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X. Yu et al.
(Figure 4B). Interaction between the LDB1 and GATA1 proteins appears already shortly before the proerythrob- last stage. It is most abundant in the P2 population (most likely colony forming unit-erythroid [CFU-E] cells) and basophilic erythroblasts but decreases during the final stages of differentiation in vivo. We applied PLA to detect the interaction between GATA1/FOG1, GATA1/LDB1, GATA1/TAL1, LDB1/LMO2 and LDB1/ETO2 in MEL cells, which mimic FL cells. Uninduced MEL cells repre- sent proerythroblasts, i.e. part of the P1 and P2 population, while induced MEL cells represent P3 and further differen- tiated populations.9, 28,29 Figure 5A shows the detection of the GATA1/FOG1 interaction by PLA using single-prima- ry antibody and secondary antibody alone as controls. Quantification of the PLA signals in nuclei show a signifi- cant increase of GATA1/FOG1 interaction after MEL cell differentiation. An additional negative control experiment to further demonstrate the specificity of the PLA assay was a TAL1/FOG1 interaction which is known not to be formed.5 Quantification of the different PLA signals con- firmed the absence of TAL1/FOG1 interaction detection in MEL cells and is comparable to the one of the single probe GATA1 only control, thereby supporting the specificity of positive PLA signals (Figure 5B). This control on non-inter- acting highly expressed proteins (TAL1 and FOG1 in MEL cells)5 also ruled out a potential threshold effect of PLA i.e. where ligations may be more likely when the relevant TF partners are expressed highly. Similar quantification of PLA signals was performed in the cytoplasm (Figure 5B). It was much lower than that observed in the nucleus and does not increase upon differentiation as seen in the nuclei. Of note the increase of signal was not due to an increase of signal volume between the differentiated and
undifferentiated states (Figure 5C). In MEL cells, the LDB1 complex binds its target genes during erythroid differenti- ation,23 e.g. the α- and β-globin locus bind the LDB1 com- plex in differentiated cells resulting in upregulation,24-27 due to the loss of the repressor ETO2 (encoding by Cbfa2t3) from the complex.9 We quantified the PLA signals for GATA1/LDB1, LDB1/LMO2 and LDB1/ETO2 interactions (Figure 5D-F) and confirmed not only an increase of both GATA1/LDB1, and LDB1/LMO2 interactions upon differ- entiation but further confirmed that the LDB1/ETO2 interaction is lost during differentiation in MEL9 (Figure 5F), supporting its role as a negative regulator during ery- throid differentiation where ETO2 and IRF2BP2 with the NCOR1/SMRT co-repressor complex suppress the expres- sion of typical erythroid genes such as Klf1 which is need- ed to express β-globin and Gypa genes.8,26
In conclusion, the GATA1/LDB1 complex starts to be formed just before the proerythroblast stage and activates erythroid specific genes of erythroid differentiation in vivo, when it looses ETO2.
LDB1 KD results in loss of the erythroid cell population
We examined the importance of the GATA1/LDB1 com- plex in fetal erythropoiesis at E12.5 by three independent (partial) KD rather than a (lethal) KO using anti-LDB1 shRNA (shLDB1_1 and shLDB1_2) and an anti-GATA1 shRNA (shGATA1). Treatment with an empty vector pLL3.7 shRNA or scrambled shRNA (Scr) was used as the controls. The level of LDB1 or GATA1 protein relative to valosin containing protein (VCP) decreased in the FL cells from D1 to D3 in the KD (Online Supplementary Figure S4). On D3, the cells were sorted by fluorescence-activated cell
AB
Figure 4. GATA1/LDB1 proximity ligation assay on sorted fetal liver cells. (A) GATA1/LDB1 proximity ligation assay (PLA) on the four sorted cell populations as described in Figure 3A. PLA signal is in red and nucleus in blue. Scale bar represents 20 μm. (B) Quantification of GATA1/LDB1 PLA signals from three biological replicates (total number of dots in one cell vs. nucleus area) and boxplot comparison among the sorted four cell populations. ****Indicates the significance (P≤0.0001) between any of two cell populations. The statistical signifi- cance is determined by Kruskal-Wallis test (Tukey method).
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haematologica | 2020; 105(7)