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January 13, 2007

Characterization of hAF9: Role in Hematopoietic
Regulation

Jessica Gonzalez

Abstract

Chromosomal translocations are often the cause of hematological diseases, such as leukemia.  These translocation often result in the aberrant recruitment of factors, which are normally only active during hematopoietic development.   For example, transcription factors are known to play a key role in embryonic development as agents of critical cell fate decisions.  hAF9  aknown developmental regulator of anterior/posterior axis patterning, is a commonly translocated to the mixed-lineage leukemia (MLL) gene.  The MLL-AF9 fusion product is usually expressed in acute myeloid leukemia (AML), while MLL-AF4 is predominantly found in tumors of B-cell lineage.  This observation indicates that the identity of the fusion partner plays an important role in the phenotypic identity of this disease.  Previous studies indicate that Cdx genes regulate Hox genes, which are also involved in the regulation of hematopoietic development.   The similarities found amongst mice containing Af9 null mutations and those carrying Cdx null mutations, suggest that Af9 may also have a role in the same Cdx/Hox pathway.  Furthermore, null Af9 mutant mice display a significantly more severe phenotype, suggesting a potential role as a master regulator of this pathway. Our goal is to investigate the putative role of hAF9 in hematopoietic development by using differentiation of mouse embryonic stem cells (mESC) as an in-vitro model of development in concert with in vitro assays for hematopoietic activity and real time PCR to assess hematopoietic gene expression. hAF9 was engineered into a doxycycline dependent system.  It was found that two of the three-engineered clones contained the hAF9 insert and the validity of the insert was confirmed by PCR and sequencing.  It was found that both clones were responsive to doxycycline, but to different extents.  ES cells of one clone had an approximate 45 fold increase in hAF9 expression upon addition of 0.5 mg/mL doxycycline, while the other underwent a mere 5 fold increase with the same concentration.  At low concentrations of doxycycline we observed increased formation of colony forming units, while at higher concentrations suppression took place, such that is evident that that increased expression of hAF9 has a suppression effect on hematopoietic development. Surprisingly, increased hAF9 expression resulted in the up regulation of various hematopoietic transcription factors.  These data is in accordance with the promotion of hematopoietic colony formation observed in our functional assays. In summation, these data suggest that hAF9 plays an important role in hematopoietic development.  Over expression of hAF9 leads to suppression of hematopoietic colony forming units and to the up regulation of hematopoietic transcription factors. Future studies will focus on refining the dose of doxycycline applied to each clone and address directly whether hAF9’s effect on hematopoietic colony formation is mediated via the Cdx/Hox pathway.  Determining the role of hAF9 in hematopoietic development may increase our understanding of and ability to treat hematopoietic malignancies.

 


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