Table 1.
Primers for qPCR.
Fig 1.
The reprogramming factors promote the expression of the core subunits of Set1/Mll complexes.
(A) MEFs were infected with increasing dose of FUW-M2rtTA and TetO-FUW-OSKM viruses in two independent infection experiments, and treated with doxycycline for 2 days. The mRNA levels of Set1/Mll complex subunits was determined by RT-qPCR and normalized by Gapdh. Average ± range of values from duplicate assays are plotted. (B) MEFs were infected with increasing dose of FUW-M2rtTA and TetO-FUW-OSKM viruses and treated with doxycycline for 2 days, and the expression of core subunits and global H3K4me3 were examined by western blotting. (C and D) MEFs were infected with virus expressing indicated individual reprogramming factor together with FUW-M2rtTA virus. MEFs infected with only FUW-M2rtTA virus was used as control (Con), and some MEFs were co-infected with TetO-FUW-Oct4 and TetO-FUW-Sox2 viruses (OS), in addition to FUW-M2rtTA. After induction with doxycycline for 2 days, the mRNA (C) and protein (D) levels of indicated Set1/Mll complex subunits were determined by RT-qPCR and normalized by Gapdh (C) and western blotting (D). Average ± SD from 3 independent infections are plotted in (C). (E and F) MEFs were infected with viral mixes that expressed individual Oct4, Sox2, Klf4 (OSK) or Oct4, Sox2, Klf4, and c-Myc (OSKM), in addition to FUW-M2rtTA, and MEFs infected with only FUW-M2rtTA virus was used as control. After induction with doxycycline for 2 days, the mRNA levels (E) of indicated core subunits were determined by RT-qPCR and normalized by Gapdh (E, left) or Actb (E, right). Average ± range of values from 2 independent infections are plotted. The indicated proteins were determined by western blotting (F). (G) Structure of human and mouse Wdr5 genes showing the highly conserved intronic sequences flanking the canonic E box. Identical residuals between human and mouse are in red. (H) Left: P493-6 cells were cultured in the absence (-Tet, Myc on) or presence of (+Tet, Myc off) tetracycline, and used for Myc ChIP followed by qPCR on indicated loci. Middle: MEFs were infected with only FUW-M2rtTA virus (control) or with FUW-M2rtTA and TetO-FUW-OSKM viruses (OSKM), induced with doxycycline, and used for Myc ChIP followed by qPCR on indicated loci. Right: MEFs were infected with FUW-M2rtTA and TetO-FUW-Myc virus (Myc) or FUW-M2rtTA and TetO-FUW-HA-Myc viruses (HA-Myc), induced with doxycycline, and used for HA ChIP followed by qPCR on indicated loci. Wdr5 dst, a Wdr5 downstream region. Cad is a previously established Myc target [63] and used as a positive control. Note that there are E boxes at +10088 and +23063 bp in the Dpy30 gene body. Average ± SD from triplicate assays are plotted, except for Myc ChIP in MEFs, for which Average ± range of values from duplicate assays are plotted. The differences between blue and red bars in all three panels are statistically significant (P<0.05 in 2-tailed Student’s t-test) for all loci except for the “Wdr5 dst” and the “Intergenic” sites.
Fig 2.
Dynamic recruitment of Dpy30 during cellular reprogramming.
MEFs were infected with FUW-M2rtTA and TetO-FUW-OSKM viruses and cultured under the reprogramming condition. At days 0, 4 and 11 during reprogramming, cells were used for H3K4me3 and Dpy30 ChIP followed by qPCR on indicated loci. Oct4-A and Oct4-B are two different primers at the Oct4 promoter [61]. Average ± range of values from duplicate assays are plotted, and results are representative of 3 independent biological repeats.
Fig 3.
Interaction of the Set1/Mll complex core subunits with the reprogramming factors.
(A) MEFs were infected with only FUW-M2rtTA virus (control), or with FUW-M2rtTA and virus expressing the indicated FLAG-HA-tagged individual reprograming factor. After induction with doxycycline for 2 days, co-immunoprecipitation was performed by anti-FLAG M2 resin. Inp, input (4%); F↓, anti-FLAG immunoprecipitation. (B) 293 cells were stably transfected with empty vector (con) or plasmid expressing the indicated FLAG-HA-tagged individual reprograming factor. Co-immunoprecipitation was performed by anti-FLAG M2 resin. Input (2.5%) was loaded.
Fig 4.
The HMG domain of Sox2 mediates its direct binding to Ash2l of Set1/Mll complexes.
(A) Diagram for truncation mutants of FLAG-HA-tagged Sox2 showing their domain structures and whether the protein binds to Ash2l as summarized from (B). (B) 293T cells were transfected with indicated FLAG-HA-tagged Sox2 mutants, and co-IP was performed by anti-FLAG M2 resin. Wdr5 blotting for the right panel is not shown due to a fortuitous cross-reactivity with ΔHMG. TAD: transcriptional activation domain. Inp, input (2.5%); F↓, anti-FLAG immunoprecipitation. (C) Purified proteins were examined by SDS-PAGE along with the molecular weight ladder on the left, and visualized by coomassie blue staining. (D) In vitro binding assay for Ni bead-bound His-Sox2 with FLAG-Ash2l and FLAG-Wdr5, and examined by western blotting using anti-FLAG antibody. Ni-beads that was not used for His-Sox2 purification was used as the mock control in the binding assay. Input (20%) was loaded.
Fig 5.
The Set1/Mll complex core subunits are required for efficient reprogramming to pluripotency.
(A) Oct4-GFP MEFs stably depleted of Rbbp5 or Dpy30 were used in reprogramming for 16 days, and imaged under microscope. (B) GFP positive clones per field, as averaged ± SD from 10 random fields, were quantified by counting under microscope. Results are representative of over 5 biological repeats. *P<0.05 in 2-tailed Student’s t-test between control and each knockdown. (C) The relative mRNA levels of indicated genes were determined by RT-qPCR after reprogramming, and normalized by Gapdh. Dpy30 shRNA#1 was used for KD. Results are representative of over 5 biological repeats. (D) Western blotting for MEFs after stable knockdown. (E) Growth curves of the control and Dpy30 KD MEFs as measured by described method [27]. Results are representative of two biological repeats, and average ± SD from triplicate measurements are plotted. *P<0.05 in 2-tailed Student’s t-test between control and each knockdown.
Fig 6.
Dpy30 is required for efficient recruitment of Oct4 to its genomic targets.
MEFs infected with control shRNA or Dpy30 shRNA#1 (KD) were used in reprogramming for 7 days. (A) Expression of Dpy30 and OSKM was determined by RT-qPCR and normalized by Gapdh. Average ± SD from 3 independent infection and reprogramming assays are plotted. (B) ChIP assays for Oct4 and H3K4me3 were performed followed by qPCR on indicated gene promoters or an intergenic site. Average ± range of values from duplicate assays are plotted, and results are representative of 3 independent infection and reprogramming assays.
Fig 7.
A model for the role of the core subunits of Set1/Mll complexes in cellular reprogramming.
This model depicts two different levels of mechanisms by which the reprogramming factors coordinate with the core subunits of Set1/Mll complexes to facilitate cellular reprogramming of differentiated cells to pluripotency. See Discussion for details.