A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes

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A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes
¨nesdogan, Herbert Ja ¨ckle & Alf Herzig+ Ufuk Gu
¨ ¨ ¨ Abteilung fur molekulare Entwicklungsbiologie, Max-Planck-Institut fur biophysikalische Chemie, Gottingen, Germany
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Despite the fundamental role of canonical histones in nucleosome structure, there is noexperimental system for higher eukaryotes in which basic questions about histone function can be directly addressed. We developed a new genetic tool for Drosophila melanogaster in which the canonical histone complement can be replaced with multiple copies of experimentally modified histone transgenes. This new histone-replacement system provides a well-defined and direct cellular assay system for histonefunction with which to critically test models in chromatin biology dealing with chromatin assembly, variant histone functions and the biological significance of distinct histone modifications in a multicellular organism. Keywords: histone deletion; histone transgenes; functional assay; Drosophila melanogaster
EMBO reports (2010) 11, 772–776. doi:10.1038/embor.2010.124

INTRODUCTION
Eukaryoticgenomes are packaged into arrays of nucleosomes composed of 147-base-pair DNA intervals wrapped around an octameric complex of the core histones H2A, H2B, H3 and H4 (Luger et al, 1997). Together with the H1-type linker histones, these proteins are termed canonical histones, as they constitute the vast majority of histone proteins in chromatin (Marzluff et al, 2008). Concomitant with genomeduplication during S-phase of the cell cycle, disassembled parental nucleosomes and de novo synthesized histones are used as distinct sources for replicationcoupled nucleosome assembly (Corpet & Almouzni, 2009). In a subset of nucleosomes, the canonical histones are subsequently replaced by histone variant proteins by replication-independent processes, which can specify functionally distinct chromatinregions (Henikoff & Ahmad, 2005). The complexity of chromatin
¨ ¨ Abteilung fur molekulare Entwicklungsbiologie, Max-Planck-Institut fur ¨ biophysikalische Chemie, Am Fassberg 11, Gottingen 37077, Germany + Corresponding author. Tel: þ 49 551 201 1798; Fax: þ 49 551 201 1755; E-mail: aherzig1@gwdg.de
Received 14 January 2010; revised 13 July 2010; accepted 26 July 2010; published online 3September 2010

diversification is further increased by the many post-translational modifications of histones, which are thought to constitute an epigenetic ‘histone code’ (Jenuwein & Allis, 2001; Kouzarides, 2007). Despite the central role of histones in chromatin assembly and diversification, no experimental tools have been developed so far to directly address canonical histone divergence fromreplacement variants and the post-translational modifications in multicellular organisms. In fact, genetic analysis has been limited to lower eukaryotes, such as the yeast Saccharomyces cerevisiae, in which histone genes are encoded by tandem repeats (Marzluff et al, 2008). In all higher eukaryotes, however, canonical histones are encoded by multiple gene units of between 10 and 400 copies that aremostly distributed over several chromosomes (Marzluff et al, 2008). The high number of histone genes and their distribution within the genome prevent straightforward functional genetics and explains the correlative nature of many previous studies in the field (Kouzarides, 2007). As the yeast model system is clearly not suitable to address chromatin diversification and its function in the context of...