Charles E. Foulds

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68−/− and p72−/− embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72−/− MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.

RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators

The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.

Structure of a Biologically Active Estrogen Receptor- Coactivator Complex on DNA

Estrogen receptor (ER/ESR1) is a transcription factor critical for development, reproduction, metabolism, and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement preexisting and sometimes controversial biochemical information is lacking. Here, we use cryoelectron microscopy (cryo-EM) to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment.

Research resource: expression profiling reveals unexpected targets and functions of the human steroid receptor RNA activator (SRA) gene.

The human steroid receptor RNA activator (SRA) gene encodes both noncoding RNAs (ncRNAs) and protein-generating isoforms. In reporter assays, SRA ncRNA enhances nuclear receptor and myogenic differentiation 1 (MyoD)-mediated transcription but also participates in specific corepressor complexes, serving as a distinct scaffold. That SRA RNA levels might affect some biological functions, such as proliferation, apoptosis, steroidogenesis, and myogenesis, has been reported. However, the breadth of endogenous target genes that might be regulated by SRA RNAs remains largely unknown. To address this, we depleted SRA RNA in two human cancer cell lines with small interfering RNAs and then assayed for changes in gene expression by microarray analyses. The majority of significantly changed genes were reduced upon SRA knockdown, implicating SRA RNAs as endogenous coactivators. Unexpectedly, only a small subset of direct estrogen receptor-alpha target genes was affected in estradiol-treated MCF-7 cells. Eight bona fide SRA downstream target genes were identified (SLC2A3, SLC2A12, CCL20, TGFB2, DIO2, TMEM65, TBL1X, and TMPRSS2), representing entirely novel SRA targets, except for TMPRSS2. These data suggest unanticipated roles for SRA in glucose uptake, cellular signaling, T(3) hormone generation, and invasion/metastasis. SRA depletion in MDA-MB-231 cells reduced invasiveness and expression of some genes critical for this process. Consistent with the knockdown data, overexpressed SRA ncRNA coactivates certain target promoters and may enhance the activity of some coregulatory proteins. This study is a valuable resource because it represents the first genome-wide analysis of a mammalian RNA coregulator.

ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion

In contrast to the well-studied classic MAPKs, such as ERK1/2, little is known concerning the regulation and substrates of the atypical MAPK ERK3 signaling cascade and its function in cancer progression. Here, we report that ERK3 interacted with and phosphorylated steroid receptor coactivator 3 (SRC-3), an oncogenic protein overexpressed in multiple human cancers at serine 857 (S857). This ERK3-mediated phosphorylation at S857 was essential for interaction of SRC-3 with the ETS transcription factor PEA3, which promotes upregulation of MMP gene expression and proinvasive activity in lung cancer cells. Importantly, knockdown of ERK3 or SRC-3 inhibited the ability of lung cancer cells to invade and form tumors in the lung in a xenograft mouse model. In addition, ERK3 was found to be highly upregulated in human lung carcinomas. Our study identifies a previously unknown role for ERK3 in promoting lung cancer cell invasiveness by phosphorylating SRC-3 and regulating SRC-3 proinvasive activity by site-specific phosphorylation. As such, ERK3 protein kinase may be an attractive target for therapeutic treatment of invasive lung cancer.

Reprogramming of the Epigenome by MLL1 Links Early-Life Environmental Exposures to Prostate Cancer Risk.

Tissue and organ development is a time of exquisite sensitivity to environmental exposures, which can reprogram developing tissues to increase susceptibility to adult diseases, including cancer. In the developing prostate, even brief exposure to endocrine-disrupting chemicals (EDCs) can increase risk for developing cancer in adulthood, with disruption of the epigenome thought to play a key role in this developmental reprogramming. We find that EDC-induced nongenomic phosphoinositide 3-kinase; (PI3K) signaling engages the histone methyltransferase mixed-lineage leukemia 1 (MLL1), responsible for the histone H3 lysine 4 trimethylation (H3K4me3) active epigenetic mark, to increase cleavage and formation of active MLL1 dimers. In the developing prostate, EDC-induced MLL1 activation increased H3K4me3 at genes associated with prostate cancer, with increased H3K4me3 and elevated basal and hormone-induced expression of reprogrammed genes persisting into adulthood. These data identify a mechanism for MLL1 activation that is vulnerable to disruption by environmental exposures, and link MLL1 activation by EDCs to developmental reprogramming of genes involved in prostate cancer.

Endocrine-disrupting chemicals and fatty liver disease

A growing epidemic of nonalcoholic fatty liver disease (NAFLD) is paralleling the increase in the incidence of obesity and diabetes mellitus in countries that consume a Western diet. As NAFLD can lead to life-threatening conditions such as cirrhosis and hepatocellular carcinoma, an understanding of the factors that trigger its development and pathological progression is needed. Although by definition this disease is not associated with alcohol consumption, exposure to environmental agents that have been linked to other diseases might have a role in the development of NAFLD. Here, we focus on one class of these agents, endocrine-disrupting chemicals (EDCs), and their potential to influence the initiation and progression of a cascade of pathological conditions associated with hepatic steatosis (fatty liver). Experimental studies have revealed several potential mechanisms by which EDC exposure might contribute to disease pathogenesis, including the modulation of nuclear hormone receptor function and the alteration of the epigenome. However, many questions remain to be addressed about the causal link between acute and chronic EDC exposure and the development of NAFLD in humans. Future studies that address these questions hold promise not only for understanding the linkage between EDC exposure and liver disease but also for elucidating the molecular mechanisms that underpin NAFLD, which in turn could facilitate the development of new prevention and treatment opportunities.

The Dual Estrogen Receptor α Inhibitory Effects of the Tissue-Selective Estrogen Complex for Endometrial and Breast Safety

The conjugated estrogen/bazedoxifene tissue-selective estrogen complex (TSEC) is designed to minimize the undesirable effects of estrogen in the uterus and breast tissues and to allow the beneficial effects of estrogen in other estrogen-target tissues, such as the bone and brain. However, the molecular mechanism underlying endometrial and breast safety during TSEC use is not fully understood. Estrogen receptor α (ERα)–estrogen response element (ERE)–DNA pull-down assays using HeLa nuclear extracts followed by mass spectrometry–immunoblotting analyses revealed that, upon TSEC treatment, ERα interacted with transcriptional repressors rather than coactivators. Therefore, the TSEC-mediated recruitment of transcriptional repressors suppresses ERα-mediated transcription in the breast and uterus. In addition, TSEC treatment also degraded ERα protein in uterine tissue and breast cancer cells, but not in bone cells. Interestingly, ERα-ERE-DNA pull-down assays also revealed that, upon TSEC treatment, ERα interacted with the F-box protein 45 (FBXO45) E3 ubiquitin ligase. The loss-of- and gain-of-FBXO45 function analyses indicated that FBXO45 is involved in TSEC-mediated degradation of the ERα protein in endometrial and breast cells. In preclinical studies, these synergistic effects of TSEC on ERα inhibition also suppressed the estrogen-dependent progression of endometriosis. Therefore, the endometrial and breast safety effects of TSEC are associated with synergy between the selective recruitment of transcriptional repressors to ERα and FBXO45-mediated degradation of the ERα protein.

Acetylation on histone H3 lysine 9 mediates a switch from transcription initiation to elongation

The transition from transcription initiation to elongation is a key regulatory step in gene expression, which requires RNA polymerase II (pol II) to escape promoter proximal pausing on chromatin. Although elongation factors promote pause release leading to transcription elongation, the role of epigenetic modifications during this critical transition step is poorly understood. Two histone marks on histone H3, lysine 4 trimethylation (H3K4me3) and lysine 9 acetylation (H3K9ac), co-localize on active gene promoters and are associated with active transcription. H3K4me3 can promote transcription initiation, yet the functional role of H3K9ac is much less understood. We hypothesized that H3K9ac may function downstream of transcription initiation by recruiting proteins important for the next step of transcription. Here, we describe a functional role for H3K9ac in promoting pol II pause release by directly recruiting the super elongation complex (SEC) to chromatin. H3K9ac serves as a substrate for direct binding of the SEC, as does acetylation of histone H4 lysine 5 to a lesser extent. Furthermore, lysine 9 on histone H3 is necessary for maximal pol II pause release through SEC action, and loss of H3K9ac increases the pol II pausing index on a subset of genes in HeLa cells. At select gene promoters, H3K9ac loss or SEC depletion reduces gene expression and increases paused pol II occupancy. We therefore propose that an ordered histone code can promote progression through the transcription cycle, providing new mechanistic insight indicating that SEC recruitment to certain acetylated histones on a subset of genes stimulates the subsequent release of paused pol II needed for transcription elongation.

SRC-2 orchestrates polygenic inputs for fine-tuning glucose homeostasis

Significance Maintenance of glucose concentrations within a homeostatic range is essential for preserving the function of glucose-sensitive tissues. Perturbations in the mechanisms that control this homeostasis give rise to a continuum of glucopathologies associated with aberrant carbohydrate metabolism. Here we show Steroid Receptor Coactivator 2 (SRC-2) to be an integral coregulator that couples gene output with energetic demand by stabilizing and amplifying transcriptional complexes. This study highlights the collective importance of transcriptional coregulators for coordination of gene expression events and may provide insight for understanding components of polygenic diseases such as type 2 diabetes mellitus. Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.