title: Repression of cardiac phospholamban gene expression is mediated by thyroid hormone receptor-{alpha}1 and involves targeted covalent histone modifications.
abstract: Phospholamban (PLB) is a critical regulator of Ca(2+) cycling in heart muscle cells, and its gene expression is markedly down-regulated by T(3). Nonetheless, little is known about the molecular mechanisms of T(3)-dependent gene silencing in cardiac muscle, and it remains unclear whether thyroid hormone receptors (TRs) directly bind at the PLB gene in vivo and facilitate transcriptional repression. To investigate the regulatory role of TRs in PLB transcription, we used a physiological murine heart muscle cell line (HL-1) that retains cardiac electrophysiological properties, expresses both TRalpha1 and TRbeta1 subtypes, and exhibits T(3)-dependent silencing of PLB expression. By performing RNA interference assays with HL-1 cells, we found that TRalpha1, but not TRbeta1, is essential for T(3)-dependent PLB gene repression. Interestingly, a PLB reporter gene containing only the core promoter sequences -156 to +64 displayed robust T(3)-dependent silencing in HL-1 cells, thus suggesting that transcriptional repression is facilitated by TRalpha1 via the PLB core promoter, a regulatory region highly conserved in mammals. Consistent with this notion, chromatin immunoprecipitation and in vitro binding assays show that TRalpha1 directly binds at the PLB core promoter region. Furthermore, addition of T(3) triggered alterations in covalent histone modifications at the PLB promoter that are associated with gene silencing, namely a pronounced decrease in both histone H3 acetylation and histone H3 lysine 4 methylation. Taken together, our data reveal that T(3)-dependent repression of PLB in cardiac myocytes is directly facilitated by TRalpha1 and involves the hormone-dependent recruitment of histone-modifying enzymes associated with transcriptional silencing.
authors.affiliation: Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854, USA.
authors.forename: Madesh
authors.initials: M
authors.lastname: Belakavadi
authors.forename: Jason
authors.initials: J
authors.lastname: Saunders
authors.forename: Noah
authors.initials: N
authors.lastname: Weisleder
authors.forename: Preethi S
authors.initials: PS
authors.lastname: Raghava
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Fondell
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D018274:Electroporation; D015870:Gene Expression / Q000187:drug effects; D006657:Histones / Q000378:metabolism*; D015151:Immunoblotting; D051379:Mice; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D034622:RNA Interference; D047490:Retinoid X Receptor alpha / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism; D037021:Thyroid Hormone Receptors alpha / Q000235:genetics / Q000378:metabolism*; D014284:Triiodothyronine / Q000494:pharmacology*; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D006657:Histones; D047490:Retinoid X Receptor alpha; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D037021:Thyroid Hormone Receptors alpha; D014284:Triiodothyronine; D000099211:Phospholamban
references.citation: Nature. 2007 Apr 19;446(7138):882-7
references.pmid: 17392792
references.citation: Cell. 2004 Dec 29;119(7):941-53
references.pmid: 15620353
references.citation: Trends Endocrinol Metab. 2000 Jan-Feb;11(1):6-10
references.pmid: 10652499
references.citation: J Mol Cell Cardiol. 2007 Jun;42(6):1026-35
references.pmid: 17509612
references.citation: Annu Rev Physiol. 2000;62:439-66
references.pmid: 10845098
references.citation: J Mol Cell Cardiol. 1999 Dec;31(12):2137-53
references.pmid: 10640442
references.citation: Proc Natl Acad Sci U S A. 1989 Dec;86(23):9114-8
references.pmid: 2480596
references.citation: J Physiol. 2009 Mar 15;587(Pt 6):1319-29
references.pmid: 19171649
references.citation: EMBO J. 1998 Jan 15;17(2):455-61
references.pmid: 9430637
references.citation: Cell Calcium. 2007 Dec;42(6):548-55
references.pmid: 17412417
references.citation: Mol Cell. 2003 Jan;11(1):139-50
references.pmid: 12535528
references.citation: Vitam Horm. 2004;68:93-122
references.pmid: 15193452
references.citation: Nature. 2003 Apr 17;422(6933):735-8
references.pmid: 12700765
references.citation: Genes Dev. 2000 Jan 15;14(2):121-41
references.pmid: 10652267
references.citation: Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):7934-9
references.pmid: 12034878
references.citation: Mol Cell Biol. 1997 May;17(5):2642-8
references.pmid: 9111334
references.citation: Mol Endocrinol. 1995 May;9(5):540-50
references.pmid: 7565802
references.citation: Circ Res. 1994 Aug;75(2):245-51
references.pmid: 8033338
references.citation: Gene. 1997 Dec 12;203(2):199-207
references.pmid: 9426251
references.citation: Mol Cell. 2005 Sep 2;19(5):631-42
references.pmid: 16137620
references.citation: Mol Endocrinol. 2000 Jul;14(7):947-55
references.pmid: 10894146
references.citation: Mol Endocrinol. 2007 Apr;21(4):865-84
references.pmid: 17244762
references.citation: Thyroid. 2000 Jan;10(1):41-52
references.pmid: 10691312
references.citation: N Engl J Med. 2001 Feb 15;344(7):501-9
references.pmid: 11172193
references.citation: J Mol Biol. 2005 Nov 4;353(4):763-71
references.pmid: 16213525
references.citation: Biochem Biophys Res Commun. 1997 Apr 28;233(3):838-43
references.pmid: 9168944
references.citation: Eur J Endocrinol. 2001 Jul;145(1):59-64
references.pmid: 11415853
references.citation: J Mol Cell Cardiol. 1994 Sep;26(9):1145-54
references.pmid: 7815458
references.citation: Physiol Rev. 2001 Jul;81(3):1097-142
references.pmid: 11427693
references.citation: Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):2979-84
references.pmid: 9501201
references.citation: Thyroid. 2002 Jun;12(6):453-7
references.pmid: 12165106
references.citation: EMBO J. 1999 Oct 1;18(19):5389-98
references.pmid: 10508171
references.citation: J Endocrinol. 2006 May;189(2):231-45
references.pmid: 16648291
references.citation: Biochem Biophys Res Commun. 1992 Oct 30;188(2):927-33
references.pmid: 1445334
references.citation: Thyroid. 2007 Dec;17(12):1211-23
references.pmid: 18177256
references.citation: Recent Prog Horm Res. 2004;59:31-50
references.pmid: 14749496
references.citation: Oncogene. 2001 May 28;20(24):3014-20
references.pmid: 11420716
references.citation: Circ Res. 1991 Aug;69(2):266-76
references.pmid: 1830516
references.citation: Mol Endocrinol. 2004 Apr;18(4):863-73
references.pmid: 14715929
references.citation: Proc Natl Acad Sci U S A. 1989 Apr;86(8):2966-70
references.pmid: 2523077
references.citation: Endocr Rev. 1987 Aug;8(3):288-308
references.pmid: 3308445
references.citation: Endocrinology. 2001 Feb;142(2):544-50
references.pmid: 11159823
references.citation: J Mol Endocrinol. 2004 Aug;33(1):21-33
references.pmid: 15291740
references.citation: J Biol Chem. 1989 Sep 5;264(25):14840-7
references.pmid: 2768243
references.citation: J Mol Cell Cardiol. 1999 Mar;31(3):679-92
references.pmid: 10198197
references.citation: J Biol Chem. 2004 Sep 10;279(37):38797-802
references.pmid: 15231818
references.citation: Front Biosci. 2006 Jan 01;11:344-55
references.pmid: 16146736
references.citation: Nature. 2007 May 24;447(7143):407-12
references.pmid: 17522673
references.citation: Methods Mol Biol. 2002;202:195-214
references.pmid: 12094605
references.citation: Endocr Rev. 2005 Aug;26(5):704-28
references.pmid: 15632316