CAPER is vital for energy and redox homeostasis by integrating glucose-induced mitochondrial functions via ERR-α-Gabpa and stress-induced adaptive responses via NF-κB-cMYC.

Ever since we developed mitochondria to generate ATP, eukaryotes required intimate mito-nuclear communication. In addition, since reactive oxygen species are a cost of mitochondrial oxidative phosphorylation, this demands safeguards as protection from these harmful byproducts. Here we identified a critical transcriptional integrator which eukaryotes share to orchestrate both nutrient-induced mitochondrial energy metabolism and stress-induced nuclear responses, thereby maintaining carbon-nitrogen balance, and preserving life span and reproductive capacity. Inhibition of nutrient-induced expression of CAPER arrests nutrient-dependent cell proliferation and ATP generation and induces autophagy-mediated vacuolization. Nutrient signaling to CAPER induces mitochondrial transcription and glucose-dependent mitochondrial respiration via coactivation of nuclear receptor ERR-α-mediated Gabpa transcription. CAPER is also a coactivator for NF-κB that directly regulates c-Myc to coordinate nuclear transcriptome responses to mitochondrial stress. Finally, CAPER is responsible for anaplerotic carbon flux into TCA cycles from glycolysis, amino acids and fatty acids in order to maintain cellular energy metabolism to counter mitochondrial stress. Collectively, our studies reveal CAPER as an evolutionarily conserved 'master' regulatory mechanism by which eukaryotic cells control vital homeostasis for both ATP and antioxidants via CAPER-dependent coordinated control of nuclear and mitochondrial transcriptomic programs and their metabolisms. These CAPER dependent bioenergetic programs are highly conserved, as we demonstrated that they are essential to preserving life span and reproductive capacity in human cells-and even in C. elegans.

TRAP/SMCC/mediator-dependent transcriptional activation from DNA and chromatin templates by orphan nuclear receptor hepatocyte nuclear factor 4.

The orphan nuclear receptor hepatocyte nuclear factor 4 (HNF-4) regulates the expression of many liver-specific genes both during development and in the adult animal. Towards understanding the molecular mechanisms by which HNF-4 functions, we have established in vitro transcription systems that faithfully recapitulate HNF-4 activity. Here we have focused on the coactivator requirements for HNF-4, especially for the multicomponent TRAP/SMCC/Mediator complex that has emerged as the central regulatory module of the transcription apparatus. Using a system that has been reconstituted from purified transcription factors, as well as one consisting of unfractionated nuclear extract from which TRAP/SMCC/Mediator has been depleted by specific antibodies, we demonstrate a strong dependence of HNF-4 function on this coactivator. Importantly, we further show a TRAP/SMCC/Mediator-dependence for HNF-4 transcriptional activation from chromatin templates. The latter involves cooperation with the histone acetyltransferase-containing coactivator p300, in accord with a synergistic mode of action of the two divergent coactivators. We also show that HNF-4 and TRAP/SMCC/Mediator can interact physically. This interaction likely involves primary HNF-4 activation function 2 (AF-2)-dependent interactions with the TRAP220 subunit of TRAP/SMCC/Mediator and secondary (AF-2-independent) interactions with TRAP170/RGR1. Finally, recruitment experiments using immobilized templates strongly suggest that the functional consequences of the physical interaction probably are manifested at a postrecruitment step in the activation pathway.

E2/Estrogen receptor/sjogren syndrome-associated autoantigen relieves coactivator activator-induced G1/S arrest to promote breast tumorigenicity.

Coactivator activator (CoAA) is a dual-functional coregulator that regulates steroid receptor-mediated transcription and alternative splicing. Previously, we have shown that CoAA has tumor-suppressive potential in tumorigenic human kidney cells. Here, we uncover a molecular mechanism by which Sjogren syndrome-associated autoantigen (SSA), an estrogen receptor (ER) coactivator, induces MYC oncogene by removing repressive CoAA through E2-dependent degradation of CoAA and promotes G(1)/S transition of the cell cycle as well as anchorage-independent growth capability of breast cancer cells. We also show that E2 and ER enhance the E3 ligase activity of SSA to modulate CoAA through splicing isoform-selective ubiquitylation. We propose this as one potential molecular basis for the reduced tumor incidence in autoimmune disease patients and suggest SSA as a potential therapeutic target to treat breast cancer.

Dual roles for coactivator activator and its counterbalancing isoform coactivator modulator in human kidney cell tumorigenesis.

Coactivator activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein, we show that CoAA is a dual-function coregulator that inhibits G(1)-S transition in human kidney cells and suppresses anchorage-independent growth and xenograft tumor formation. Suppression occurs in part by down-regulating c-myc and its downstream effectors ccnd1 and skp2 and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, coactivator modulator (CoAM), antagonizes CoAA-induced G(1)-S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma compared with normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice isoform. This is, thus far, the only example of a nuclear receptor coregulator involved in suppression of kidney cancer and suggests potentially significant new roles for coregulators in renal cancer biology.

Human Mediator enhances basal transcription by facilitating recruitment of transcription factor IIB during preinitiation complex assembly.

The multisubunit Mediator is a well established transcription coactivator for gene-specific activators. However, recent studies have shown that, although not essential for basal transcription by purified RNA polymerase II (pol II) and general initiation factors, Mediator is essential for basal transcription in nuclear extracts that contain a more physiological complement of factors (Mittler, G., Kremmer, E., Timmers, H. T., and Meisterernst, M. (2001) EMBO Rep. 2, 808-813; Baek, H. J., Malik, S., Qin, J., and Roeder, R. G. (2002) Mol. Cell. Biol. 22, 2842-2852). Here, mechanistic studies with immobilized DNA templates, purified factors, and factor-depleted HeLa extracts have shown (i) that Mediator enhancement of basal transcription correlates with Mediator-dependent recruitment of pol II and general initiation factors (transcription factor (TF) IIB and TFIIE) to the promoter; (ii) that Mediator and TFIIB, which both interact with pol II, are jointly required for pol II recruitment to the promoter and that TFIIB recruitment is Mediator-dependent, whereas Mediator recruitment is TFIIB-independent; (iii) that a high level of TFIIB can bypass the Mediator requirement for basal transcription and pol II recruitment in nuclear extract, thus indicating a conditional restriction of TFIIB function and a key role of Mediator in overcoming this restriction; and (iv) that an earlier rate-limiting step involves formation of a TFIID-Mediator-promoter complex. These results support a stepwise assembly model, rather than a preformed holoenzyme model, for Mediator-dependent assembly of a basal preinitiation complex and, more important, identify a step involving TFIIB as a key site of action of Mediator.

The TRAP/Mediator coactivator complex interacts directly with estrogen receptors alpha and beta through the TRAP220 subunit and directly enhances estrogen receptor function in vitro.

Target gene activation by nuclear hormone receptors, including estrogen receptors (ERs), is thought to be mediated by a variety of interacting cofactors. Here we identify a number of nuclear extract-derived proteins that interact with immobilized ER ligand binding domains in a 17beta-estradiol-dependent manner. The most prominent of these are components of the thyroid hormone receptor-associated protein (TRAP)/Mediator coactivator complex, which interacts with ERalpha and ERbeta in both unfractionated nuclear extracts and purified form. Studies with extracts from TRAP220(-/-) fibroblasts reveal that these interactions depend on TRAP220, a TRAP/Mediator subunit previously shown to interact with ER and other nuclear receptors in a ligand-dependent manner. The physiological relevance of the in vitro interaction is documented further by the isolation of an ERalpha-TRAP/Mediator complex from cultured cells expressing an epitope-tagged ERalpha. Finally, the complete TRAP/Mediator complex is shown to enhance ER function directly in a highly purified cell-free transcription system. These studies firmly establish a direct role for TRAP/Mediator, through TRAP220, in ER function.

Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes.

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones.