Figure 5. Negative Regulation of Dot1l by Cdk1.

(A) In vitro kinase reactions with the indicated wild-type cyclin-Cdk kinases using Dot1l as a substrate. Lower panel, kinase reactions with histone H1 were used to ensure the comparable specific activity of all cyclin-Cdk kinases used. Lanes marked ‘−’ indicate incubation of Dot1l or histone H1 with [γ³²P]-ATP without any added kinase. In the upper right panel, the middle lanes were spliced out (indicated by a vertical line).

(B) Amino acid sequence of Dot1l containing the bipartite predicted nuclear localization signal (in bold). Cdk1-dependent phosphorylation site (S1105) is in red. Below, changes in the phosphorylation status of the indicated Dot1l phosphopeptides upon an acute 30 min Cdk1 inhibition. Note that the most affected peptide (>40% decrease in phosphorylation), contains the S1105 phosphosite.

(C) Cdk1 or Cdk2 were immunoprecipitated from human breast cancer MCF7 or wild-type ES (ES-V6.5) cells and used for in vitro kinase reactions with histone H1 as a substrate. For control, immunoprecipitates from ES cells with normal IgG (IgG) were used.

(D) MCF7 cells were transfected with constructs encoding Flag-tagged wild-type Dot1l or a phosphomimetic S1105 Dot1l mutant. The localization of Dot1l was assessed by immunostaining with an anti-Flag antibody. DAPI was used to visualize cell nuclei. Scale bar, 10 μm.

(E and F) Cdk1^AS/AS ES cells were transduced with lentiviruses encoding Flag-tagged wild-type Dot1l (E), or phosphomimetic S1105 Dot1l mutant (F). Cells were treated for 6 h with vehicle (DMSO), or 3-MB-PP1, and Dot1l localization was assessed by immunostaining with an anti-Flag antibody. DAPI was used to visualize cell nuclei. Scale bar, 5 μm.

(G) Cdk1^AS/AS ES cells were cultured in the presence of vehicle (DMSO) or 3-MB-PP1, together with retinoic acid, and immunostained with an antibody against H3K79me2; DAPI was used to visualize cell nuclei. Scale bar, 10 μm.

(H) Distribution of H3K79me2 peak widths in Cdk1^AS/AS ES cells threated with vehicle (DMSO), or 3-MB-PP1.

(I) H3K79me2 ChIP-sequencing signal height and position relative to transcription start sites (TSS) for all genes containing this mark in Cdk1^AS/AS ES cells treated with DMSO or 3-MB-PP1.

(J) ChIP-sequencing density heatmap of H3K79me2 enrichment in DMSO- and 3-MB-PP1-treated Cdk1^AS/AS ES cells, within 20 kb around the transcription start site. Gene order was arranged from highest to lowest density.

(K) Examples of H3K79me2 modification profiles in Cdk1^AS/AS ES cells treated with DMSO or with 3-MB-PP1. Shown are genome browser tracks for H3K79me2 ChIP-sequencing.

See also Figure S5, Tables S6, S7.