Identification and Characterization of Histone H3K36 Demethylases in Drosophila melanogaster

Lin, Chia-Hui (2011). Identification and Characterization of Histone H3K36 Demethylases in Drosophila melanogaster. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f18c

Abstract

Covalent modifications of histones, such as acetylation, methylation, phosphorylation and ubiquitination, have an important role in regulating gene expression. Histone methylation is implicated in both gene activation and repression depending on the methylation site and the state of methylation (Li et al., 2007a). Historically, histone methylation was considered to be a static modification. Recent discoveries of histone demethylases demonstrate that histone methylation is reversible. Numerous studies have shown that dynamic regulation of histone methylation plays an important role in many cellular processes (Cloos et al., 2008). However, mechanisms governing the targeting and regulation of histone demethylation remain elusive.

In this thesis, I identified two Drosophila melanogaster JmjC domain-containing proteins, dKDM4A and dDKM4B, which are histone H3K36 demethylases. Affinity purification and mass spectrometry analysis revealed that Heterochromatin Protein 1a (HP1a) associates with dKDM4A. I found that the chromoshadow domain of HP1a and a HP1-interacting motif within dKDM4A are responsible for this interaction. HP1a stimulates the histone H3K36 demethylation activity of dKDM4A and this stimulation depends on HP1a binding to the H3K9me. Loss of HP1a leads to increased level of histone H3K36me3. By chromatin immunoprecipitation using an antibody against H3K36me3 in wild type and dKDM4A mutant embryos, I identified candidate target genes of dKDM4A. A subset of dKDM4A target genes are also shown to be bound by HP1a, suggesting dKDM4A-HP1a complex may function in regulating H3K36 levels at these genes.