Title: Developmental Regulation Of Erythropoietin Gene Expression Through M Sally Palmisano, MS III¹, Mabel Padilla¹, Aihua Dai, MD¹, William Palmisano, PhD². , and Robin K Ohls, MD ethylation
¹. ¹Pediatrics, University of New Mexico, Albuquerque, NM, and ²SoBran, Inc., Cincinnati, Ohio.
Background: Erythropoiesis in the fetus is marked by a constant and significant need for increased red blood cell numbers. Epo producing tissues in the fetus vary significantly in the amount of protein produced, however it is unclear what mechanism of gene regulation is occurring during gestation.
Objective: In order to determine if methylation plays a role in the differential gene expression of Epo in Epo-producing tissues during development, we investigated the methylation patterns of the enhancer region of the Epo gene in the mid-trimester human fetal liver and kidney using combined bisulfite restriction analysis (COBRA).
Design/Methods: DNA was isolated from fetal liver and kidney at 10-22 weeks gestation and bisulfite-modified. For comparison, DNA was also isolated from Hep3B cells (as positive control). The methylation status of the Epo enhancer region was determined using primers that recognize the bisulfite-modified DNA template but did not discriminate between methylated and unmethylated alleles. Sequences were amplified, purified from gel, and ligated into a PCR II vector. The 191 bp PCR product from each tissue sample was cloned and sequenced to determine if methylation occurred in the vital portion of the enhancer region known as the hypoxia response element (HRE). The DNA from 5 clones of each tissue type were isolated, the sequences determined, and the % methylation quantified.

Results: The enhancer region of the Epo gene from Hep3B cells was mostly unmethylated (only 6% of potential CGs were methylated). Early gestation fetal liver showed 36% methylation, while early kidney showed 60% methylation. Later gestation fetal tissue showed a greater degree of methylation [figure1]. These data were consistent with tissue hypoxia experiments, where early gestation tissue responded to hypoxia better than late gestation tissue, and liver responded better than kidney.

Conclusions: Methylation in the enhancer region of the fetal Epo gene was greater in kidney than in liver, and was greater in fetal tissues than in Hep3B cells. Methylation might inhibit full upregulation during hypoxic stimulus in vivo.