Smad4 Inhibits Nup98-Hoxa9 Transcriptional Activity by Interacting at Hox Homeodomain.
Authors: Wang Ning , Wan Mei , Tang Yi , Kim Hyung G. , Cotta Claudia , Klug Christopher A. , Cao Xu 
Published: April 2020

Abstract
Homeobox (Hox) genes play crucial roles in hematopoiesis. Alterations of Hox gene expressions are found in patients with hematological malignancy. Nup98-Hoxa9, a fusion protein combining the N-terminus of Nup98 and the C-terminus of Hoxa9 resulting from t (7; 11) chromosomal translocation, is the first evidence of Hox gene involvement in human leukemia. We have shown that BMP specific R-Smads and Smad4 interact with Hox proteins including Hoxa9, and the interaction blocks binding of Hox proteins to their DNA elements. Here, we show that Smad4, the common signal transducer of both TGF-β and BMP, interacts with the homeodomain of Nup98-Hoxa9 in a coimmunoprecipitation assay. Furthermore, this interaction abolished the DNA binding ability of Nup98-Hoxa9 to DNA in both EMSA using purified GST fusion protein and in in vivo chromatin immunoprecipitation assays. Subsequent mapping data revealed the MH1 domain of Smad4 mediated the interaction. These findings suggest that Smad4 could be used to inhibit Hox-induced leukemia.
We then attempted to examine whether Smad4 could inhibit the downstream target gene by Nup98-Hoxa9. Although Nup98-Hoxa9 is believed to function as an aberrant DNA-binding transcriptional activator, downstream genes that contribute to leukemogenesis are largely unknown. It has been reported that Nup98-Hoxa9 induces Hoxa9 expression in murine primary bone marrow cells. Accumulating evidence suggests that Hoxa9 plays important roles in normal and malignant hematopoiesis. Enforced expression of Hoxa9 causes proliferative expansion of primitive hematopoietic stem/progenitor cells and subsequently inhibits their differentiation. Aberrant expression of HOXA9 due to retroviral integration induced acute myeloid leukemia (AML) in mice. By visually scanning the sequence of the Hoxa9 promoter, we found many TGAT and TTA (T/C) consensus sequences, which are preferentially recognized by the heterodimer of PBX protein and the proteins of the Abd-B-like gene family, including Hoxa9, respectively. To test whether Hoxa9 could be a direct transcriptional target for Nup98-Hoxa9, we used a Hoxa9 promoter/luciferase reporter construct along with the expression plasmid pcDNA-Nup98/Hoxa9 to transiently transfect NIH/3T3 fibroblast cells and Ba/F3 murine hematopoietic progenitor cells. Nup98-Hoxa9 showed substantial transcription activation on Hoxa9 promoter in these cell lines. This activation is further enhanced by cotransfection of PBX1a, which stabilizes the binding of Nup98-Hoxa9 to DNA. Nup98-Hoxa9 also bound to radiolabelled DNA oligo from the Hoxa9 promoter in vitro by EMSA assay. These data indicate that Nup98-Hoxa9 directly activates the Hoxa9 promoter. Interestingly, Smad4 removed the binding of Nup98-Hoxa9 to the Hoxa9 promoter. Co-overexpression of Smad4 inhibited the transactivation activity of Nup98-Hoxa9 on the Hoxa9 promoter regardless of the presence of PBX1a. In NIH/3T3 cells, Smad4 inhibited Nup98-Hoxa9-induced Hoxa9 expression both at the mRNA and protein levels. In summary, we show that Smad4 inhibited DNA binding activity of Nup98-Hoxa9 by interacting at Hox homeodomain. This interaction abolished the transcription activity of Nup98-Hoxa9 on its target gene, thus preventing Nup98-Hoxa9 from working as a transcription factor, which could lead to a new therapeutic target for the treatment of myeloid leukemia caused by Nup98-Hoxa9.