G9a has a dual role in regulating expression of β-globin genes. (A) The expression of G9a and GLP was analyzed by Western blot after Dox-induced KD of G9a for two independent MEL clones (cl.1 and cl.2) targeting different regions of the G9a transcript. (B) Bulk cellular levels of H3K9me2, H3K27me2, and other histone modifications were analyzed by Western blot before and after G9a KD as indicated. (C) Relative enrichment of H3K9me2 and H3K27me2 as analyzed by Western blot in (B) are quantitated. Average ± SD represent three independent experiments. (D) Schematic representation of the murine β-globin locus. Shaded triangles represent the β-like globin genes. The white triangle represents the inactive olfactory receptor gene. Probes used to detect transcripts on the β-globin locus by RT-qPCR are labeled according to the variation of the transcripts levels upon G9a KD as indicated. (E) Transcription at the indicated genes was assessed by RT-qPCR after differentiation in G9a-depleted (Dox) vs. normal (No Dox) MEL cells. Transcripts are expressed relative to GAPDH with the highest ratio set to 1 or as a percentage of the E^y transcript relative to the β^maj transcript as indicated. Average values ± SD represent three independent experiments. (F) ChIPs were performed before (Nondiff.) and after induction of differentiation in G9a-depleted (Diff. G9a KD) vs. normal (Diff.) MEL cells to analyze the binding of the RPB1 subunit of Pol II. ChIPs were revealed by qPCR using TaqMan probes located within the promoter (prom), exon 2 (ex2), and exon 3 (ex3) of the globin genes as indicated. Values are expressed as a function of the highest enrichment and represent average of at least two replicates ± SD.

NF-E2/p45 interacts with the H3K9 MT complex G9a/GLP. (A) Western blot analysis of endogenous proteins immunoprecipitated from an erythroid nuclear extract via Abs against NF-E2/p45, GLP and G9a. Abs used for Western blot are indicated on the right. Asterisk indicates Ab heavy chain. (B) Recombinant UBC4 or NF-E2/p45 proteins were incubated with recombinant G9a protein before G9a IP. (C) Histone H3 previously acetylated with p300 (+p300) or not (-p300) was used as a substrate for methylation by NF-E2/p45-interacting proteins and submitted to Edman degradation sequencing. The incorporated [³H]-methyl at each amino acid is indicated in cpm.

G9a-dependent histone methylation on the β-globin locus. (A) ChIPs were performed before (Nondiff.) and after differentiation in G9a-depleted (Diff. G9a KD) vs. normal (Diff.) MEL cells to analyze the binding of NF-E2/p45 and G9a as well as the enrichment of H3K9me2 and H3K27me2. ChIPs were revealed by qPCR using indicated probes. Values are expressed as a function of the highest enrichment and represent average of at least two replicates ± SD. (B) RT-qPCR analysis of the embryonic E^y and adult β^maj globin genes was performed upon differentiation in G9a KD cells after transfection of DNA constructs expressing WT G9a (WT) or a MT-defective G9a mutant (Mut.). These constructs were rendered resistant to shRNA-mediated KD of G9a via silent mutations (sequences available in SI Text). Transcripts values are expressed relative to GAPDH with the highest ratio set to 1. Average values ± SD represent three independent experiments. ChIPs were performed in the same conditions to analyze the enrichment of the H3K9me2 and H3K27me2 on the E^y globin promoter. ChIPs were revealed by qPCR. Values are expressed as a function of the highest enrichment and represent average of at least two replicates ± SD. (C) UTX and TFIIHp89 proteins levels were analyzed by Western blot after Dox-induced KD of UTX in differentiating MEL cells. Transcription at the E^y and β^maj-globin genes was assessed by RT-qPCR after differentiation in UTX-depleted (Diff.UTX KD) vs. normal (Diff.) MEL cells. Transcripts values are expressed relative to GAPDH with the highest ratio set to 1. ChIPs were performed after differentiation in UTX-depleted vs. normal MEL cells to analyze the binding of UTX and the enrichment of H3K27me2. ChIPs were revealed by qPCR using probes located at the promoters of the E^y and β^maj globin genes. Values are expressed as a function of the highest enrichment and represent average of at least two replicates ± SD.