Dnmt2-deficiency affects the P-TEFb complex.

<p>(A) Cdk9 expression in Dnmt2-deficient and control ES cells was analyzed by quantitative RT-PCR (left) and Western blot assays (right). (B) Cdk9 expression in Dnmt2-deficient and control hearts was investigated by quantitative RT-PCR (left) (<i>n</i> = 5) and Western blot assays (right). (C) Quantitative RT-PCR for Ctip expression in the hearts of Dnmt2^-/- and Dnmt2^+/+ mice (<i>n</i> = 6). Northern blot assay (D) and quantitative RT-PCR (E) for Rn7sk (7SK) expression in the hearts of Dnmt2^-/- and Dnmt2^+/+ mice. (F) RNA immunoprecipitation using a 5-methyl Cytidine antibody with RNA extracted from Dnmt2^-/- and Dnmt2^+/+ hearts, followed by RT-PCR for Rn7sk (<i>n</i> = 6). Note that Rn7sk is significantly less methylated in Dnmt2^-/- hearts. (G) PTEF-b immunoprecipitation using an antibody against Cdk9 on lysates from Dnmt2- mutant and control ES cells followed by RT-PCR for Rn7sk (<i>n</i> = 3) and subsequent quantification. An anti Cyp2 antibody served as negative control. Note that less Rn7sk is associated to the P-TEFb complex in Dnmt2-deficient cells.</p

Dnmt2-deficiency is associated with increased phosphorylation of RNA polymerase II.

<p>Representative Western Blots for RNA polymerase II (RNA Pol II, upper panels) and quantification of the ratio of phosphorylated to non-phosphorylated RNA Pol II (lower panels) in mouse hearts (A) and ES cells (B) with knockout of Dnmt2 and controls. β-actin was used as a standard. Data are mean ± SEM. *p < 0.05.</p

Primers for quantitative RT-PCR analysis.

<p>Primers for quantitative RT-PCR analysis.</p

Schematic representation of Dnmt2-mediated RNA polymerase II transcriptional activity in cardiac growth.

<p>Storage of inactive protein in a complex with 7SK RNA and inhibitory proteins, release of active kinase (P-TEFb) in Dnmt2 knock out conditions. The phosphorylation of a C-terminal domain of the polymerase by P-TEFb allows elongation of the transcripts. RNAPII CTD phosphorylation increases mRNA and protein expression, which mediates cardiac growth in Dnmt2-deficient mice.</p