Histones and their posttranslational modifications influence the regulation of many DNA-dependent processes. Although an essential role for histone-modifying enzymes in these processes is well established, defining the specific contribution of individual histone residues remains a challenge because many histone-modifying enzymes have nonhistone targets. This challenge is exacerbated by the paucity of suitable approaches to genetically engineer histone genes in metazoans. Here, we describe a platform in Drosophila for generating and analyzing any desired histone genotype, and we use it to test the in vivo function of three histone residues. We demonstrate that H4K20 is neither essential for DNA replication nor for completion of development, unlike inferences drawn from analyses of H4K20 methyltransferases. We also show that H3K36 is required for viability and H3K27 is essential for maintenance of cellular identity but not for gene activation. These findings highlight the power of engineering histones to interrogate genome structure and function in animals.

Introduction

Margueron and Reinberg, 2010 Margueron R.

Reinberg D. Chromatin structure and the inheritance of epigenetic information. Rothbart and Strahl, 2014 Rothbart S.B.

Strahl B.D. Interpreting the language of histone and DNA modifications. Shogren-Knaak et al., 2006 Shogren-Knaak M.

Ishii H.

Sun J.M.

Pazin M.J.

Davie J.R.

Peterson C.L. Histone H4-K16 acetylation controls chromatin structure and protein interactions. Lachner et al., 2001 Lachner M.

O’Carroll D.

Rea S.

Mechtler K.

Jenuwein T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Margueron and Reinberg, 2010 Margueron R.

Reinberg D. Chromatin structure and the inheritance of epigenetic information. During animal development, a single genome gives rise to a wide diversity of cells. Each cell type differentially regulates genome activity to accurately execute a particular program of gene expression, cell-cycle progression, and DNA replication. Failure of this execution can lead to developmental defects or disease states that reduce organismal fitness. Because the genome sequence is essentially identical in most cell types, epigenetic mechanisms have been proposed to bring about cell-type specific regulation of genome activity (). Such mechanisms require a substrate that carries regulatory information and a means of propagating this information over time. Histone proteins are particularly attractive candidates for carriers of epigenetic information because they can fulfill both of these criteria. First, histone proteins have the potential to be dynamic regulators of genome activity because they are subject to a broad range of posttranslational modifications (PTMs), including phosphorylation, acetylation, and methylation (). Histone PTMs are thought to contribute to regulation of genome activity by controlling chromatin packaging (), and by serving as binding sites for protein complexes that control a variety of DNA-dependent processes including transcription, replication, and repair (). Second, histone proteins provide a potential means of propagating information over time through their partitioning to daughter cells during each cell division ().

Marzluff et al., 2008 Marzluff W.F.

Wagner E.J.

Duronio R.J. Metabolism and regulation of canonical histone mRNAs: life without a poly(A) tail. Marzluff et al., 2002 Marzluff W.F.

Gongidi P.

Woods K.R.

Jin J.

Maltais L.J. The human and mouse replication-dependent histone genes. Lifton et al., 1978 Lifton R.P.

Goldberg M.L.

Karp R.W.

Hogness D.S. The organization of the histone genes in Drosophila melanogaster: functional and evolutionary implications. Günesdogan et al., 2010 Günesdogan U.

Jäckle H.

Herzig A. A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes. Hödl and Basler, 2012 Hödl M.

Basler K. Transcription in the absence of histone H3.2 and H3K4 methylation. Pengelly et al., 2013 Pengelly A.R.

Copur Ö.

Jäckle H.

Herzig A.

Müller J. A histone mutant reproduces the phenotype caused by loss of histone-modifying factor Polycomb. A particularly powerful approach for studying the biological function of specific histone PTMs is to change the acceptor residue to an amino acid that cannot be appropriately modified and then to engineer a complete gene replacement for phenotypic analysis. Implementing this strategy in animals is technically challenging because metazoan histones are typically encoded by gene clusters found at multiple chromosomal locations (). For example, the human genome has 64 histone genes, clustered at three different loci (). In contrast, the Drosophila replication-dependent histone genes are found at a single locus (). Recently, Herzig and colleagues created a system for complementing deletion of the endogenous Drosophila histone gene cluster with plasmid-based transgenes (), allowing for the first analysis of histone residue function in animal development (). However, a minimum of four transgenes was required to rescue the histone locus deletion phenotype, limiting the ease with which this strategy can be used in combination with other genetic tools to study histone gene function in Drosophila.

Here, we present a BAC-based platform that can rescue deletion of the endogenous Drosophila histone locus with a single transgenic insertion, allowing us to study not only the regulation of histone genes themselves, but also the specific contribution of histones to the regulation of DNA-dependent processes. After demonstrating its in vivo functionality, we used this platform to directly test the function during animal development of three posttranslationally modified histone residues: H3K36, H3K27, and H4K20. Unlike results obtained in yeast, we show that H3K36 is required for viability in Drosophila. Consistent with current models, we find that H3K27 is required for the maintenance of Polycomb target gene repression, demonstrating that histone residues can perform an essential function in gene regulation. These results underscore the essential roles played by these two histone residues in gene expression and animal development. Finally, in contrast to current models, we show that a modifiable H4K20 residue is neither required for DNA replication nor for completion of Drosophila development. Together, these studies demonstrate the importance of directly testing the function of individual histone residues in animal development, and highlight the potential of this approach to test the role of histones in metazoan genome structure and function.