Abstract
The role of DNA methylation in gene inactivation has been studied extensively in vertebrates but it is not clear whether it serves similar functions in other organisms. We devised a novel approach to induce hypermethylation of both endogenous and injected DNA in the sea urchin Lytechinus pictus in order to study the effect of DNA methylation on gene expression in this invertebrate. By injecting 5-methyl dCTP either alone or together with a cloned DNA construct into fertilized sea urchin eggs, replicating DNA became hypermethylated from the random incorporation of the methylated nucleotide in place of cytosine during DNA synthesis. During subsequent rounds of replication, the injected 5-methyl dCTP became depleted but methylation at CpG sites was still elevated presumably due to the action of a methyltransferase enzyme. Using this approach, we studied the effect of hypermethylation on two members of the sea urchin multigene family, the early H2B and the late H2B genes. De novo methylation was shown to occur at known cis-regulatory regions of the genes. The effect of methylation on gene activity was probed using RNase protection assay. Methylation resulted in increased early H2B histone gene expression but had no effect on late H2B histone gene expression. These results demonstrate that methylation does not necessarily inactivate genes in the sea urchins as previously thought. Interestingly, the development of embryos injected with 5-methyl dCTP typically was arrested at the blastula stage, and analysis of the genomic DNA extracted from injected embryos showed a significant increase in the endogenous methylation content. These data suggest that perturbation of methylation patterns in developing sea urchin embryos may be responsible for the developmental arrest through altering the gene expression pattern.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.