Targets of the Tal1 Transcription Factor in Erythrocytes

Abstract

The Tal1 transcription factor is essential for the development of the hematopoietic system and plays a role during definitive erythropoiesis in the adult. Despite the importance of Tal1 in erythropoiesis, only a small number of erythroid differentiation target genes are known. A chromatin precipitation and cloning approach was established to uncover novel Tal1 target genes in erythropoiesis. The BirA tag/BirA ligase biotinylation system in combination with streptavidin chromatin precipitation (Strep-CP) was used to co-precipitate genomic DNA bound to Tal1. Tal1 was found to bind in the vicinity of 31 genes including the E2-ubiquitin conjugase UBE2H gene. Binding of Tal1 to UBE2H was confirmed by chromatin immunoprecipitation. UBE2H expression is increased during erythroid differentiation of hCD34 ؉ cells. Tal1 expression activated UBE2H expression, whereas Tal1 knock-down reduced UBE2H expression and ubiquitin transfer activity. This study identifies parts of the ubiquitinylation machinery as a cellular target downstream of the transcription factor Tal1 and provides novel insights into Tal1-regulated erythropoiesis.

Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart

“…Our HL1 screen using in vivo biotinylated TF allowed us to use uniform, stringent pulldown conditions to perform ChIP and circumvented limitations of currently available antisera. The in vivo biotinylation approach has been validated in prior studies (11)(12)(13)(14)(15), and current data suggest that it does not alter TF activity. Limitations of our HL1 screen are that only a subset of the binding sites identified in this system may correspond to binding sites present in vivo at different stages of development and disease and that some in vivo binding sites may not be captured in this in vitro system.…”

“…S1A), permitting factor pulldown on streptavidin under stringent, uniform conditions and independent of antibodies (11). The use of this approach to tag TFs was validated previously for genome-wide ChIP analysis without reported effect on the factor's protein interactions or DNA binding properties (11)(12)(13)(14)(15). We expressed the biotinylated factors in the HL1 cardiomyocyte cell line, which expresses signature cardiac genes, forms organized sarcomeres, responds to β-adrenergic agonists, and exhibits spontaneous beating (16).…”

PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation

“…Erythroleukemia cells harbouring the birA-ligase together with a bir-tagged Tal1 (bir-Tal1) were grown in heavy SILAC medium (H)10. In vivo biotinylated bir-Tal1 protein was affinity purified using streptavidine beads and mixed in equimolar amounts with a control sample from birA-ligase cells, which were grown in light SILAC medium (L).…”

“…For biotin purification of bir-Tal1, we used nuclear extracts of 1 × 10 8 K562-BirA control and K562-BirA-Tal1 cells10. Extracts were essentially prepared as described63.…”

Stem Cell Leukemia: how a TALented actor can go awry on the hematopoietic stage

“…During mouse erythropoiesis, Tal1 regulates expression of the red blood cell membrane components, such as Glycophorin A 17 and protein 4.2 18 , and ubiquitination machinery components, such as the E2-ubiquitin conjugase UBE2H 19 . Their promoter activation is dependent on the assembly of a multifactorial transcriptional complex containing TAL1, E47, Sp1, Ldb1, LMO2 and GATA1 10,17,18 .…”