title: DNA methylation and differentiation: HOX genes in muscle cells.
abstract: BACKGROUND
  Tight regulation of homeobox genes is essential for vertebrate development. In a study of genome-wide differential methylation, we recently found that homeobox genes, including those in the HOX gene clusters, were highly overrepresented among the genes with hypermethylation in the skeletal muscle lineage. Methylation was analyzed by reduced representation bisulfite sequencing (RRBS) of postnatal myoblasts, myotubes and adult skeletal muscle tissue and 30 types of non-muscle-cell cultures or tissues.
  
  
  RESULTS
  In this study, we found that myogenic hypermethylation was present in specific subregions of all four HOX gene clusters and was associated with various chromatin epigenetic features. Although the 3' half of the HOXD cluster was silenced and enriched in polycomb repression-associated H3 lysine 27 trimethylation in most examined cell types, including myoblasts and myotubes, myogenic samples were unusual in also displaying much DNA methylation in this region. In contrast, both HOXA and HOXC clusters displayed myogenic hypermethylation bordering a central region containing many genes preferentially expressed in myogenic progenitor cells and consisting largely of chromatin with modifications typical of promoters and enhancers in these cells. A particularly interesting example of myogenic hypermethylation was HOTAIR, a HOXC noncoding RNA gene, which can silence HOXD genes in trans via recruitment of polycomb proteins. In myogenic progenitor cells, the preferential expression of HOTAIR was associated with hypermethylation immediately downstream of the gene. Other HOX gene regions also displayed myogenic DNA hypermethylation despite being moderately expressed in myogenic cells. Analysis of representative myogenic hypermethylated sites for 5-hydroxymethylcytosine revealed little or none of this base, except for an intragenic site in HOXB5 which was specifically enriched in this base in skeletal muscle tissue, whereas myoblasts had predominantly 5-methylcytosine at the same CpG site.
  
  
  CONCLUSIONS
  Our results suggest that myogenic hypermethylation of HOX genes helps fine-tune HOX sense and antisense gene expression through effects on 5' promoters, intragenic and intergenic enhancers and internal promoters. Myogenic hypermethylation might also affect the relative abundance of different RNA isoforms, facilitate transcription termination, help stop the spread of activation-associated chromatin domains and stabilize repressive chromatin structures.
authors.affiliation: Hayward Human Genetics Program and Tulane Cancer Center, Tulane Health Sciences Center, New Orleans LA, USA. ehrlich@tulane.edu.
authors.forename: Koji
authors.initials: K
authors.lastname: Tsumagari
authors.forename: Carl
authors.initials: C
authors.lastname: Baribault
authors.forename: Jolyon
authors.initials: J
authors.lastname: Terragni
authors.forename: Sruti
authors.initials: S
authors.lastname: Chandra
authors.forename: Chloe
authors.initials: C
authors.lastname: Renshaw
authors.forename: Zhiyi
authors.initials: Z
authors.lastname: Sun
authors.forename: Lingyun
authors.initials: L
authors.lastname: Song
authors.forename: Gregory E
authors.initials: GE
authors.lastname: Crawford
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan
authors.forename: Michelle
authors.initials: M
authors.lastname: Lacey
authors.forename: Melanie
authors.initials: M
authors.lastname: Ehrlich
references.citation: Genome Res. 2012 May;22(5):837-49
references.pmid: 22391556
references.citation: Dev Dyn. 2000 Oct;219(2):261-76
references.pmid: 11002345
references.citation: J Biol Chem. 2010 May 21;285(21):16135-44
references.pmid: 20348100
references.citation: Mech Dev. 2009 Jul;126(7):517-22
references.pmid: 19393316
references.citation: Cell. 2009 Jun 26;137(7):1194-211
references.pmid: 19563753
references.citation: Dev Dyn. 2012 Apr;241(4):792-802
references.pmid: 22374744
references.citation: Dev Biol. 2007 Apr 15;304(2):633-51
references.pmid: 17292343
references.citation: PLoS Genet. 2011 Jun;7(6):e1002154
references.pmid: 21731508
references.citation: PLoS Genet. 2013;9(1):e1003184
references.pmid: 23359544
references.citation: Am J Hum Genet. 2007 Dec;81(6):1304-15
references.pmid: 17999367
references.citation: Nat Biotechnol. 2011 Nov 13;30(1):99-104
references.pmid: 22081020
references.citation: Genome Res. 2010 Mar;20(3):320-31
references.pmid: 20133333
references.citation: Biol Reprod. 2004 Jan;70(1):39-45
references.pmid: 12954737
references.citation: Am J Pathol. 2013 Jan;182(1):64-70
references.pmid: 23141928
references.citation: Cell. 2011 Apr 29;145(3):423-34
references.pmid: 21496894
references.citation: Biochemistry. 2010 Apr 13;49(14):2999-3008
references.pmid: 20210320
references.citation: Epigenetics. 2011 Jun;6(6):740-51
references.pmid: 21562367
references.citation: Genome Res. 2011 Dec;21(12):2026-37
references.pmid: 21873453
references.citation: Nat Protoc. 2012 Mar 01;7(3):562-78
references.pmid: 22383036
references.citation: PLoS One. 2010 Feb 11;5(2):e9163
references.pmid: 20161795
references.citation: Ann Anat. 2010 Sep 20;192(5):261-74
references.pmid: 20739155
references.citation: Epigenetics. 2013 Mar;8(3):317-32
references.pmid: 23417056
references.citation: Nature. 2008 Aug 7;454(7205):766-70
references.pmid: 18600261
references.citation: Mol Cell Biol. 2009 Nov;29(22):6074-85
references.pmid: 19703992
references.citation: Proc Natl Acad Sci U S A. 2009 Jan 20;106(3):671-8
references.pmid: 19139413
references.citation: Curr Opin Clin Nutr Metab Care. 2010 May;13(3):243-8
references.pmid: 20098319
references.citation: Development. 2005 May;132(9):2215-23
references.pmid: 15829525
references.citation: Breast Cancer Res Treat. 2012 Dec;136(3):875-83
references.pmid: 23124417
references.citation: Curr Opin Genet Dev. 2007 Aug;17(4):359-66
references.pmid: 17644373
references.citation: BMC Med Genomics. 2011 Sep 27;4:67
references.pmid: 21951698
references.citation: Am J Pathol. 2001 Mar;158(3):955-66
references.pmid: 11238043
references.citation: Genome Res. 2013 Mar;23(3):555-67
references.pmid: 23325432
references.citation: Am J Obstet Gynecol. 2006 Apr;194(4):1100-7; discussion 1107-9
references.pmid: 16580301
references.citation: Bioinformatics. 2005 Mar 1;21(5):671-3
references.pmid: 15284102
references.citation: Epigenetics. 2009 Nov 16;4(8):537-40
references.pmid: 19923920
references.citation: Cell. 2007 Jun 29;129(7):1311-23
references.pmid: 17604720
references.citation: Genes Dev. 1995 Jan 1;9(1):108-22
references.pmid: 7828847
references.citation: Cell Growth Differ. 1993 Aug;4(8):665-9
references.pmid: 8104467
references.citation: Science. 2011 Oct 14;334(6053):222-5
references.pmid: 21998387
references.citation: Genome Res. 2011 Oct;21(10):1757-67
references.pmid: 21750106
references.citation: Nat Commun. 2012 May 08;3:818
references.pmid: 22569366
references.citation: PLoS One. 2012;7(2):e31621
references.pmid: 22355382
references.citation: Nature. 2011 Apr 7;472(7341):120-4
references.pmid: 21423168
references.citation: Dev Cell. 2010 Apr 20;18(4):662-74
references.pmid: 20412780
references.citation: Epigenetics Chromatin. 2009 Jun 08;2(1):7
references.pmid: 19505295
references.citation: Nature. 2010 Sep 16;467(7313):338-42
references.pmid: 20720541
references.citation: Proc Natl Acad Sci U S A. 2007 Sep 4;104(36):14366-71
references.pmid: 17726103
references.citation: Physiol Genomics. 2006 Feb 14;24(3):264-75
references.pmid: 16291736
references.citation: J Biol Chem. 2011 Feb 4;286(5):3250-60
references.pmid: 21087926
references.citation: Cell Rep. 2013 Feb 21;3(2):567-76
references.pmid: 23352666
references.citation: PLoS Genet. 2006 Jul;2(7):e119
references.pmid: 16895450
references.citation: PLoS Biol. 2008 Jan;6(1):e22
references.pmid: 18232738
references.citation: J Invest Dermatol. 1994 Sep;103(3):341-6
references.pmid: 7915745
references.citation: EMBO J. 2011 Aug 16;30(20):4198-210
references.pmid: 21847099
references.citation: Epigenetics Chromatin. 2012 Jan 09;5(1):1
references.pmid: 22230046
references.citation: Genome Biol. 2013 Mar 25;14(3):R25
references.pmid: 23531360
references.citation: Nature. 2011 May 5;473(7345):43-9
references.pmid: 21441907
references.citation: RNA. 2007 Feb;13(2):223-39
references.pmid: 17185360
references.citation: Int J Dev Biol. 2009;53(8-10):1404-19
references.pmid: 19247930
references.citation: Epigenetics. 2010 Jan 1;5(1):41-6
references.pmid: 20083893
references.citation: Gene. 2003 Jan 2;302(1-2):65-72
references.pmid: 12527197
references.citation: Genome Biol. 2011;12(3):R22
references.pmid: 21410973
references.citation: Biochem Biophys Res Commun. 2007 Jun 8;357(3):724-30
references.pmid: 17445766
references.citation: Nucleic Acids Res. 2001 Nov 15;29(22):4598-606
references.pmid: 11713309
references.citation: Dev Growth Differ. 2003 Oct-Dec;45(5-6):485-98
references.pmid: 14706073
references.citation: PLoS One. 2010 May 12;5(5):e10600
references.pmid: 20485555
references.citation: PLoS Biol. 2011 Apr;9(4):e1001046
references.pmid: 21526222
references.citation: Dev Biol. 2010 Aug 1;344(1):7-15
references.pmid: 20435029
references.citation: Nature. 2011 Nov 3;479(7371):74-9
references.pmid: 21964334
references.citation: Annu Rev Genet. 1996;30:529-56
references.pmid: 8982464
references.citation: Genome Biol. 2011 Jun 20;12(6):R54
references.pmid: 21689397
references.citation: Annu Rev Genet. 2011;45:145-66
references.pmid: 22060042
references.citation: PLoS One. 2012;7(6):e38829
references.pmid: 22701719
references.citation: Cell. 2005 Jan 28;120(2):169-81
references.pmid: 15680324
references.citation: PLoS One. 2010 Dec 23;5(12):e15367
references.pmid: 21203455
references.citation: Nature. 2010 Jul 8;466(7303):253-7
references.pmid: 20613842
references.citation: Bioinformatics. 2010 Mar 15;26(6):841-2
references.pmid: 20110278
references.citation: PLoS One. 2007 Jan 24;2(1):e175
references.pmid: 17245451
references.citation: Dev Dyn. 1999 Dec;216(4-5):385-97
references.pmid: 10633858
references.citation: PLoS One. 2013;8(1):e53880
references.pmid: 23326524
references.citation: Mol Cell Biol. 2003 Jun;23(11):3872-83
references.pmid: 12748289
references.citation: Mech Dev. 1993 Dec;44(2-3):139-54
references.pmid: 7908827
references.citation: Atherosclerosis. 2007 Dec;195(2):e50-60
references.pmid: 17586512