Fig 1.
A) Eye color of representative flies of enhancer-blocking assays using the transgenic line B727.1 that contains the Fab7 insulator element in wild-type and heterozygous line 86 background. B) Eye color of representative flies of enhancer-blocking assays using the transgenic line F72.5 that contains the Fab7 insulator and PRE elements in haspin RNAi mutant background. C) Quantitative analysis of eye pigment in flies with the F72.5 construct and the genotypes indicated. n = 3, significant differences between wild-type and the different mutant backgrounds as determined by Student’s t-test (p<0.001). D) Quantitative analysis of eye pigment in flies with constructs containing Fab6, Fab7 and Fab8 insulator elements (see S1 Fig for details of the constructs) in wild-type and haspin128 heterozygous mutant background (n = 3). Statistical significance (**p<0.01 and ***p<0.001) was determined by Student’s t-test.
Fig 2.
Haspin kinase phosphorylates H3T3 in mitosis and interphase and it is required for chromatin binding of Pds5.
A) Immunolocalization of H3T3ph (green) and Cenp-C (red) in mitotic cells of wild-type and haspin128 mutant Drosophila larval brains. DNA is stained with DAPI (blue). Scale bars are 2μm. B) Representative polytene chromosome spreads from wild-type and haspin86 mutant salivary glands of third-instar larvae immunostained with antibodies against H3T3ph (green). DNA is stained with DAPI (blue). C) Representative polytene chromosome spreads from salivary glands of third-instar larvae that express Pds5-HA under the control of nubbin promoter in GFP (upper panels) or haspin (lower panels) RNAi backgrounds, immunostained with antibodies against HA (red) and CP190 (green). DNA is stained with DAPI (blue). D) Pds5-HA/CP190 immunofluorescence intensity ratio in polytene chromosome spreads from salivary glands of third-instar larvae that express Pds5-HA under the control of nubbin promoter in GFP or haspin RNAi backgrounds. n = 5, means and s.d. are shown. ***p<0.001 was determined by Student’s t-test.
Fig 3.
Modulation of nuclear architecture by haspin kinase.
A) Immunostaining of Drosophila salivary glands with antibodies against lamin Dm0 in wild-type third-instar larvae (upper panel) and haspin86 mutant background (lower panel). DNA is stained with DAPI. In the overlap (right panels), lamin Dm0 is shown in green and DAPI in blue. Scale bars are 50 μm. B) Box plot showing quantification of DAPI signals of Drosophila salivary gland nuclei in wild-type (WT), haspin mutant background (haspin86), overexpression of haspin under control of its own promoter (haspinPROM), overexpression of haspin in a mutant background (haspin86; haspinPROM) and in haspin, CP190, and CTCF RNAis under the control of nubbin promoter (nub > haspinRNAi, nub > CP190RNAi, and nub > CTCFRNAi). Nuclear size was determined in around 50 nuclei for each condition (n≥3). The p values as determined by Wilcoxon test of the different genetic backgrounds respect to WT are indicated (*** p < 0.001). C) Box plot showing quantification of DAPI signal of Drosophila salivary gland nuclei in wild-type (WT) and overexpression under the control of 69B of tagged versions of either haspin wild-type (haspin-HA) or haspin with a mutated kinase domain (haspinH420A-HA and haspinK282M-HA). Nuclear size was determined in around 50 nuclei for each condition (n≥3). Significant differences between wild-type and mutated kinase domains as determined by Wilcoxon test (*** p<0.001).
Fig 4.
Haspin is a suppressor of position-effect variegation.
A) ChIP-seq data for H3T3ph genome-wide localization. Enriched regions of HP1a (modENCODE data, see S1 Text) are depicted below. B) ChIP-seq data for H3T3ph over a region of 500 kb in chromosome 3 in Drosophila S2 cells. Binding profiles of CP190 and Ibf2 [66], Pol II S5ph and HP1a (modENCODE data) are shown. H3T3ph peaks are depicted in last row. C) Effect of haspin128 mutation on position-effect variegation in In(1)wm4 males (n = 3). D) Representative ChIP-seq data for H3T3ph over a 400 kb region of chromosome 3R in Drosophila S2 cells. Binding profiles of CP190 and Ibf2 [66], Pol II S5ph, H3K27me3 and HP1a (modENCODE data, see S1 Text) and lamin Dm0 [69] are shown. H3T3ph peaks are depicted in last row.
Fig 5.
Haspin is required for Polycomb-dependent homeotic gene silencing.
A) Abdominal-B transcriptional levels normalized to Actin5C in wild-type or haspin128 mutant Drosophila larval brains. n = 4, means and s.d. are shown. B) ChIP-qPCR using an antibody against Polycomb and primers for pointed as a control negative region, Mcp, Fab7 and Fab8 regulatory elements and Abd-B promoters A and B. Average enrichments (normalized to the input sample) are plotted as the ratio of precipitated DNA in either wild-type or haspin128 Drosophila larval brains relative to the control negative region. n = 3, means and s.e.m. are shown. C) Representative examples of homeotic transformations observed: second to first leg (L2-L1), third to first leg (L3-L1) and wing to haltere (W-H). A red circle marks sex combs that appear in the second and third leg of male flies. D) Frequencies of homeotic transformations in flies heterozygous for Pc1, Pc3 or Pc15 mutations in either wild-type or homozygous haspin128 mutant backgrounds. n = 3, over 20 individuals scored. E) Eye color of representative flies of a transgenic line P714 containing a homozygous PRE-miniwhite in a wild-type or haspin128 mutant background (n = 3). F) Frequencies of homeotic transformations in flies heterozygous for Pc3 mutation in either wild-type or combinations of heterozygous mutant backgrounds for haspin128 and cohesin complex components. n = 4, over 50 individuals scored. Statistical significance (*p<0.05, **p<0.01 and ***p<0.001) was determined by Student’s t-test (A and B) or z-test (D and F).