Functional Analysis Of TGIF1 In Erythroid Differentiation

Hematopoietic stem cells (HSCs) differentiate into committed progenitor, burst-forming unit erythroid (BFU-e), the colony-forming unit-erythroid (CFU-e), and give rise to proerythroblasts, the reticulocyte, and finally create red cells. Erythropoiesis is a complex and delicate biological process that each stage is maintained by spatial and temporal gene regulation. Gene regulation at transcriptional level in erythroid differntiation is one of the most well-studied and understood mechanisms. Transcription factors play important roles in erythroid differentiation, by activating or repressing a set of target genes to maintain complex regulation network. TGIF1 was determined as a potential erythorid transcription factor by our trancriptome analyses, and validate it by functional studies using in vitro models, investigate the mechnism behind and complex regulations with other transcription factors in erythroid differentiation.We performed Differential Gene Expression analysis in our transcriptome data of HSC and 4 different stages of erythroid cells induced from HSC. TGIF1 was determined as a potential erythroid transcription factor as its consistent expression along with erythroid differentiation. It has been reported that TGIF1 is a negative regulator in brain development. However its functions in hematopoiesis remain unclear. Combined with TGIF1 expression patterns and a variety of functional studies, we concluded that TGIF1 play important role in erythroid differentation. First, we knockdown TGIF1 in TF1 and K562 cell lines and it reduces expression of globin genes, GATA1 and KLF1. Conversely when we overexpressed TGIF1 in TF1 and K562 cell line, we got the opposite data.Then we performed RNA-seq of TGIF1-knockdown TF-1 cells. The transcription analysis reveal that, GATA1 and ALAS2 are two of most significant down-regulated genes and the expression of Smad family genes is upregulated. Thus, TGIF1 may play a role in erythropoiesis through upregulating expression of GATA1 and ALAS2 or repressing TGF-β signaling.In cancer and brain development, it has been shown that TGIF1 inhibits expression of Smad family to repress TGF-P signaling. In our trancriptome data, we also found that TGIF1 knockdown lead to upregulation of Smad family. These results suggest that TGIF1 may play an important role in erythoroid differentiation through TGF-β signaling.Our study helps to understand the complex regulation in erythoroid differentiation and provide new information for pathogenesis and treatments of diseases caused by erythropoiesis defects.