PKA Mediated Phosphorylation of TAL1 Regulates Its Interaction with LSD1 During Hematopoiesis.

Abstract 2599

TAL1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We showed that TAL1 dynamically interacts with LSD1 complex containing both histone H3K4 demethylase and deacetylase activities during hematopoiesis (Proc. Natl. Acad. Sci. USA 106: 10141–10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated by Protein kinase A (PKA) during the transcriptional activation of TAL1. The PKA inhibitor, H89, stimulated TAL1 interaction with LSD1 in hematopoietic cells, while Forskolin, an activator of PKA, completely abolished TAL-LSD1 interaction. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1^Ser172Ala mutant remains interacted with LSD1, TAL1^Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 by PKA destabilizes the TAL1 and LSD1 interaction. Our previous results indicate that LSD1 inhibits TAL1 mediated erythroid differentiation. To further test whether the activity of TAL1 is mediated through an interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 leads to a promotion of erythroid differentiation and H3K4 hypermethylation of the P4.2 promoter. In contrast, the expression of the TAL1^Ser172Ala mutant and TAL1-LSD1 chimerical fusion enhanced cellular proliferation and colony formation ability of the hematopoietic progenitor cells while these constructs inhibited erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs.