Loss of CARM1 affects the immune-reactivity of an antibody specific for methylated histone H3 (α-methyl[R17]H3). (A) Immunoblot of purified calf thymus histone H3 before and after incubation with AdoMet and recombinant GST-CARM1 (10-s exposure). (B) Immunoblot of total protein lysates prepared from E12.5 and E18.5 embryos by using α-methyl[R17]H3 (Upper) and α-FBP21 (Lower, loading control; ref. 39). (C) Acid-purified core histones from MEF cell lines 13^+/+, 20^–/–, and 20.3 (rescued) Coomassie-stained as a loading control (Left) and an immunoblot by using α-methyl[R17]H3 (7-min exposure; Right). (D) Immunohistochemical localization of CARM1 and α-methyl[R17]H3 reactivity in Carm1^+/+ and Carm1^–/– embryos at day E18.5 viewed at ×100 magnification. This view represents the primordium of the upper incisor tooth.

CARM1 is involved in estrogen receptor-mediated transactivation of a luciferase reporter in MEF cells and endogenous targets in embryos. (A) A panel of MEFs was tested for their ability to transactivate a vitellogenin estrogen response element. Cell lines were transient transfected with estrogen receptor (ERα), vitERE-TK-Luc reporter, and renilla (control). After transfection, cells were grown in charcoal striped serum and treated with 10 nM estradiol. Relative activity of firefly luciferase was normalized against renilla luciferase activity, and the results were expressed as fold induction of luciferase activity on treatment with hormone. The results are expressed as the mean ± SD of three independent experiments done in triplicate. (B) Immunoblot of total protein lysates prepared from E18.5 embryos by using α-complement C3 (Top), α-STC2 (Middle), and α-FBP21 (Bottom, loading control). Sizes of immune-reactive bands are labeled in kDa.

Loss of PABP1 and p300 methylation in the absence of CARM1. (A) Methylated proteins in wild-type (13^+/+) and Carm1 mutant (20^–/–) MEF cell lines were labeled in vivo (31). Immunoprecipitations (IP) were performed with an αPABP antibody. The ³H-labeled proteins were visualized by fluorography (Left), and the same membrane was immunoblotted with αPABP antibodies (Right). Protein A/G-horseradish peroxidase (HRP) was used as the secondary reagent. (B) Comparison of global protein methylation from wild-type (13^+/+) and Carm1 mutant (20^–/–) MEF cell lines. Total protein (200 μg) from in vivo-labeled MEFs was subjected to 2D-gel analysis, over a 6–11 pH gradient, followed by fluorography. The framed region focuses on the location of PABP1. The location of PABP1 (arrowhead) in the 20^–/– line was determined by probing the fluorographed membrane with αPABP antibodies. (C) The CARM1 substrates p300 and PABP1 cannot be methylated when using CARM1 knockout cell extracts as an enzyme source. Wild-type (lines 13 and 4) and rescued (20.3) MEF extracts, but not extracts from knockout lines (lines 20 and 3), were able to methylate recombinant CARM1 substrates, GST-p300, and GST-PABP1. Both CARM1 knockout and wild-type cell line extracts methylated the PRMT1 substrate, GST-GAR.

Targeted disruption of Carm1 gene. (A) Strategy for homologous recombination of the Carm1 genomic locus. Position of the external probe is shown. A neo cassette flanked by frt sites was introduced into the intronic region. Two Carm1 exons, which encode a portion of the substrate-binding region, were floxed. Arrowheads depict loxP sites, solid boxes depict exons, and hatched boxes depict frt sites. Filled arrows depict direction of transcription, and open arrows the position of PCR primers used to generate the arms of the targeting construct. (B) A litter of E12.5 embryos was analyzed for gene targeting. Genomic DNA was isolated from embryo trunk, digested with BamHI, and analyzed by Southern blotting by using the 3′ external probe (Upper). Protein was extracted from the heads of the same embryo set and subjected to Western blot analysis by using an αCARM1 antibody, demonstrating the absence of CARM1 immunoreactive staining in mutant embryos (Lower). (C) Embryonic (E18.5) size difference of wild-type (+/+) and Carm1 mutant (–/–) mice. (D) Subcellular localization of CARM1 protein in mutant (–/–) and wild-type (+/+) cells. Confocal microscope image of wild-type (13^+/+), Carm1 mutant (20^–/–), and Flpase rescued (20-1) MEF cell lines processed for immunostaining with αCARM1 primary antibody, an FITC-conjugated secondary antibody and Texas red-conjugated phalloidin.