O’Malley 1
PMCID: PMC8845409  NIHMSID: NIHMS1776909  PMID: 34061186

Abstract

Steroid receptors activate gene transcription through recruitment of a number of coregulators to facilitate histone modification, chromatin remodeling, and general transcription machinery stabilization. Understanding the structures of full-length steroid receptor and coregulatory complexes has been difficult due to their large molecular sizes and dynamic structural conformations. Recent developments in cryo-electron microscopy (cryoEM) technology and proteomics have advanced the structural studies of steroid receptor complexes. Here, we will review the insights we learned from cryoEM studies of the estrogen and androgen receptor transcriptional complexes. Despite similar domain organizations, the two receptors have different coregulator interaction modes. The cryoEM structures now have revealed the fundamental differences between the two receptors and their functional mechanisms.

Introduction

Ligand-dependent nuclear receptors (NRs) are a family of transcription factors that regulate the expression of target genes upon sensing cognate ligands, usually small lipophilic molecules, such as steroid, retinoid, and thyroid hormones. There are 48 members in the human NR family. These receptors regulate a variety of physiological functions, including genetic development, reproduction, metabolism, inflammation, and homeostasis. Key elements in NR-mediated transcription include the regulatory hormone-responsive elements embedded in the promoter/enhancer regions of DNA, epigenomic marks on the chromatin, and binding of NRs to the DNA regulatory elements in response to ligand stimulation. These elements together still are not sufficient to induce gene transcription. Transcriptional coregulators are required for NRs to activate transcription (Figure 1). They are essential partners recruited by NRs to the target gene promoter/enhancer regions and are critical for NR activity. Coregulators can be coactivators, which usually are associated with agonist-bound receptors to enhance gene transcription, or corepressors that repress transcription either in the absence of NR agonists or in the presence of NR antagonists. More than 300 coregulators have been identified [1] since the first cloning of coactivator and corepressor proteins in 1995 [2–4]. These coregulators have diverse functions including bridging between the NRs and the general transcription machinery, such as the mediator complex, scaffolding proteins to recruit additional coregulators to NR binding sites, such as steroid receptor coactivators (SRCs), histone modifiers to add epigenetic marks to histones, such as histone acetyltransferase CBP/p300, and ATP-dependent chromatin remodelers to promote chromatin reorganization, such as SWI/SNF. They engage in various steps of the initiation of the transcription. Ordered and dynamic interactions between NRs and different types of coregulators are a central theme of ligand-stimulated NR activation. Understanding the fundamental structural basis of different NR/coregulator complex formations provides valuable insights for understanding the coordinate and effective regulation of transcription by multiple coregulators.

Figure 1. Coregulators are essential components of nuclear receptor-activated transcription.

Figure 1.

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NR-mediated transcription involves regulatory hormone-responsive DNA elements, epigenomic marks at the chromatin, and the binding of NRs to the regulatory elements in response to ligand stimulation. In addition, NR coregulators are also indispensable in forming a functional NR transcriptional complex. TF, transcription factor.

Over the last 30 years, structural studies of NR functional domains, particularly the ligand-binding and the DNA-binding domains, have significantly enriched our understanding of how NRs bind agonists and antagonists, recognize specific DNA sequences, and interact with coregulator peptides [5]. Coregulator structures were less studied in the past due to their relatively large sizes (and formation of multi-component complexes) and their dynamic conformations (often containing multiple intrinsic disordered regions). Recent advances in cryoEM imaging and data processing make solving large NR and coregulator complex structures feasible. These cryoEM studies provide valuable structural information and illustrate the challenges that traditional structure approaches or biochemical methods are facing. The breakthroughs in analyses of large megadalton coregulator complexes such as that of mediator and SWI/SNF complex made via cryoEM have been reviewed elsewhere [6–8]; how these complexes interact with NRs has remained elusive. Here, we review our recent progress in steroid receptor (SR) cryoEM structural studies focusing on the mechanisms by which SRs interact with coactivators at specific DNA elements (HREs).

Estrogen receptor complexes

The estrogen receptor (ER) is a key member of the steroid receptor superfamily of NRs. ER contains several major domains like other NRs [9] (Figure 2A). The N-terminal domain (A/B region) is highly variable among NRs, and it often presents a ligand-independent activation function (AF-1). The central domain (C region) is a highly conserved DNA-binding domain (DBD). A flexible hinge region (D region) connects the DBD to the C-terminal ligand-binding domain (LBD, E/F region). The LBD is responsible for ligand binding and contains a ligand-dependent activation function (AF-2). Upon binding estrogen, the ER undergoes a conformational change and recruits a number of primary coactivators, such as SRCs, through its AF-2. The LXXLL motifs residing in the receptor interaction domain of SRCs interact with a hydrophobic cleft on the surface of ER LBD [10,11]. SRCs in turn recruit secondary coactivators, such as CBP/p300 that is capable of acetylating histones. Aside from the LBD, interactions of the N-terminal domain with both SRCs and p300 have been suggested [12–14]. In most promoter contexts, AF-1 and AF-2 act synergistically to contribute to full ER transcriptional activity [15,16]. In some studies, ligand-induced functional synergism of AF1 and AF2 is mediated by binding of coactivators to both [12,14,17]; some even suggest that the two domains interact directly [18]. While LBD and DBD structures of ERs have been extensively studied by crystallography and NMR [19–22], the spatial organization of ER N- and C-terminal functional domains in the context of full-length receptor and their contributions to coactivator recruitment have not been well-defined until recently when a cryoEM approach was implemented for the determination of DNA-bound ERα/coactivator complex structures [23,24]. Our cryoEM structure also addressed other controversies in the literature, for example, whether ER binds one or two SRCs or whether CBP/p300 directly binds to ER or DNA.

Figure 2. The structural domain organization of estrogen and androgen receptor.

Figure 2.

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(A) Schematic representation of estrogen receptor functional domains. (B) The structural organization of DNA-bound ERα dimer from the ERα/SRC-3/p300 complex density map. The LBDs and DBDs form the ERα dimer interface and the two NTDs are far apart and located at each side of the LBD. The ERα LBD (PDB ID: 3ERD) and DBD (PDB ID: 1HCQ) crystal structures are docked into the ERα cryoEM density map. Left panel: side view of the structure; right panel: top view of the structure. Bottom panel: the cartoon illustration of the ERα dimer structure. (C) The structural organization of DNA-bound AR dimer. AR dimerization follows in a unique head-to-head and tail-to-tail manner with all functional domains (NTD, DBD and LBD) involved. The NTDs wrap around the LBDs and make large areas of LBDs inaccessible to the surface. Left panel: side view of the structure; right panel: top view of the structure. Bottom panel: the cartoon illustration of the AR dimer structure; DBD, DNA-binding domain; H, Hinge region; LBD, ligand-binding domain; NTD, N-terminal domain.

Our study of ERα/coactivator complex structure used a biotinylated ERE (estrogen-responsive element) containing DNA to assemble the DNA-bound ERα/SRC-3/p300 complex. All recombinant proteins were purified from baculoviruses and were tested for their transcription activation functions using a cell-free transcription assays before employment in structural studies. The complex was then assembled in the presence of estrogen and purified using magnetic streptavidin beads followed by restriction enzyme digestion to release the complex from the beads. Final structures were confirmed using mutations and domain antibodies [23,24].

In the ERE DNA/ERα/SRC-3/p300 complex cryoEM density (a dimension of 220 × 260 × 320 Å), four protein components were found, including one ERα dimer, two SRC-3s (a and b), and one p300 [23]. Each ERα monomer interacts with one SRC-3, and the two SRC-3s comprise multiple contact regions for p300 to stabilize its binding to the complex (Figure 3A). The ERα dimer density in the complex structure reveals the spatial organization of ERα functional domains (Figure 2B). The LBD and DBD are located at the center and form a dimer interface. On both sides of the dimer, the NTDs occur and bind the LBDs directly. This architecture supports one previous finding that the two domains can have direct interactions [18]. The two SRC-3s, although of similar shapes and scales, do not have identical densities and do not interact with ERα and p300 in the same way. The SRC-3a has a larger contact area with ERα compared with SRC-3b. It contacts both ERα AF-2 and AF-1 regions while the SRC-3b mainly interacts with the AF-2. Consequently, the ERα AF-1-specific antibody is not able to bind the precise ERα monomer with which SRC-3a interacts due to the steric hindrance. The cooperative function of AF-2 and AF-1 in recruiting SRC-3 reveals how the N- and C-terminal domains activate ERα transcriptional activity synergistically. SRC-3a also has a stronger interaction with p300 compared with SRC-3b. P300 is a multi-domain interacting protein. Its C-terminus contains an SRC-interacting domain (SRCID). The SRC-3a mainly interacts with this SRCID while SRC-3b contacts multiple other regions of p300. P300 itself does not directly interact with ERα. Deleting the region in SRC-3 that is responsible for p300 recruitment completely abolishes p300 binding to the ERα complex. Interestingly, there was an apparent conformational change seen in p300 when comparing the p300 density in the complex to free p300, either with or without antibody binding. The functional consequence of this conformational change is that p300 has higher HAT activity to acetylate histone H3 when recruited by ERα and SRC-3 comparing to the free p300. The ERα/SRC-3/p300 cryoEM structure gives us a first overview of how ER functional domains work together to employ primary and secondary coactivators and to address several controversies which have not yet been resolved.

Figure 3. The estrogen and androgen receptor/coactivator complex structural organization.

Figure 3.

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(A) The structural assembly of DNA-bound ERα, SRC-3 and p300 complex. ERα recruits two SRC-3s (−a and −b) and the two SRC-3s interact with different regions of p300 to bring in p300 to the ERα-binding site. (B) The structural assembly of DNA-bound AR, SRC-3, and p300 complex. AR interacts directly with one SRC-3 and one p300. The AR NTD-b is solely responsible for SRC-3 recruitment. Both AR NTDs are major contributors for p300 recruitment. Small areas of LBDs also interact with p300. The arrows indicate the interaction between different proteins.

Sequential coactivator recruitment to the ERα complex

In addition to p300, SRC-3 also sequentially recruits a secondary coactivator CARM1 (coactivator arginine methyltransferase 1) to the ER complex. CARM1 belongs to the PRMT (protein arginine methyltransferase) family. It promotes arginine asymmetric dimethylation at histone H3 R17, R26, and R42 residues [25,26]. CARM1 synergizes with SRCs and p300 to activate ER transcriptional activity [24,27,28]. Its recruitment to the ER complex follows after the SRC and p300 recruitments [24,29]. The ERE-bound ERα/SRC-3/p300/CARM1 cryoEM structure surprisingly reveals that recruitment of CARM1 to the ERα/SRC-3/p300 complex is not a simple add-on of a coactivator protein to the complex [24], and addition of CARM1 does not increase the size of the complex. Three of the components (ERα dimer, SRC-3a, and p300) are still present while the SRC-3b is replaced by CARM1 (Figure 4A). Although CARM1 occupies the same position as SRC-3b in the ERα/SRC-3/p300 complex, there are several major differences between them. First, CARM1 does not directly interact with ER while SRC-3b contacts the ERα LBD. Second, CARM1 and SRC-3b interact with different regions of p300. Finally, the p300 conformation undergoes significant changes in complexes with or without CARM1 recruitment. This CARM1 binding-induced p300 conformational change results in an additional impact on p300 HAT activity. In vitro HAT assays demonstrate that p300 activity for auto-acetylation and histone H3 acetylation (H3K18 acetylation specifically) is boosted with the recruitment of CARM1. CARM1-targeted methylation site H3R17 is adjacent to the p300-targeted acetylation site at K18. There is cooperative cross-talk between the two coactivators and histone epigenetic modifications. It was reported that CARM1 recruitment and R17 methylation follows prior CBP/p300 recruitment and K18 acetylation [29]. K18 acetylation also dramatically increases CARM1 methyltransferase activity on R17 [30]. The cryoEM architecture changes demonstrate that the cross-talk is bidirectional. The knockdown of CARM1 also reduces the level of H3K18 acetylation in ER-targeted gene promoter/enhancer in breast cancer cells [24]. We proposed a working model for sequential recruitment of CARM1. Since CARM1 contacts different regions of p300 than SRC-3, p300 adopts a different conformation to accommodate CARM1 binding. The conformational change increases p300-mediated H3K18 acetylation and in turn enhances CARM1 activity on H3R17 methylation. The histone methylation mark further recruits reader proteins (e.g., Tudor domain containing protein TDRD3 [31,32] and the ‘transcription elongation’ associated PAF1 complex (PAF1c) [33]) to promote subsequent transcription elongation.

Figure 4. The structural organization of DNA-bound ERα/SRC-3/p300/CARM1 complex.

Figure 4.

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(A) Three subclasses of structural assembly of DNA-bound ERα/SRC-3/p300/CARM1 complex with SRC-3b binding (left panel), with CARM1 binding (right panel), or without any protein binding to one ERα monomer (middle panel), representing before (left) and after (right) CARM1 binding complex states, and a possible intermediate state (middle panel). (B) CARM1 exists as a dimer in the ERα complex and the N-terminal region of CARM1 is involved in interacting with p300. Shown is a CARM1 antibody labeling of the DNA-bound ERα/SRC-3/p300/CARM1 structure. Two CARM1 N-terminal domain-specific Fabs (yellow color) bind to the region near CARM1-p300 interaction surface. (CryoEM structure images were adapted from [24]).

The structure also reveals that CARM1 forms a dimer in the complex. When the complex was incubated with the CARM1 N-terminal domain-specific Fab, two Fab densities were found binding to the CARM1 (Figure 4B). The major functions of CARM1, including catalytic core, SRC-binding activity of CARM1, and oligomerization interface, are located in the central domain [34]. However, the N-terminal domain also is essential for CARM1 to synergize with p300 to activate ER transcription [34]. This underlying mechanism previously was not clear until our ERα/coactivator complex structure was solved [24]. The N-terminal domain presents a Pleckstrin Homology (PH) domain-like fold when isolated, but it is not visible in the crystal structure when fused to the central domain, indicating its position inside full-length CARM1 is flexible [35]. The PH domain is found in various proteins and is often involved in protein binding [36]. This domain is found to be immobilized in the cryoEM structure and it directly contacts p300 when CARM1 binds to the ERα complex. It is indispensable for CARM1 to regulate p300 activity since deletion of this domain abolishes the ability of CARM1 to increase p300-mediated H3 acetylation.

Overall, the arrangement indicates that sequential coactivator recruitment is an ordered process. The structural changes associated with each coactivator binding are closely correlated with the specific transcriptional functions of each complex.

Androgen receptor and coactivator complex structures

The androgen receptor (AR) was thought to contain a similar domain organization as ERα. However, the size of AR (110 kDa) is much larger than ERα (66 kDa) due to a long N-terminal domain (NTD). Unlike ERα, the AR NTD contains a ‘dominant AF-1 function’ while its LBD has a weak AF-2 function. Deletion of the NTD virtually abolishes AR transcriptional activity [37,38]. The very N-terminus of the NTD has a FXXLF motif resembling the LXXLL motif found in SRCs. Like the LXXLL motif, the FXXLF motif contains an amphipathic α-helix structure, and it binds the LBD hydrophobic groove with higher affinity than the LXXLL peptide [39]. Androgen binding induces a unique FXXLF motif-mediated N- and C-terminal interaction [40]. This N/C interaction is needed for AR to activate certain target gene transcriptions [41,42]. Both intramolecular N/C interaction within the AR monomer and intermolecular N/C interaction between the two AR monomers have been observed [43,44]. It was believed that AR forms an anti-parallel dimer to allow the intermolecular N/C interaction to occur [40,44,45].

Our recent AR dimer cryoEM structure demonstrates that it is distinct from the ER dimer structure (Figure 2C) [46]. It does not adopt a typical symmetrical dimer conformation; nor does it form an anti-parallel dimer. It follows in a unique head-to-head and tail-to-tail manner with all domains involved. The DBD and LBD are located at the center of the dimer interface. The overall size and shape of the LBDs fit well with previously published AR LBD dimer crystal structure [47]. This DBD and LBD structural organization is similar to ERα and other NRs [23,48,49]. The most noticeable distinction between AR and ERα structures is the NTD arrangement. The NTD structural information has been lacking in other full-length NR structures [48–51]. The two NTDs within the AR dimer surprisingly adopt slightly different conformations. Instead of positioning far apart and side-by-side with the LBD as in the ERα dimer, the two NTDs connect to each other. They wrap around and make contacts with both LBDs. This arrangement allows both intra- and inter-molecular N/C interactions to occur. It also results in a large part of LBDs being buried inside while the NTDs expose large areas to the surface. This could be the fundamental structural basis for the differential activation domain preferences observed in the two sex hormone receptors.

The composition of the AR/SRC-3/p300 assembled complex also is different from that of the ERα/SRC-3/p300 complex (Figure 3B). AR recruits only one SRC-3 and one p300. The one SRC-3 corresponds to the SRC-3a as in the ERα complex. However, it exclusively contacts the NTD of AR while the same SRC-3a interacts with both the LBD and NTD of ERα. Biochemical analyses also demonstrate that SRC-3 utilizes separate regions to interact with AR and ERα. The LXXLL motif essential for SRC-3 recruitment to the ERα LBD does not contribute to its binding to AR NTD. The SRC-3 contact site is in close proximity to the FXXLF motif where the N/C interaction occurs. Androgen-dependent N/C interaction likely induces a NTD conformational change that is optimal for SRC-3 recruitment. This explains why androgen stimulates SRC-3 recruitment even though SRC-3 does not interact with the LBD. In the AR complex, the p300 directly contacts both SRC-3 and AR, while it only interacts with two SRC-3s in the ERα complex. SRC-3 stabilizes the interaction between p300 and AR. The AR NTD also is the major p300 interaction region. This AR/coactivator complex structure underscores the importance of targeting AR NTD to therapeutically inhibit AR activity, especially in prostate cancer when androgen antagonists encounter eventual resistance.

Conclusion and perspectives

Upon activation of a nuclear receptor, it initiates a series steps of coregulator association and dissociation, chromatin remodeling, and RNA polymerase recruitment at its genomic binding sites. The sophistication of this transcription mechanism involves a thorough picture of how numerous coregulators communicate with NR, with each other, with the chromatin and the general transcription machinery to induce a burst of target gene transcription. The single particle cryoEM analyses reviewed here provide a first glimpse on how functional domain organization of ER/AR contributes to their interaction with the core coactivators and how different coactivators communicate with each other cooperatively to modify histones and activate transcription.

The insights we learned from these studies are the following: (1) ERα forms a symmetric tail-to-tail dimer with the two NTDs far apart and located at each side of the LBDs. AR forms an asymmetrical head-to-head and tail-to tail dimer with large areas of LBDs buried inside the NTDs, and the two NTDs, which adopt different conformations, have direct contacts to contribute to dimerization. (2) ERα recruits two SRC-3s and one p300 to form a core initiation complex while it only retains one SRC-3 and one p300 following sequential CARM1 recruitment during elongation. AR recruits only one SRC-3 and one p300 for initiation. (3) ERα interacts with SRC-3 mainly through its AF-2 domain located in the LBD. The NTD domain directly contacts and cooperates with the LBD to participate in the SRC-3 recruitment. AR mainly utilizes its NTD to interact with SRC-3 and p300. (4) P300 does not directly interact with ERα or DNA while it contacts large surfaces of the two AR NTDs and some areas of AR LBDs and DNA. (5) SRC-3 uses different domains to interact with AR and ER. LXXLL motifs in the SRC-3 RID are important for interacting with ERα but not AR. (6) SRC-3 not only interacts with the SRCID domain of p300 but also contacts multiple other regions. The two SRC-3s recruited by ERα also contact different areas of p300 to stabilize the p300 binding.

In addition to revealing new structural insights, defining the coactivator interaction sites via complex structures also offers potential alternate targets for regulation of NR function. For example, the ER core complex structure reveals that the p300 is recruited to the ERα complex only by connections with SRCs. The p300 binding to the ER and its complex HAT activity are effectively minimized with the SRC-specific small molecular inhibitor SI-2 [52] (unpublished data). The AR/coactivator complex structure also highlights the importance of NTD in recruiting coactivators. Current AR-targeting therapies mainly target the LBD. However, all patients responding to these therapies eventually develop resistance. Our cryoEM structure indicates that both SRC-3 and p300 directly contact the AR NTD [46]. SRC-3 interacts with the region close to the N-terminal FXXLF motif while p300 mainly interacts with the AF-1 region. Future drug design to prevent recruitment of both coactivators may be necessary to efficiently inhibit AR function.

The NR-coactivator complex structures have not yet reached a near atomic resolution and certain of the protein–protein contact details remain unclear. The major challenges are the complex heterogeneity and conformational dynamics during each stage of transcription. NRs and most coactivators contain intrinsic disordered regions. In such areas, a particular configuration is frequently adopted upon contacting an interaction partner [53]. This ‘induced-fit’ structural feature is common to many transcription factors and scaffolding coactivators to allow specific protein–protein interaction. With respect to ERα, its flexible NTD is poorly structured and not observable by X-ray analysis [54]. However, cryoEM shows that the NTD is immobilized and visible when contacting SRC-3 [23]. In contrast, one of the NTD domain densities is missing due to flexibility in the ERα/SRC-3/p300/CARM1 complex when CARM1 is not contacting the ERα NTD [24]. ERα can form a large complex with multiple additional coactivators that is stable and resistant to high salt and high concentrations of urea [55]. In the future forming a larger NR/coactivator complex could limit the issue of dynamic conformations of a NR complex. New algorithms to process heterogeneous mixtures [56], and new structural approaches in mass spectrometry-based proteomics [57,58] also will improve the detailed structural characterization of heterogeneous NR complex assembly, and ultimately the reconstruction of NR complex structures inside the target cells [59].

Summary.

  • Estrogen receptor recruits two SRC-3s and one p300 to form a transcriptionally active complex.

  • Sequential recruitment of CARM1 to the ER/coactivator complex alters p300 conformation and enhances p300 HAT activity as well as CARM1 HMT activity to activate transcription.

  • Androgen receptor recruits one SRC-3 and one p300 mainly through its N-terminal domains.

Funding

This work is supported by NIH [grant numbers NIH-NICHD HD008818 and HD007857 (to B.W. O.)]; CPRIT [grant number RP150648 (to B.W.O.)]; the Robert Welch Foundation [grant number Q-1967-20180324 (to Z.W.)]; and BCM BMB department seed funds (to Z.W.).

Abbreviations

cryoEM

cryo-electron microscopy

DBD

DNA-binding domain

LBD

ligand-binding domain

NTD

N-terminal domain

ER

estrogen receptor

AR

androgen receptor

NR

nuclear receptor

SRC

steroid receptor coactivator

SRCID

SRC-interacting domain

CARM1

coactivator associated arginine methyltransferase1

SR

steroid receptor

Footnotes

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

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