Fig. 7

The SNAG-LSD1 interaction is critical for the regulation of primitive hematopoiesis

(A) Sequence of the SNAG domain peptide with dimethylated Lys 8 indicated in red.

(B and C) Single-cell zebrafish embryos from a kdm1a(23095) heterozygous incross were injected with both zlsd1-3XFLAG mRNA and the SNAG peptide. WISH was then performed at 24 hpf for gata1 (B) and gfi1ab (C) expression.

(D and E) Single-cell zebrafish embryos from a kdm1a(23095) heterozygous incross were injected with zlsd1-ΔTower mRNA. WISH was then performed at 24 hpf for gata1 (D) and gfi1ab (E) expression. Representative images are shown with the number of animals with similar staining patterns for the indicated genotype shown in the upper right corner. The lower right corner indicates the mRNA injection conditions. Embryos were imaged, blindly scored by expression and then genotyped by HRMA. Carons indicate ICM expression; closed arrowheads indicate posterior blood island/caudal hematopoietic tissue; narrow triangles indicate inner-hair-cell expression. Scale bar, 200 μm.

(F) Model: LSD1-dependent hematopoietic phenotypes require SNAG-domain binding. Under normal conditions, a SNAG-domain protein recruits LSD1 and a regulatory complex (e.g., CoREST) to target specific loci to silence the endothelial/stem program and promote hematopoietic cell differentiation. (II) In the absence of LSD1, SNAG-target loci are not transcriptionally repressed and an endothelial/stem cell fate is permitted. (III) An SNAG-domain protein is still able to recruit a demethylase-deficient LSD1 (K661A) and a regulatory complex to target the genetic loci that enable primitive hematopoietic gene expression. (IV) LSD1 SNAG-domain-binding mutations, such as A539E or D555K, prevent LSD1 from binding SNAG-domain-containing proteins. These LSD1 mutants are therefore not recruited to SNAG-protein target genes, such as endothelial/stem genes, which remain expressed and permit an endothelial/stem cell fate.