338. METHYL BINDING DOMAIN PROTEIN (MBD1) IN THE NUCLEUS OF THE MOUSE ZYGOTE

Abstract

MBD1 is one of five proteins which bind methylated DNA and regulate gene transcription. The binding of these proteins, particularly MBD1, is commonly used as a proxy measurement of global CpG methylation. Since methylation is reported to be highly dynamic during the first cell-cycle, with reported asymmetric global demethylation of the paternal and maternal genomes by the time of syngamy, we were interested to assess the pattern of staining of the MBD1 during this stage of development. A specific antibody to MBD1 was shown by Western analysis to detect in zygotes a protein of predicted mass. Using immunolocalization, however, we found no staining in pronuclei. Brief acid treatment (10min, 4M HCl) followed by immunolabelling revealed strong pronuclear MBD1 staining throughout the maturation of the zygote and on metaphase chromosomes, indicative of epitope masking under normal staining conditions. Upon unmasking by acid treatment zygotes collected fresh from the oviduct did not show consistent differences in MBD1 staining between the maternal or paternal chromosomes or pronuclei, but for those embryos produced by IVF we found more MBD1 staining in the male paternal pronucleus. Brief treatment with trypsin caused a marked loss of MBD1 staining and this treatment increased the extent of staining of 5-methylcytosine. These results show that MBD1 antigen persists on DNA after treatments normally used for the detection of 5-methylcytosine. MBD1 at least partially masks methylcytosine from immunological detection and the results therefore raise the possibility that the reported changes in genome methylation in the zygote are a consequence of the binding of MBD1. If MBD1 binding is truly a proxy for methylation, the persistence of high levels of MBD1 throughout the first cell-cycle questions the current paradigm of global demethylation during zygote maturation.