Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Differential gene regulation by the SRC family of coactivators Hua Zhang,1 Xia Yi,1 Xiaojing Sun, Na Yin, Bin Shi, Huijian Wu, Dan Wang, Ge Wu, and Yongfeng Shang2 Department of Biochemistry and Molecular Biology, Peking University Health Science Center, Beijing 100083, China SRCs (steroid receptor coactivators) are required for nuclear receptor-mediated transcription and are also implicated in the transcription initiation by other transcription factors, such as STATs and NF␬B. Despite phenotypic manifestations in gene knockout mice for SRC-1, GRIP1, and AIB1 of the SRC (Steroid Receptor Coactivator) family indicating their differential roles in animal physiology, there is no clear evidence, at the molecular level, to support a functional specificity for these proteins. We demonstrated in this report that two species of SRC coactivators, either as AIB1:GRIP1 or as AIB1:SRC-1 are recruited, possibly through heterodimerization, on the promoter of genes that contain a classical hormone responsive element (HRE). In contrast, on non-HRE-containing gene promoters, on which steroid receptors bind indirectly, either GRIP1 or SRC-1 is recruited as a monomer, depending on the cellular abundance of the protein. Typically, non-HRE-containing genes are early genes activated by steroid receptors, whereas HRE-containing genes are activated later. Our results also showed that SRC proteins contribute to the temporal regulation of gene transcription. In addition, our experiments revealed a positive correlation between AIB1/c-myc overexpression in ER+ breast carcinoma samples, suggesting a possible mechanism for AIB1 in breast cancer carcinogenesis. [Keywords: Gene regulation; coactivators; steroid receptors] Supplemental material is available at http://www.genesdev.org. Received February 16, 2004; revised version accepted May 21, 2004. Gene transcription activation by transcription factors which were shown to enhance nuclear receptor-medi- such as nuclear receptors is accomplished through the ated gene transcription as well as gene transactivation by recruitment of a series of coactivators. Coactivator pro- other transcription factors. teins fulfill their functions through protein–protein in- The physiological significance of the SRC family and teractions that bridge transcription factors and the basal the functional specificity of these proteins have been an transcription machinery and through their chromatin re- area of extensive study. In this context, AIB1 has been modeling activities that facilitate the assembly of a tran- found to be strongly amplified and/or overexpressed in scription initiation complex (Glass and Rosenfeld 2000). 64% of primary breast cancers as well as in some pri- Several general coactivators, such as CBP/p300 and mary ovarian tumors (Anzick et al. 1997; Bautista et al. p300/CBP-associated protein factor (pCAF), are recruited 1998); and translocation between the GRIP1 gene and by transcription factors to mediate their transcriptional the MOZ (monocytic zinc finger) gene encoding a HAT activities (Glass and Rosenfeld 2000). These proteins are protein (Champagne et al. 2001) has been identified in known to possess intrinsic histone acetyltransferase human acute myeloid leukemia (Carapeti et al. 1998), (HAT) activity and are capable of chromatin modifica- suggesting important functions of SRC proteins in physi- tion by histone acetylation. The general coactivators can ology. Genetic studies with gene ablation showed that, also be recruited by interaction with more specific coac- whereas both male and female SRC1-null mice are viable tivators, including members of the SRC (Steroid Recep- and fertile, they exhibit partial resistance to several hor- tor Coactivator) family (also referred to as p160 family) mones, including estrogen, progestin, androgen, and thy- SRC-1, TIF2/GRIP1/SRC-2 (hereafter referred to as roid hormones (Xu et al. 1998; Weiss et al. 1999); elimi- GRIP1), and pCIP/ACTR/AIB1/RAC3/TRAM1/SRC-3 nation of AIB1 has revealed that it is required for normal (hereafter referred to as AIB1; Glass and Rosenfeld 2000), mouse growth (Wang et al. 2000; Xu et al. 2000), as well as for some female reproductive functions (Xu et al. 2000); and, in contrast to SRC-1 and AIB1 knockout 1These authors contributed equally to this work. mice, there is impairment of fertility in both male and 2Corresponding author. female GRIP1-null mice (Gehin et al. 2002). A recent E-MAIL [email protected]; FAX 86-10-82801355. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ study showed that GRIP-null mice are protected against gad.1194704. obesity and display enhanced adaptive thermogenesis, GENES & DEVELOPMENT 18:1753–1765 © 2004 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/04; www.genesdev.org 1753 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Zhang et al. whereas SRC-1-null mice are prone to obesity due to quickly activated and include c-Myc (Dubik and Shiu reduced energy expenditure (Picard et al. 2002). 1992) and IGF-I (Umayahara et al. 1994), whose promot- Despite the variation in phenotypic manifestations of ers do not contain a classical HRE. Analogously, genes these SRC-null mice, which suggests differential biologi- regulated through direct AR binding, such as PSA (pros- cal activities of the SRC proteins, there is as yet no clear tate specific antigen), typically harbor HREs with a con- evidence, at the molecular level, to indicate functional sensus sequence of 5Ј-GGTACAnnnTGTTCT-3Ј in their specificities for the three members of the SRC family. promoters (Cleutjens et al. 1996, 1997) and need a longer Structurally, SRC-1, GRIP1, and AIB1 share 40% overall time to be activated; genes regulated by binding AR in- sequence identity, and they all contain, in the N termi- directly are early genes such as c-Myc (Bieche et al. 2001; nus, a basic helix–loop–helix (bHLH) domain that is Silva et al. 2001), which does not contain a classical HRE known as a dimerization and DNA interaction domain in its promoter. In previous studies, we and others have as well as a PAS domain, which is known to be involved shown that SRC-1, GRIP1, and AIB1 are all implicated in in protein–protein interaction. Three LXXLL motifs, also both ER- and AR-mediated gene transcription (Shang et known as NR boxes (for nuclear receptor binding), are al. 2000, 2002; Louie et al. 2003). centrally located in all three proteins (Heery et al. 1997; To investigate whether there are any coactivation Darimont et al. 1998; McInerney et al. 1998). Although specificities for these three coactivators in ER- and AR- various biochemical approaches have implied that an in- mediated gene transcription, we first sought to investi- dividual member of the SRC family may possess func- gate the pattern of SRC-1, GRIP1, and AIB1 recruitment tional specificities in a manner of propensity or selectiv- on different gene promoters. The estrogen-dependent hu- ity for different types or isotypes of nuclear receptors or man mammary carcinoma cell line T-47D and the hu- other transcription factors (Kraichely et al. 2000; Issa et man endometrial carcinoma cell line ECC-1, and an an- al. 2001; Barkhem et al. 2002; Beischlag et al. 2002; Bom- drogen-responsive human prostate carcinoma cell line mer et al. 2002), most in vitro studies and transfection LnCAP, were used for this experiment. EBAG9 and c- experiments indicate that each member of the SRC fam- Myc were chosen to represent HRE-containing target ily is capable of interaction with multiple nuclear recep- genes and non-HRE-containing target genes, respec- tors, and that a particular nuclear receptor is able to in- tively, in ER-mediated transcription, and PSA and c-Myc teract with all three members of the SRC family. were chosen to represent HRE-containing target genes Nuclear receptors, especially steroid receptors such as and non-HRE-containing target genes, respectively, in ER and androgen receptor (AR), exhibit a bimodal pattern AR-mediated transcription. The temporal difference in of gene regulation in the presence of their ligands. Some steroid-induced expression of these genes was confirmed genes are activated quickly and others are only activated first in our experimental system. In these experiments, by prolonged stimulation with the ligands. In addition, T-47D cells and ECC-1 cells were grown in the absence tissue-specific activities have been well recognized for of estrogen, and LnCAP cells were grown in the absence nuclear receptors, and these clearly are a reflection of of androgen, for at least 3 d, followed by no treatment or their differential gene regulation in different tissues. treatment with saturating levels of 17␤-estradiol (E2) or However, how the precise regulation of gene expression dihydrotestosterone (DHT) for 6 h, and the mRNA ex- is achieved, and, as important players in nuclear receptor pression of c-Myc, EBAG9, and PSA was analyzed at dif- signaling, how SRC proteins, as a whole family, contrib- ferent times of treatments by real-time RT–PCR. These ute to the temporal regulation of transcription and to the experiments indicated that c-Myc activation occurred tissue-specific functions of nuclear receptors, are not un- much earlier and peaked at1hofE2orDHTtreatment derstood. in these cells, whereas EBAG9 and PSA activation de- In this report, we investigated the functional specific- veloped later and peaked at 4–6 h of E2 or DHT treat- ity of all three members of the SRC family and provided ment (Supplementary Fig. 1). We then investigated the the mechanistic and pathobiological insights into the coactivator recruitment on the promoter of these genes. temporal gene regulation by steroid receptors. In these experiments, T-47D cells and ECC-1 cells were grown in the absence of estrogen, and LnCAP cells were grown in the absence of androgen, for at least 3 d, fol- Results lowed by no treatment or treatment with saturating lev- Steroid receptors such as ER and AR regulate gene tran- els of E2 or DHT for 45 min.