The zinc finger protein Kox1 is encoded by the ZNF10 gene located on chromosome 12 (12q24.33). Despite being cloned in 1990, genes whose expression are regulated by this putative transcription factor are yet to be identified. Earlier investigations by this laboratory using differential display PCR investigation of in vitro hematopoietic progenitor commitment identified this gene as being upregulated in response after exposure to thrombopoietin. This work has now been extended by developing specific quantitative-PCR probes and primers as well as a rabbit polyclonal antibody to be used in profiling and quantifying Kox1 expression in human tissues and cell lines. In addition, chromatin immunoprecipitation experiments are planned to identify not only interacting protein partners but also DNA binding sites. Quantitative-PCR measurement of Kox1 levels in total RNA isolated from 20 human tissues showed the greatest expression in testis and the lowest in heart. Subsequent supplementation of this data by examination of large scale microarray expression screening of numerous human tissues and cell lines at http://expression.gnf.org has simultaneously confirmed and extended these results. Expression of Kox1 in pituitary gland and CD34+Thy+ hematopoietic progenitors were shown to be even greater than in testis. In addition, cell lines have been identified which are currently being investigated as suitable in vitro model systems for further exploration of Kox1 function. Recombinant Kox1 has been expressed as fusion proteins in bacteria, purified and used to produce a rabbit polyclonal antibody. A separate fusion construct of Kox1 has also been overexpressed in bacteria and coupled to sepharose. This column has since been used to affinity purify anti-Kox1 from rabbit polyclonal serum. Western blot analysis of various human tissues has revealed agreement with the data already obtained from mRNA quantification/profiling experiments. Further, the existence of smaller isoforms of Kox1 in some tissues are suggested by this data. In the immediate future, immunohistochemical screening of tissue microarray are expected to provide information on the breadth of cell types expressing Kox1 leading to the identification of possible cell types amenable to further exploration of Kox1 function. Subsequent chromatin immunoprecipitation experiments are expected to define DNA motifs preferentially bound by Kox1 paving the way for identification of candidate genes subject to Kox1 regulation.
