Figure 2. Serine 172 is critical for regulating the recruitment of LSD1, H3K4methylation, and TAL1 mediated repression of p4.2 gene.

(A) MEL cells were stably infected with retroviruses encoding the vector control, Flag-TAL1, Flag-TAL1^Δ142-185, and Flag-TAL1^S172A. The cells were induced to differentiation by addition of 1.5% DMSO for 3 and 5 days. The cells were collected for analysis of hemoglobin expression by using benzidine staining and counting consecutive 200 cells in the field. The data were collected from three independent experiments. (B) The levels of endogenous and exogenous TAL1 and their mutants expressed in MEL cells were determined by Western Blotting analysis (Top). The levels of PCNA (Bottom) were shown as a loading control. (C) Schematic representation of the mouse p4.2 gene locus. (D and E) Cross-linked chromatins from MEL cells infected with retrovirus encoding Flag-TAL1 and its mutants were precipitated with LSD1 (D) and H3K4me2 (E) antibodies. The precipitated chromatins were purified and analyzed by real-time PCR. The recruitment of LSD1 or enrichment of H3K4me2 is significantly difference in the Flag-TAL1Δ142-185 expressed cells in comparison to that of the MSCV, Flag-TAL1, and Flag-TAL1^S172A expressed cells. (F) Total RNA from retrovirus infected MEL cells were isolated and analyzed for P4.2 expression by RT-qPCR. *P<0.05; **P<0.01 by student t test. Shown are the mean ± SEM of at least 3 independent experiments. The red dotted line represents background levels of signal.