Evolution of NCoR-1 and NCoR-2 corepressor alternative mRNA splicing in placental mammals.

ObjectiveThe NCoR-1 and NCoR-2 corepressors are products of an early gene duplication near the beginning of vertebrate evolution and play both overlapping and divergent roles in development and physiology. Alternative-splicing of NCoR-1 and NCoR-2 further customizes their functions. To better understand the evolutionary basis of this phenomenon we extended our prior study of NCoR-1 and NCoR-2 alternative-splicing to an expanded series of species.ResultsAlternative-splicing of NCoR-2 was observed in all vertebrates examined whereas alternative-splicing of NCoR-1 was largely limited to placental mammals. Notably the most prominent of the NCoR-1 alternative-splicing events specific to the placental lineage, in exon 37 that plays a key role in murine metabolism, mimics in many features an analogous alternative-splicing event that appeared in NCoR-2 much earlier at the beginning of the vertebrate radiation. Detection of additional alternative-splicing events, at exons 28 in NCoR-1 or NCoR-2, was limited to the Rodentia or Primates examined, indicating both corepressor paralogs continued to acquire additional splice variations more recently and independently of one another. Our results suggest that the NCoR-1/NCoR-2 paralogs have been subject to a mix of shared and distinct selective pressures, resulting in the pattern of divergent and convergent alternative-splicing observed in extant species

Corepressor diversification by alternative mRNA splicing is species specific.

BackgroundSMRT and NCoR are corepressor paralogs that help mediate transcriptional repression by a variety of transcription factors, including the nuclear hormone receptors. The functions of both corepressors are extensively diversified in mice by alternative mRNA splicing, generating a series of protein variants that differ in different tissues and that exert different, even diametrically opposite, biochemical and biological effects from one another.ResultsWe report here that the alternative splicing previously reported for SMRT appears to be a relatively recent evolutionary phenomenon, with only one of these previously identified sites utilized in a teleost fish and a limited additional number of the additional known sites utilized in a bird, reptile, and marsupial. In contrast, extensive SMRT alternative splicing at these sites was detected among the placental mammals. The alternative splicing of NCoR previously identified in mice (and shown to regulate lipid and carbohydrate metabolism) is likely to have arisen separately and after that of SMRT, and includes an example of convergent evolution.ConclusionsWe propose that the functions of both SMRT and NCoR have been diversified by alternative splicing during evolution to allow customization for different purposes in different tissues and different species

Phosphorylation of PML by mitogen-activated protein kinases plays a key role in arsenic trioxide-mediated apoptosis

AbstractThe promyelocytic leukemia (PML) protein is a potent growth suppressor and proapototic factor, whereas aberrant fusions of PML and retinoic acid receptor (RAR)-α are causal agents in human acute promyelocytic leukemia. Arsenic trioxide (As2O3) treatment induces apoptosis in acute promyelocytic leukemia cells through an incompletely understood mechanism. We report here that As2O3 treatment induces phosphorylation of the PML protein through a mitogen-activated protein (MAP) kinase pathway. Increased PML phosphorylation is associated with increased sumoylation of PML and increased PML-mediated apoptosis. Conversely, MAP kinase cascade inhibitors, or the introduction of phosphorylation or sumoylation-defective mutations of PML, impair As2O3-mediated apoptosis by PML. We conclude that phosphorylation by MAP kinase cascades potentiates the antiproliferative functions of PML and helps mediate the proapoptotic effects of As2O3

An improved high throughput protein-protein interaction assay for nuclear hormone receptors

The Glutathione-S-Transferase (GST) “pulldown” assay has been used extensively to assay protein interactions in vitro. This methodology has been especially useful for investigating the interactions of nuclear hormone receptors with a wide variety of their interacting partners and coregulatory proteins. Unfortunately, the original GST-pulldown technique relies on multiple binding, washing and elution steps performed in individual microfuge tubes, and requires repeated centrifugation, aspiration, and suspension steps. This type of batch processing creates a significant liquid handling bottleneck, limiting the number of sample points that can be incorporated into one experiment and producing inherently less efficient washing and elution than would a flow-through methodology. In this manuscript, we describe the adaptation of this GST-pulldown assay to a 96-well filter plate format. The use of a multi-well filter plate makes it possible to assay more samples in significantly less time using less reagents and more efficient sample processing than does the traditional single tube assay

Corepressors: custom tailoring and alterations while you wait

A diverse cadre of metazoan transcription factors mediate repression by recruiting protein complexes containing the SMRT (silencing mediator of retinoid and thyroid hormone receptor) or N-CoR (nuclear receptor corepressor) corepressors. SMRT and N-CoR nucleate the assembly of still larger corepressor complexes that perform the specific molecular incantations necessary to confer transcriptional repression. Although SMRT and N-CoR are paralogs and possess similar molecular architectures and mechanistic strategies, they nonetheless exhibit distinct molecular and biological properties. It is now clear that the functions of both SMRT and N-CoR are further diversified through alternative mRNA splicing, yielding a series of corepressor protein variants that participate in distinctive transcription factor partnerships and display distinguishable repression properties. This review will discuss what is known about the structure and actions of SMRT, N-CoR, and their splicing variants, and how alternative splicing may allow the functions of these corepressors to be adapted and tailored to different cells and to different developmental stages

Nuclear Receptor Coactivator ACTR Is a Novel Histone Acetyltransferase and Forms a Multimeric Activation Complex with P/CAF and CBP/p300

AbstractWe report here the identification of a novel cofactor, ACTR, that directly binds nuclear receptors and stimulates their transcriptional activities in a hormone- dependent fashion. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, we show that purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex

Atrophin proteins: an overview of a new class of nuclear receptor corepressors

The normal development and physiological functions of multicellular organisms are regulated by complex gene transcriptional networks that include myriad transcription factors, their associating coregulators, and multiple chromatin-modifying factors. Aberrant gene transcriptional regulation resulting from mutations among these elements often leads to developmental defects and diseases. This review article concentrates on the Atrophin family proteins, including vertebrate Atrophin-1 (ATN1), vertebrate arginine-glutamic acid dipeptide repeats protein (RERE), and Drosophila Atrophin (Atro), which we recently identified as nuclear receptor corepressors. Disruption of Atrophin-mediated pathways causes multiple developmental defects in mouse, zebrafish, and Drosophila, while an aberrant form of ATN1 and altered expression levels of RERE are associated with neurodegenerative disease and cancer in humans, respectively. We here provide an overview of current knowledge about these Atrophin proteins. We hope that this information on Atrophin proteins may help stimulate fresh ideas about how this newly identified class of nuclear receptor corepressors aids specific nuclear receptors and other transcriptional factors in regulating gene transcription, manifesting physiological effects, and causing diseases

Temporal Comparison of PBDEs, OH-PBDEs, PCBs, and OH-PCBs in the Serum of Second Trimester Pregnant Women Recruited from San Francisco General Hospital, California

Prenatal exposures to polybrominated diphenyl ethers (PBDEs) can harm neurodevelopment in humans and animals. In 2003–2004, PentaBDE and OctaBDE were banned in California and phased-out of US production; resulting impacts on human exposures are unknown. We previously reported that median serum concentrations of PBDEs and their metabolites (OH-PBDEs) among second trimester pregnant women recruited from San Francisco General Hospital (2008–2009; n=25) were the highest among pregnant women worldwide. We recruited another cohort from the same clinic in 2011–2012 (n=36) and now compare serum concentrations of PBDEs, OH-PBDEs, polychlorinated biphenyl ethers (PCBs) (structurally similar compounds banned in 1979), and OH-PCBs between two demographically similar cohorts. Between 2008–2009 and 2011–2012, adjusted least square geometric mean (LSGM) concentrations of ΣPBDEs decreased 65% (95% CI: 18, 130) from 90.0 ng/g lipid (95% CI: 64.7,125.2) to 54.6 ng/g lipid (95% CI: 39.2, 76.2) (p=0.004); Σ OH-PBDEs decreased six-fold (p<0.0001); and BDE-47, -99, and -100 declined more than BDE-153. There was a modest, non-significant (p=0.13) decline in LSGM concentrations of ΣPCBs and minimal differences in ΣOH-PCBs between 2008–2009 and 2011–2012. PBDE exposures are likely declining due to regulatory action, but the relative stability in PCB exposures suggests PBDE exposures may eventually plateau and persist for decades