Figure 2.

The MENIN/MLL2 H3K4 Methyl Transferase Is Cell-Cycle Regulated and Controls the Activation of Bivalent Genes in G1

(A) qChIP of Fucci hESC cell-cycle fractions examining levels of MLL2 and WDR5 on the GATA6 and SOX17 promoters. Data are the average of three independent replicates.

(B) qChIP assays for MLL2 in untreated or MI-2 (25 μM for 24 hr)-treated WA09 ESCs at the indicated promoters. Data are the average of three independent replicates.

(C) qChIP assays for MLL2 in ESCs of pluripotency genes. Data are the average of three independent replicates.

(D) qRT-PCR transcript analysis of Fucci-sorted hESCs after treatment with the MLL/MENIN inhibitor, MI-2 (25 μM for 24 hr). Data are the average of three independent replicates.

(E) Immunoblot analysis of WA09 hESC and DE (2 days) lysates (20 μg per lane) with or without MI-2 (25 μM).

(F) Immunostaining for SOX17 (top) and FOXA2 (bottom) following the infection of GFP-control or MENIN shRNA lentivirus in WA09 hESCs or cells differentiated to DE for 3 days. Cells are co-stained with DAPI to visualize nuclei. Micron bar represents 50 μm.

(G) Quantitation of immunostaining represented in (E) for three independent fields, n > 1,000. Data are representative of three independent experiments.

(H) CXCR4 flow cytometry analysis of WA09 hESCs and DE (3 days differentiation) transduced with MENIN or GFP shRNA lentivirus. The percentage of CXCR4⁺ cells in each condition is indicated.

(I) qRT-PCR transcript analysis of WA09 hESCs and DE (3 days differentiation) following lentiviral infections with MENIN or GFP (control [C]) shRNA. Data are the average of three independent replicates.

^∗p < 0.05. Error bars in this figure represent the SEM. See also Figure S3.