A genetic system to assess in vivo the functions of histones and histone modifications in higher eukaryotes - PubMed (original) (raw)

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

Ufuk Günesdogan et al. EMBO Rep. 2010 Oct.

Abstract

Despite the fundamental role of canonical histones in nucleosome structure, there is no experimental 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 histone function 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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Df(2L)His C deletes all canonical histone genes. (A) Schematic representation of the canonical histone gene organization in Drosophila melanogaster. A total of 23 histone gene repeat units, each containing a single His1, His2B, His2A, His4 and His3 gene (His-GU), are clustered in the histone complex. The deletion of the histone complex includes the region between the distal rearrangement screen element CB-5033-3 and the proximal rearrangement screen element 5-HA-1581. Neighbouring genes are nrv3 and CG3305. (B–D) Wild-type histone H1 (His1) expression in S-phase 15 (S15). Cyclin B labels cells in G214; cells in early S15 show low or absent Cyclin B staining (B). S15 cells expressed His1 mRNA as detected by in situ hybridization (C). Expression was absent from G2 cells (D, Cyclin B red, His1 green). (E–G) Corresponding staining showed that His1 expression was undetectable in homozygous His C mutant embryos (His C /His C). Wild type refers to internal control. Scale bar, 100 μm; anterior, left. mRNA, messenger RNA.

Figure 2

Figure 2

Rescue of His C by 12 His-GUs. (A–C) Representative embryos from time-matched collections 4.5–5 h after egg laying stained for Cyclin B. Wild-type embryos undergo M15 in the dorsal epidermis (A), whereas His C mutant embryos are blocked with high levels of Cyclin B before M15 (B). In the presence of 12 transgene-based His-GUs, His C mutant embryos display a wild-type M15 pattern (C); wild type refers to _w_− control. (D) The percentage of embryos that completed S15 (S15 exit) or progressed into M15 (M15 entry) in the dorsal epidermis was determined. Embryonic collections were time matched for the indicated time interval after egg laying. The left set of columns are wild type. The middle display His C homozygotes rescued by 12 His-GUs and the right display embryos from the same collection, which were not homozygous His C mutant. These embryos acted as internal controls to ensure reproducible timing of the collections. For details on classification of embryos see supplementary Fig S5 online. Scale bar, 100 μm (A–C); anterior left (A–C), His-GU; histone gene repeat unit; M15, mitosis 15; n, number of embryos.

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