Alternative linker histone permits fast paced nuclear divisions in early Drosophila embryo

Abstract

In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis.

Figure 1.

CRISPR/Cas9-guided generation of chimeric BigH1/H1 alleles. (A) Schematic view of the BigH1 genomic region with the indicated target sites of the gRNAs used for CRISPR/Cas9 gene editing. (B) Schematic map of the donor plasmid for generating the 3F allele. The donor plasmid carries upstream and downstream homology arms, dsRed marker gene surrounded by LoxP sites and the modified BigH1 encoding sequence. Manipulated sequences were inserted between BstZ17I and BglII restriction sites. (C) Genotypes of BigH1 mutant animals, abbreviated name and general structure of generated BigH1 alleles.

Figure 2.

BigH1 mutant embryos showed nuclear fallout phenotype and cold sensitivity. (A) Hatching rate of wild type and BigH1 mutant embryos at 25°C. (B) Nuclear fallout phenotype observed in wild type and BigH1 mutant embryos. (C) Immunostaining of typical wild type and HHH mutant embyos representing nuclear fallout phenotype. (D) PCNA staining of 3F and HHH syncytial blastoderm embryos during a mitotic wave. Arrows indicate nuclei in which the nuclear cycle cannot progress to mitosis. (E) Nuclear fallout in 3F and HHH syncytial blastoderm embryos. Eliminated nuclei do not show PCNA staining in 3F embryos (arrowheads), while in HHH mutant embryos descending nuclei are PCNA positive (arrows). Nuclei in the cortical layer are in non-replicative phase in both embryos, indicated by negative PCNA staining. Scale bar represent 50 μm (C–E). (F–G) Hatching rate of wild type and BigH1 mutant embryos at 30 and 15°C, respectively. For statistics, two-tailed, unpaired t-test was used, error bars represent SD, P-values: **<0.01; ***<0.001 (A, B, F, G).

Figure 3.

Wild type and HHH mutant embryos differ greatly in nuclear volume but only slightly in nucleosomal DNA length. (A) Nuclear volume at the nuclear cycles leading up to cellularization (NC 10–13). The diameters of nuclei were measured in H2Av-GFP expressing precellular embryos, and their volume calculated. (B) Mono- and multinucleosomal DNA size and linker DNA length compared in WT and HHH mutant, determined by running MNase digested sample DNA on a Bioanalyzer DNA 1000 chip. (C) Percentage of mono- and multinucleosomal DNA in wild type and HHH mutant embryonic chromatin, to assess the general compaction state of chromatin. For statistics, two-tailed, unpaired t-test was used. Error bars represent SD, P-values: *<0.05; **<0.01; ****<0.0001 (A–C).

Figure 4.

Nucleosome exchange is less dynamic in chromatin formed in the presence of H1 in early embryonic nuclei (A) Images of early and late S-phase in nuclei of H2Av-GFP;+ (wild type) syncytial blastoderm embryos before and after photobleaching. Scale bar represents 2 μm. Recovery of fluorescent signal originating from transgenic H2Av-GFP refers to nucleosome dynamics. (B) Nucleosome dynamics in early and late S-phase in wild type embryos. Comparison of nucleosome dynamics in wild type and HHH mutant embryos in (C) early and (D) late S-phase. Error bar: SEM. Box plots on these diagrams show values of area under curve of individual FRAP measurements in the first 40 seconds where error bars represent SD. For statistics, two-tailed, unpaired t-test was used (B–D). (E) Images of embryonic nuclei of H2Av-GFP; + (wild type) and H2Av-GFP; HHH embryos in the early S-phase of NC 10, 11 and 12 display higher number of H2Av-GFP containing LDs in HHH mutant embryos. Scale bar represents 2 μm. (F) Quantitation LD density in wild type and HHH mutant embryos in different stages. Error bars represent SD. For statistics, two-tailed, unpaired t-test was used, P values: *<0.05, ***<0.001 (B–D, F).

Figure 5.

Nucleosomes formed in embryos in the presence of H1 are less stable. (A, B) Results of two independent QINESIn experiments where nucleosome stability was calculated by the loss of H2Av-GFP signal in nuclei isolated from H2Av-GFP;+ (wild type) and H2Av-GFP;HHH (mutant) embryos. In both experiments normalized fluorescence intensity differs significantly at 800 mM NaCl treatment (nonparametric Mann-Whitney test; A: P = 0.0015; B: P = 0.0002). Error bar: SEM. (C) Combined results of experiments 1 and 2, showing the average values. In this case error bars represent the average SEM. (D) Percentage of chromatin-bound core histones (H3 and H4) after 800 mM NaCl salt elution, determined by Western blot. (E) Percentage of chromatin-bound linker histones prepared from 3F+HHH mixed and 3F/HHH heterozygous early embryonic chromatin, determined by Western blot. For statistics, two-tailed, unpaired t-test was used (D, E). (F) Percentage of chromatin-bound linker histones after 100–300 mM NaCl salt elution, prepared from 3F+HHH mixed and 3F/HHH heterozygous early embryonic chromatin, and determined by Western blot. Error bars represent SD (D, F).

Figure 6.

Comparison of H1 and BigH1 predicted globular domain structures reveals differences in presumed DNA binding loop. (A) Conserved 3D structure of the globular domains of Gallus (4QLC, 5WCU) and Xenopus (5NL0) linker histones in relation to nucleosomal DNA. (B) Structure predictions for dH1 and BigH1 indicates a shorter loop (L3) in BigH1. (C) Structural models for DNA-bound dH1 and BigH1 built on Gallus linker histone template (4QLC), with model quality indicators. (D) Comparison of the models obtained by the two indicated programs with highlighted alteration in the loop (L3) involved in DNA binding. (E) Enlarged structure of the loop (L3) involved in nucleosomal DNA binding. Two amino acids, Val87 and Ser90 (orange), described to be important in DNA binding by Zhou et al. (21) are missing from BigH1, suggesting difference between the two linker histones in DNA interactions.