title: TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing.
abstract: Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Marina
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: David
authors.initials: D
authors.lastname: Sturgill
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Bailly
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Morgan
authors.initials: M
authors.lastname: Thenoz
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Garima
authors.initials: G
authors.lastname: Varma
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Maria F
authors.initials: MF
authors.lastname: Prigge
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Kyster K
authors.initials: KK
authors.lastname: Nanan
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Shukla
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Nazmul
authors.initials: N
authors.lastname: Haque
authors.affiliation: Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA shalini.oberdoerffer@nih.gov.
authors.forename: Shalini
authors.initials: S
authors.lastname: Oberdoerffer
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism*; D017398:Alternative Splicing; D000076246:CCCTC-Binding Factor; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D049308:Dioxygenases; D006801:Humans; D006899:Mixed Function Oxygenases; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins; D044503:5-Methylcytosine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human; D049308:Dioxygenases; C541081:TET2 protein, human
keywords: CTCF; DNA methylation; TET1; TET2; alternative splicing