(a) In vitro formaldehyde release assay using histone substrates generated with the indicated methyltransferases and incubated with either recombinant Flag-Jhdm1b (empty bars) or no enzyme control (black bars). (b) In vitro formaldehyde release assay using recombinant Jhdm1b, Jhdm1b(H211A), JHDM1A and RBP2 incubated with H3K36-methylated (empty bars) or H3K4-methylated (black bars) substrates generated with SET2 or SET7(Y245A) methyltransferase, respectively. (c) HeLa cell core histones were incubated in the presence (+) or absence (−) of recombinant Jhdm1b. The resulting reaction was subjected to Western blotting with the indicated modification state-specific antibodies. (d) Jhdm1b mRNA levels from AD293 cells stably over-expressing F-Jhdm1b, F-Jhdm1b(H211A), or empty vector control cells. Samples were normalized relative to Gapdh and the Jhdm1b expression level in the empty vector control cells was arbitrarily set to 1. (e) Acid extracted histones were prepared from AD293 cells stably overexpressing F-Jhdm1b or F-Jhdm1b(H211A) and subjected to Western blotting using the indicated methyl-state specific histone antibodies. All error bars represent s.d. (n=3).

(a) Primary MEFs at passage 1 were transduced with lentiviral control (black bars), Jhdm1b (Jhdm1b KD, grey bars) and Ring1b (Ring1b KD, white bars) shRNA and further selected by puromycin for 48 hours. The expression level of the indicated genes was determined by qRT-PCR. Samples were normalized against levels of Gapdh and the expression level of each gene in empty vector control cells was arbitrarily set to 1. (b) Total protein was collected from cells prepared as in (a) and subjected to Western Blot analysis using antibodies against p15^Ink4b, p16^Ink4a, p21 and tublin. (c) Primary MEF cells isolated from p15^Ink4b null mice at passage 1 were transduced with lentiviral control (diamonds), Jhdm1b (Jhdm1b KD, squares), and Ring1b (Ring1b KD, triangles) shRNA and further selected by puromycin for 48 hours. 5×10⁵ cells were plated and cell number was counted at different time points using a hemocytometer. (d) Primary MEFs were prepared as in (c), pulse labeled with BrdU and stained with PI and FITC conjugated anti-BrdU antibody. The percentage of BrdU positive cells was determined by flow cytometry for control (black bars), Jhdm1b KD (grey bars) and Ring1b KD (empty bars). All error bars represent s.d. (n=3).

(a) Primary MEF cells were transduced with mock or F-Jhdm1b expressing lentiviral vector followed by superinfection with retroviral H-Ras12V. 5000 cells were plated on soft agar and analyzed 14 days later. Graph represents quantification of large colonies on H-Ras transduced plates. (b) Primary MEF cells were transduced with lentiviral vectors carrying control shRNA (Control shRNA) or Jhdm1b shRNA (Jhdm1b KD) followed by puromycin selection and retroviral H-Ras12V transduction. 5000 cells were plated on soft agar and analyzed 14 days later. Graph represents quantification of large colonies on H-Ras transduced plates. All error bars represent s.d. (n=3).

(a) Primary MEFs at passage 1 were transduced with lentiviral control (black bars), Jhdm1b (Jhdm1b KD, grey bars) or Ring1b (Ring1b KD, empty bars) shRNA and further selected by puromycin for 48 hours. mRNA levels were determined by quantitative real-time PCR (qRT-PCR). Samples were normalized against levels of Gapdh. The Jhdm1b or Ring1b expression level in empty vector control cells was arbitrarily set to 1. (b) Primary MEF cells transduced at passage 1 with lentiviral control (diamonds), Jhdm1b (Jhdm1b KD, squares), and Ring1b (Ring1b KD, triangles) shRNA and further selected by puromycin for 48 hours. 5×10⁵ cells were plated and cell number was counted at different time points using a hemocytometer. (c) Primary MEFs were prepared as in (b), pulse labeled with BrdU and stained with PI and FITC conjugated anti-BrdU antibody. The percentage of BrdU positive cells was determined by flow cytometry for control (black bars), Jhdm1b KD (grey bars) and Ring1b KD (empty bars). (d) Cells prepared as in (b) were split at 1:5 for 5 passages and senescence associated β-galactosidase activity at pH 6.0 was determined by cellular staining. Data is presented for passage 1 (P1) and passage 5 (P5) cells. All error bars represent s.d. (n=3).

(a) Primary MEF cells were transduced with lentiviral vectors carrying control shRNA (diamonds), Jhdm1b shRNA (Jhdm1b KD, squares), and Jhdm1b shRNA with expression of F-Jhdm1b (Jhdm1b KD/F-Jhdm1b, circles) or F-Jhdm1b(H211A) (triangles). 5×10⁵ cells were plated 48 hours after transduction and maintained in culture for 8 days. Cell number was counted using a hemocytometer at the indicated time points. (b) mRNA was collected from cells prepared as in (a) and qRT-PCR was carried out to determine p15^Ink4a expression level in each sample. Samples were normalized against levels of Gapdh and the expression level of each gene in empty vector control cells was arbitrarily set to 1. (c) Schematic representation of the p15^Ink4b locus in mouse indicating the genomic structure of the locus (exons demarcated by black boxes), as well as the location of the three amplicons studied in ChIP experiments. (d) ChIP experiment utilizing chromatin prepared from primary MEF cells transduced with retrovirus encoding F-Jhdm1b (empty bars) or control virus (black bars) were carried out using antibody against Flag. F-Jhdm1b binding was assayed via qPCR at the three genomic regions depicted in (c). (e) ChIP experiment utilizing chromatin from primary MEFs prepared as in (a) was carried out using antibody specific for H3K36me2. H3K36me2 levels were assayed via qPCR at the three genomic regions depicted in (c). Error bars represent s.d. (n=3).