Page 69 - 2020_07-Haematologica-web

Temporal and spatial emergence of GATA1 complex
S1B), most of it is in the fractions of 37-39, but some is present in fractions 22-24 indicating that some complex has formed. In MEL cells, i.e. a much later stage of devel- opment and differentiation, most LMO2 is in the fractions overlapping with LDB1 (Online Supplementary Figure S1B), showing they are in the same complex consistent with previous results.8 Although the expression of GATA1 and partners is low at D4-5 ES cell differentiation, we could detect the formation of the different complexes by PLA due to its ability to detect 102-fold or 106-fold lower pro- tein concentrations than ELISA or WB, respectively.33
GATA1/FOG1 regulates genes that are less important for erythroid specific functions in the undifferentiated stage, e.g. pro-erythroblasts. We originally postulated a switch from GATA1/FOG1, repressing alternative lineage genes to GATA1/GFI1B repressing proliferation related genes during differentiation.5 In MEL non-induced cells, GFI1B and LDB1 can also be detected in an ETO2 pull- down, indicating that LDB1 complexes containing ETO2 and/or GFI1B proteins suppress archetypical erythroid genes primed for the onset of terminal erythroid differen- tiation.26 Indeed, RNA-seq data shows that Cbfa2t3 (encoding ETO2) and Irf2bp2 expression decreased from P1 to P2 suggesting these genes mainly function in P1 to P2, similar to the stage of non-induced MEL cells. Meanwhile, Klf1 increased its expression and reached the peak at P3, together with the increase of typical erythroid specific genes (Online Supplementary Figure S3). This result indicates that clear erythroid differentiation starts at P2. Our PLA result on sorted FL cells show that the GATA1/LDB1 interaction peaks in CD71+/TER119– cells at a relatively early stage of erythropoietic differentiation, like undifferentiated MEL cells, when many erythroid genes are still suppressed, until ETO2 disappears from the complex turning on typical erythroid genes. Other LDB1 complex regulated genes such as c-myb remain suppressed, because they no longer bind the (activating) LDB1 com- plex through an as yet unresolved mechanism (Giraud et al., unpublished data).
We show that PLA can detect endogenous level of GATA1/LDB1 and GATA1/FOG1 interactions in FL tissues and we located the GATA1/LDB1 interaction in a specific cell type in situ. Whether it corresponds to E7.5 yolk-sac derived primitive cells, E8.5/E9 yolk sac and placenta derived intermediate erythroid-myeloid,34,35 or HSC derived definitive progenitors is still an open question.
From PLA images a number of transcription factor com- plexes seem to be located outside the nuclei. The studied factors are certainly formed in the cytoplasm and travel to the nuclei; whether a fraction of interacting factors
remains in the cytoplasm is a possibility. The extensive set of complementary experiments that would aim at study- ing the existence of interactions in the cytoplasm, such as cellular compartment fractionation followed by IP would be similarly questionable, as (like for PLA) it would be dif- ficult to exclude the possibility of the nuclear extract frac- tions leaking out in the cytoplasmic fractions. We per- formed an additional control by quantifying the PLA sig- nals in the cytoplasmic area of MEL cells for the positive GATA1/FOG1 and the non-existing TAL1/FOG1 interac- tions and associated negative controls (single probes and secondary antibodies only), and observed that non-exist- ing TAL1/FOG1 interaction detection presents a lower level to the existing GATA1/FOG1 and GATA1/TAL1 in the cytoplasm (Figure 5B). These differences suggest the existence of a certain level of transcription factor interac- tion in the cytoplasmic fraction and the amount of signal in TAL1/FOG1 sets the level of background of the PLA technique. It should also be noted that the level of PLA sig- nals from different interactions cannot be compared directly, since PLA is dependent on antibody quality. Therefore, like other immuno-based technic PLA present a certain level of background, easily quantifiable by using single probe, secondary antibodies only, protein mutant and/or non-existing interaction detection. PLA can detect an interaction up to a 40 nm distance36 and enables super high resolution immunofluorescence microscopy, which can distinguish molecules at a similar distance37. Although PLA cannot detect real-time protein-protein interaction, quantification of PLA signals of different GATA1 complex- es in sequential stages of ES cell differentiation improves our understanding on the temporal changes of these com- plexes.
This study therefore reveals that PLA is a powerful tool to examine dynamic protein/protein interactions and their dynamics in differentiating erythroid cells and demon- strates that it provides an excellent alternative for cells in which the abundance of proteins is too low to perform standard co-IP experiments. Our study revealed that PLA can be used to detect very low amount of essential GATA1 complexes emerging at early time point of ES cell differen- tiation and later on FL tissue, the site of definitive erythro- poiesis. In addition we show that the increase followed by a decrease of expression of GATA1 and LDB1 affects a number of genes differentially, for example the expression of the c-myb gene which is regulated by the LDB1 com- plex15 decreases during differentiation, while the expres- sion of the β-globin genes, which are also dependent on the LDB1 complex increases. Further studies will be need- ed to understand how these differences are regulated.
References
1. Dzierzak E, Speck NA. Of lineage and lega- cy: the development of mammalian hematopoietic stem cells. Nat Immunol. 2008;9(2):129-136.
4.
al. Arrest in primitive erythroid cell devel- opment caused by promoter-specific dis- ruption of the GATA-1 gene. J Biol Chem. 1997;272(19):12611-12615.
Pevny L, Lin CS, D'Agati V, Simon MC, Orkin SH, Costantini F. Development of hematopoietic cells lacking transcription factor GATA-1. Development. 1995; 121(1):163-172.
Rodriguez P, Bonte E, Krijgsveld J, et al. GATA-1 forms distinct activating and repressive complexes in erythroid cells.
EMBO J. 2005;24(13):2354-2366.
6. Snow JW, Orkin SH. Translational isoforms of FOG1 regulate GATA1-interacting com- plexes. J Biol Chem. 2009;284(43):29310-
29319.
7. Wadman I, Li J, Bash RO, et al. Specific in
vivo association between the bHLH and LIM proteins implicated in human T cell leukemia. EMBO J. 1994;13(20):4831-4839.
8. Meier N, Krpic S, Rodriguez P, et al. Novel binding partners of Ldb1 are required for haematopoietic development.
2. Medvinsky A, Rybtsov S, Taoudi S. Embryonic origin of the adult hematopoiet-
ic system: advances and questions. 5. Development. 2011;138(6):1017-1031.
3. Takahashi S, Onodera K, Motohashi H, et
haematologica | 2020; 105(7)
1811