Fig 2: Ash1L Dimethylates Lysine 36 on Histone H3 at the FSHD Locus, Related to Figure 6(A) ChIP-qPCR for enrichment on total H3 of H3K4me3, H3K36me2 and IgG, as control, performed on human chr4/CHO cells in the repressed (control) or in the de-repressed (AZA+TSA) state. ChIP material was analyzed by qPCR with primers specific for the NDE region. The error bars represent SEM.(B) ChIP-qPCR for enrichment on total H3 of H3K4me3, H3K36me2 and IgG, as control, performed in control shRNA or shAsh1L expressing cells in the de-repressed state (AZA+TSA). ChIP material was analyzed by qPCR with primers specific for the NDE region. The error bars represent SEM.(C) ChIP-qPCR for enrichment on total H3 of H3K36me2 and IgG, as control, in control shRNA or shDBE-T expressing cells in the de-repressed state (AZA+TSA). ChIP material was analyzed by qPCR with primers specific for the NDE region. The error bars represent SEM.(D) ChIP for Ash1L and IgG, as control, on human chr4/CHO cells in the repressed (control) or de-repressed (AZA+TSA) state. Analysis was performed by qPCR with primers specific for ANT1, FRG1, FRG2 promoters and NDE. Results are expressed as relative percentage of input. The error bars represent SEM.(E) ChIP-qPCR for enrichment on total H3 of H3K36me2 and IgG, as control, performed on human chr4/CHO cells in the repressed (control) or in the de-repressed (AZA+TSA) state. Analysis was performed by qPCR with primers specific for ANT1, FRG1, FRG2 promoters and NDE. Results are expressed as relative percentage of H3. The error bars represent SEM.

Fig 3: DBE-T Directly Binds the TrxG Protein Ash1L and Recruits It to the FSHD Locus(A) RNA immunoprecipitation (IP) following UV crosslinking for Ash1L or IgG on AZA+TSA treated chr4/CHO cells. DBE-T or, as control, pre-miR19A and U1 snRNA enrichments were measured by qRT-PCR. The error bars represent SEM.(B) In vitro RNA-GST pull-down assay showing the interaction between recombinant GST-fused Ash1L SET domain or GST and in vitro transcribed DBE-T. On the right, Coomassie staining of purified recombinant proteins. After RNA recovery, samples were analyzed by qRT-PCR. The error bars represent SEM.(C) Following AZA+TSA treatment, chr4/CHO cells stably expressing a nonsilencing control shRNA or shDBE-T were analyzed by ChIP for Ash1L or IgG. Enrichment for NDE was analyzed by qPCR and displayed as enrichment relative to input. The error bars represent SEM.(D) Upon AZA+TSA treatment, control shRNA, and shAsh1L cells were collected to analyze DBE-T expression by qRT-PCR. Results are expressed over ß-actin. The error bars represent SEM.See also Figure S8.

Fig 4: Tethering of ASH1L increases CENP-A levels on HAC centromeres. (A) Schematic showing quantification of CENP-A assembly on an alphoidtetO HAC. A HeLa cell line containing an alphoidtetO HAC (HeLa-HACtetO-2-4) was transfected with tetR–EYFP-fusion expression plasmids and fixed 2 d after transfection for immunostaining with anti-CENP-A antibody. The red and blue arrows indicate the effect on endogenous CENP-A (assembly or disassembly) of tetR­–EYFP-fusion tethering. (B) Example of quantification of CENP-A assembly on the HAC. HeLa-HACtetO-2-4 cells transfected with tetR–EYFP alone were stained with anti-CENP-A antibody. Scale bar: 5 µm. For quantification, CENP-A signal intensity on the HAC (green circle) is normalized by the average signal intensity of the endogenous centromeres (red circles) as described in Shono et al. (2015). (C,D) Quantification of CENP-A assembly on the HAC by tethering of the identified factors (right panel). Example images of the quantification assay are shown in the left panel (blue squares indicate the HAC, shown magnified in inset images). Scale bar: 5 µm. Fifty cells were analyzed for each assay. Blue dashed line indicates relative values to the median of control (Alone). Red lines indicate the median. **P<0.01, ***P<0.001 compared to control (Mann–Whitney test; red asterisk, increase; blue asterisk, decrease).

Fig 5: Identification of CENP-B domains that assemble ASH1L and HP1, using the FMIT assay. (A,B) Schematic drawing of FMIT assay (A) and tested tetR–EYFP–CENP-B deletion series bait (B). (C) Frequency of assembly of Halo-fused proteins on the ectopic alphoidtetO DNA integration site. The tetR–EYFP–CENP-B series, tetR­–EYFP alone and Halo-tag clone (Halo-fused gene) expression plasmids were co-transfected into the HeLa Int-03 CENP-B KO cell line. More than 50 cells were counted to obtain the frequency of Halo-positive EYFP-spots for each assay. Results are mean±s.e.m. (n=3 experiments). P-values against tetR–EYFP alone (two-tailed t-test) are indicated by asterisks. *P<0.05, **P<0.01. (D,E) Representative images of Halo–ASH1L (D) and Halo–HP1β (E) assembly in the FMIT assay with the tetR–EYFP–CENP-B series bait. Cells were stained with DAPI and Halo-TMR ligand. The yellow squares indicate tetR–EYFP–protein spots on the alphoidtetO DNA site, and are shown magnified in inset images. Scale bars: 5 µm.