Analysis of the histone code: Identification and characterization of murine histone demethylases

The structural and functional features of chromatin play an important role in the regulation of nuclear processes, like transcription, DNA replication, recombination and DNA repair. Chromatin structure and thereby function ist modulated by posttranslational modifications of its protein constitutents, the core histones. Histones are modified by phosphorylation, ubiquitination, glycosylation, ADP-ribosylation, methylation and acetylation. The latter modifications have come into the focus of current research interest. All modifications on nucleosomal histones together represent an epigenetic marker system, called the "histone code". Not only histones are modified by histone acetyltransferases, deacetylases, methyltransferases or demethylases but also nonhistone regulatory proteins. Just recently it has been recognized that the methylation of histones is less stable as has been assumed. Histone methyltransferases have been shown to play an important role for the epigenetic regulation of gene expression; moreover it has been demonstrated that histone methylation is interrelated with histone acetylation and DNA methylation. In the proposed project we will analyze cultured mouse cell lines for the presence of histone demethylase enzyme activities. We will chromatographically separate distinct enzymes, that will be characterized in biochemical, enzymatic and immunological terms. The central aim of the project is to isolate and purify a putative histone demethylase, an enzyme not identified so far. By chromatographic purification, a specific enzyme activity assay, protein microsequencing, immunological and molecular genetic methods we will purify and identify murine HDMEs. We will further (dis)prove the hypothesis that Elp3-related HATs posess demethylase activity and investigate whether HDME is part of high molecular weight complexes that contain other enzymes involved in histone modification (HATs, HDACs, HMTs). The identification of a histone demethylase would represent a milestone in our understanding of the complex epigenetic code of histone modifiations in eukaryots.

The genome of all organisms contains the entire information needed to build the organism. However, during various transitions in differentiation and development, additional epigenetic information determines the functional state of cells and tissues. This epigenetic information can be introduced by DNA methylation, chromatin remodeling, and by modifying nucleosomal histones. Therefore the terms "epigenetic histone code" or "chromatin code" in contrast to the genetic DNA code have been introduced. Posttranslational modifications of core histones represent essential elements of this "histone code". The most extensively investigated modification is acetylation, catalyzed by histone acetyltransferases and histone deacetylases. Our laboratory has been engaged in investigations of this modification since 1982. A second modification, the methylation of arginines and lysines has attracted considerable interest during the past 5 years. We have started to work on histone methylation approx. 3 years ago and the primary aim of this project was to characterize and identify enzymes that revert the methylation of histones, namely histone demethylases. When we started the project in November 2002 histone demethylases had not yet been identified. At that time we had preliminary data that strongly suggested the presence of such enzymes. Indeed, in late 2004 two histone demethylating activities have been reported, a true demethylase LSD1, which demethylates a certain lysine residue, and an arginine deiminase which converts methylated arginines into citrullines. Within our research project we established a chromatographic purification system for the purification of protein complexes of murine cells in culture that exhibited demethylase activity in an indirect, antibody-based demethylase assay. A low molecular weight peak had a substrate preference for arginines, a high molecular weight peak displayed preferential substrate specificity for lysine residues. For the latter a suitable purification scheme of 6 chromatography columns was developed that should allow the identification of the enzyme and further characterization of its biochemical and enzymatic properties. We obtained evidence that other histone modifying enzymes are associated with demethylases. We detected a 1.3 MDa complex in nucleoli that contains demethylase activity and the histone deacetylase HDAC1, underscoring the complex interrelations between different histone modifying enzymes and their marks on chromatin in the sense of a histone code.