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Thyroid Hormone Increases Bulk Histones Expression by Enhancing Translational Efficiency

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Thyroid Hormone Increases Bulk Histones Expression by Enhancing Translational Efficiency ORIGINAL RESEARCH Thyroid Hormone Increases Bulk Histones Expression by Enhancing Translational Efficiency Alberto Zambrano,* Verónica García-Carpizo,* Raquel Villamuera, and Ana Aranda Instituto de Investigaciones Biomédicas “Alberto Sols,” Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, 28029 Madrid, Spain Downloaded from https://academic.oup.com/mend/article/29/1/68/2556825 by guest on 02 October 2021 The expression of canonical histones is normally coupled to DNA synthesis during the S phase of the cell cycle. Replication-dependent histone mRNAs do not contain a poly(A) tail at their 3Ј terminus, but instead possess a stem-loop motif, the binding site for the stem-loop binding protein (SLBP), which regulates mRNA processing, stability, and relocation to polysomes. Here we show that the thyroid hormone can increase the levels of canonical histones independent of DNA replication. Incubation of mouse embryonic fibroblasts with T3 increases the total levels of histones, and expression of the thyroid hormone receptor ␤ induces a further increase. This is not restricted to mouse embryonic fibroblasts, because T3 also raises histone expression in other cell lines. T3 does not increase histone mRNA or SLBP levels, suggesting that T3 regulates histone expression by a posttranscriptional mech- anism. Indeed, T3 enhanced translational efficiency, inducing relocation of histone mRNA to heavy polysomes. Increased translation was associated with augmented transcription of the eukaryotic ␥ translation initiation factor 4 2 (EIF4G2). T3 induced EIF4G2 protein and mRNA levels and the thyroid hormone receptor bound to the promoter region of the Eif4g2 gene. Induction of EIF4G2 was essential for T3-dependent histone induction, because depletion of this factor abolished histone increase. These results point out the importance of the thyroid hormones on the posttranscriptional regulation of histone biosynthesis in a cell cycle–independent manner and also suggest the potential regulation of eukaryotic translation by the modulation of the initiation factor EIF4G2, which also operates in the translation of canonical mRNAs. (Molecular Endocrinology 29: 68–75, 2015) ucleosomes, which fold chromosomal DNA, contain Canonical histones, ie, the four core histones as well as N2 molecules each of the core histones H2A, H2B, H3, the linker H1 histone found between nucleosomes, are and H4 (1). Histones are subjected to a great variety of encoded by a family of replication-dependent histone posttranslational modifications, which normally occur genes. In most cells, the expression of cell cycle–regulated on the amino-terminal and carboxy-terminal histone histone mRNAs is tightly coupled to DNA synthesis oc- “tail” domains, which play an essential role in controlling curring during the S phase. This is achieved by the precise gene expression, DNA repair, or chromosome condensa- regulation of mRNA synthesis, processing, and stability tion (2). Maintenance of chromosomal integrity requires ac- (5, 6). Replication-dependent histone mRNAs are the curate coordination of DNA replication with histone syn- only known cellular mRNAs that do not contain a thesis (3). Both histone excess and deficiency can have poly(A) tail at their 3Ј terminus, but instead exhibit a deleterious effects, and the cells have developed multiple stem-loop structure that is highly conserved. This stem- regulatory systems to control histone levels. Thus, mamma- loop motif is the binding site for the stem-loop binding lian cells sequester the excess of histones accumulating dur- protein (SLBP), which regulates pre-mRNA processing, ing replication stress, and the histone chaperone Asf1 (anti- mRNA stability, and localization of the mRNA to polyri- silencing function 1) plays a key role in this process (4). bosomes (polysomes) (7, 8). Although histone mRNAs ISSN Print 0888-8809 ISSN Online 1944-9917 * A.Z. and V.G.-C. contributed equally to the study. Printed in U.S.A. Abbreviations: ChIP, chromatin immunoprecipitation; EIF4G, eukaryotic translation initi- Copyright © 2015 by the Endocrine Society ation factor 4 ␥; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MEFs, mouse Received July 31, 2014. Accepted November 20, 2014. embryo fibroblasts; qRT-PCR, quantitative real-time PCR; siRNA, small-interfering RNA; First Published Online November 25, 2014 SLBP, stem-loop binding protein; SLIP1 (MIF4GD), SLBP-binding protein 1; THR, thyroid hormone receptor; TRE, thyroid hormone response element. 68 mend.endojournals.org Mol Endocrinol, January 2015, 29(1):68–75 doi: 10.1210/me.2014-1235 doi: 10.1210/me.2014-1235 mend.endojournals.org 69 have a different 3Ј end than the polyadenylated mRNAs, Materials and Methods they can be efficiently translated by a similar mechanism and with a similar efficiency. At the 5Ј mRNA, the cap Cell culture, transfections, and chemical reagents Immortal mouse embryonic fibroblasts (MEFs) obtained binding subunit eukaryotic initiation factor 4E interacts from TP53-knockout mice were a gift from M. Serrano (CNIO). with the scaffold protein eukaryotic translation initiation MEFs expressing either the thyroid hormone receptor ␤1 factor 4 ␥ (EIF4G). The 3Ј histone mRNA end is the (THRB) or ␣1 (THRA) isoforms in an stable manner were ob- binding site of SLBP and SLBP-binding protein 1 (SLIP1). tained as described previously by retroviral transduction and SLIP1 interacts with EIF4G, acting as a bridge between posterior selection with puromycin (15). Spontaneously immor- talized MEFs obtained from knockout mice lacking both Thr the 2 mRNA ends mediating the mRNA circularization genes were a gift from J. Samarut (Lyon, France). These cells and thus enhancing translation efficiency (9). In addition, were also transduced with THRB or THRA (15). Parental NIH- histone mRNAs are bound to polysomes through SLBP, 3T3 cells and cells expressing THRB in a stable manner were Downloaded from https://academic.oup.com/mend/article/29/1/68/2556825 by guest on 02 October 2021 also allowing high translational efficiencies (10). described previously (16). The rat pituitary cell line GH4C1 and the human colocarcinoma cell line HCT-116 were also used. The thyroid hormones (THs) T3 and its precursor T4, are essential for growth and development. Among other Cells were grown under standard conditions in DMEM supple- mented with 10% fetal bovine serum, 2 mM glutamine, and functions, they increase the basal metabolic rate and af- antibiotics. Before hormone treatment, cells were grown in me- fect almost every aspect of cellular physiology as a result dium containing 10% TH–depleted fetal bovine serum by treat- of their actions on fat, carbohydrate, and protein metab- ment with resin AG-1-X8 (Bio-Rad). Cells were plated at a 2 olism (11). THs largely exert their actions through the density of 16 000/cm and incubated for the times indicated in the presence or absence of T or GC-1 (5 nM). siRNA transfec- binding to nuclear thyroid hormone receptors (THRs), 3 tions were performed by using the TransIT-X2 reagent (Myrus), which normally regulate gene expression by binding to following the manufacturer’s instructions. siRNA duplexes thyroid hormone response elements (TREs) located in SR30004 (control) and SR421103A, B, and C (mouse Eif4g2) regulatory regions of target genes (12, 13). There are 2 were purchased from Origene. genes encoding THRs (THRA and THRB) and 4 THR isoforms designated THRA1, THRB1, THRB2, and Protein extraction, Western blotting, and acidic THRB3 that are able to bind hormone. THRA1 is widely extraction of histones expressed, showing higher levels of expression in brain, Cells were harvested and lysed in triple-detergent lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.02% sodium cardiac, and skeletal tissue. THRB1 is predominantly ex- azide, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 pressed in brain, liver, and kidney. THRB2 expression is mM phenylmethylsulfonylfluoride, 2 ␮g/mL pepstatin, 2 ␮g/mL circumscribed to hypothalamus, pituitary, retina, and in- aprotinin, 2 ␮g/mL leupeptin, and phosphatase inhibitor cock- ner ear, and THRB3 is predominantly expressed in kid- tails 2 and 3 (Sigma-Aldrich). Next, 10 ␮g of protein lysates ney, liver, and lungs (12–14). were mixed with Laemmli sample buffer, boiled, and loaded onto 8% or 12% SDS-PAGE. Western blotting and protein Although the regulation of DNA synthesis and cell detection were performed as described previously (15). Anti- growth by THs has been known for decades, little is bodies used were tubulin (clone DM1A; Sigma-Aldrich T6199), known about their actions on histone expression during H3 and H4, (Abcam ab1791 and ab10158), EIF4G2 (donated or outside the S phase of the cell cycle. Here we describe by C. de Haro (CBM, Madrid, Spain), and anti-SLBP (donated the effects of TH on bulk histone levels in various cell by W.F. Marzluff, University of North Carolina, Chapel Hill, North Carolina). Acid extraction of histones and electrophore- models. TH treatment and the overexpression of THRB1 sis were performed as described previously (17). induced an increase in histone levels not coupled to DNA synthesis. Regulation occurred by a posttranscriptional Polysome separation mechanism. TH treatment did not change the amount of Separation of free and ribosome-bound mRNA was per- histone mRNAs but increased the amount of histone mR- formed by centrifugation in linear sucrose gradients. Cells were NAs bound to polysomes. TH induced the expression of harvested, washed with 1ϫ PBS, and lysed in a mixture contain- ing 1 mL of ice-cold NP-40 buffer (10 mM Tris-HCl [pH 8], 140 EIF4G2 (also known as aging-associated protein 1), 1 of ␮ mM NaCl, 1.5 mM MgCl2, and 0.5% NP-40), 20 Lof1M the 2 isoforms of EIF4G with essential roles in histone dithiothreitol, 10 ␮Lof40U/␮L RNasin (Invitrogen), and 100 biosynthesis. The analysis of the Eif4g2 promoter by ␮L of 5% sodium deoxycholate. Lysates were centrifuged dur- chromatin immunoprecipitation (ChIP) showed the spe- ing 10 seconds at 13 000 rpm, and supernatants were trans- cific binding of THRB to a region upstream to the tran- ferred to a new tube containing 13.3 ␮L of heparin (50 mg/mL), ␮ ␮ scriptional initiation site of the Eif4g2 gene.
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