JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila

Abstract

In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1's targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome.

Fig. 1

JIL-1’s C-terminal domain interacts with JASPer’s LEDGF domain to form the JJ-complex. a Western blot analysis with α-JASPer and α-JIL-1 antibodies of co-IP from nuclear embryo extracts. Co-IP was performed with two different monoclonal α-JASPer antibodies containing culture supernatants and culture medium as control. The corresponding unbound fractions are loaded next to each IP. Molecular weight markers are shown to the left. Source data are provided as a Source Data file. b SDS-PAGE with Coomassie staining of recombinant JJ-complex purification from Sf21 cells using a baculovirus dual expression system of FLAG-JIL-1 and untagged JASPer. Molecular weight markers are shown to the left. A contaminant band is marked by asterisk. Source data are provided as a Source Data file. c JIL-1 and JASPer domain architecture drawn to scale. In JIL-1, PEST domains are highlighted in black, kinase domains in dark gray and a predicted prion-like domain in white. In JASPer, PWWP and LEDGF domains are highlighted in dark gray and conserved region in intermediate gray. C-terminal truncation breakpoints a–g for JIL-1 and N-terminal (N1-N3) and C-terminal truncation breakpoints (C1-C3) for JASPer used in d and e are indicated. Δ denotes the deletion in JASPer-ΔLEDGF. d Western blot analysis using α-JIL-1 and α-JASPer antibodies of co-IP experiments with extracts from Sf21 cells expressing wild type, untagged JASPer and various FLAG-JIL-1 C-terminal deletion mutants. Co-IP was performed with α-FLAG beads. Source data are provided as a Source Data file. e Western blot analysis as in d of co-IP experiments with extracts from Sf21 cells expressing various untagged JASPer deletion mutants and FLAG-JIL-1. Uninfected Sf21 cell extract was used as control (NV = no virus). Co-IP was performed with α-FLAG beads. Source data are provided as a Source Data file.

Fig. 2

JIL-1 is unstable in absence of JASPer in the JASPer^cw2/cw2 mutant and in cell lines. a Gene model for P-element excision in the JASPer locus to generate JASPer^cw2 allele. The mRNA isoforms RA and RB are shown below. The excised genomic portion is marked in white and EP denotes the position of the excised P-element in EP-element line GS3268. b Western blot analysis of salivary gland extracts from L3 larvae of homozygous JASPer^cw2/cw2 and JIL-1^z2/z2 mutants and wild type larvae as control. Western blots using α-JIL-1, α-JASPer, α-H3S10ph, and α-H3K9me2 antibodies are shown, western blot with α-tubulin antibody was used as loading control. c Immunofluorescence microscopy of polytene chromosome squashes from L3 larvae of homozygous JASPer^cw2/cw2 and wild type larvae as control. From left to right, staining for JASPer, JIL-1, merged images and DNA are shown. The X chromosome is marked by arrow heads. Source data are provided as a Source Data file. d Immunofluorescence microscopy of polytene chromosome spreads from L3 larvae of homozygous JASPer^cw2/cw2 and wild type larvae as control. From left to right, staining for H3S10ph, H3K9me2, merged images and DNA are shown. The X chromosome is marked by arrow head and the chromocenter is labeled with “CC”. e Table summarizing viability of male and female JASPer^cw2/cw2 mutant flies. f Representative western blot analysis using α-JASPer and α-JIL-1 antibodies on whole cell extracts from S2 cells (left panel) and Kc cells (right panel) after jasper or jil-1 RNAi treatment, as used for RNA-seq experiments. A cross-reacting band is marked by asterisk. Source data are provided as a Source Data file. g Bar chart showing mean log₂ fold-change of normalized mean RNA-seq counts for JIL-1 and JASPer RNAi. Left panel, JIL-1 mRNA mean log₂ fold-change upon jasper RNAi (S2 n = 4 and Kc n = 4). Right panel, JASPer mRNA mean log₂ fold-change upon jil-1 RNAi (S2 n = 5 and Kc n = 4). Error bars represent standard error of the mean.

Fig. 3

The JJ-complex binds H3K36me3 nucleosomes in vitro and in vivo, and is enriched on the male X chromosome. a Bar chart of mean enrichment (n = 3 independent experiments with 2 different protein preparations) of nucleosome library pull-down with JASPer-FLAG (left panel) and aromatic cage mutant (right panel) relative to unmodified nucleosome, which is set to 1. Error bars represent standard error of the mean. b Bar chart of mean enrichment (n = 3 independent experiments) of nucleosome library pull-down with JJ-complex (FLAG-JIL-1 and untagged JASPer) (left panel) and aromatic cage mutant (right panel) relative to unmodified nucleosome, which is set to 1, as in a. Error bars represent standard error of the mean. c Genome browser profile showing mean H3K36me3 (upper panel, n = 4 independent experiments), JASPer (second upper panel, n = 4 independent experiments with 2 different antibodies), JIL-1 (second lower panel, n = 5 independent experiments with 2 different antibodies) and MSL3 (lower panel, n = 3 independent experiments) MNase ChIP-seq normalized coverage along representative 200 kb windows on chromosome 2 R and X in male S2 cells. HAS are marked by red bars above the gene models in gray. d Box plot showing mean H3K36me3 (left panel, n = 4), JASPer (second left panel, n = 4), JIL-1 (second right, n = 5) and MSL3 (right, n = 3) MNase ChIP-seq normalized coverage, as in c, at active (tpm > 1) and inactive (tpm ≤ 1) genes on the autosomes (n = 5785 and n = 8726, respectively) and X chromosome (n = 1214 and n = 1407, respectively) in male S2 cells. Box plot elements are defined as center line marking the median, box limits are the upper and lower quartiles, whiskers extend maximally 1.5-times the interquartile range and outliers are removed.

Fig. 4

JIL-1 and not H4K16ac is responsible for the enrichment of JASPer at the male X chromosome. a Genome browser profile showing mean (n = 3, for MSL3 n = 2) spike-in ChIP-seq normalized coverage in control male S2 cells and after jil-1 RNAi treatment from top to bottom for JASPer, MSL3, H4K16ac and H3K9me2 along representative 200 kb windows on chromosome 2R and X. HAS are marked by red bars above the gene models in gray. b Genome browser profile as in a showing mean (n = 3, for MSL3 n = 2) spike-in ChIP-seq normalized coverage in control male S2 cells and after jil-1 RNAi treatment, from top to bottom for JASPer, MSL3, H4K16ac, and H3K9me2 along a representative 200 kb window on chromosome X. Signal overlay is marked in grey. c Density plot showing mean (n = 3, for MSL3 n = 2) spike-in ChIP-seq normalized coverage in control male S2 cells and after jil-1 RNAi treatment at active (tpm > 1) genes for JASPer (top left), MSL3 (top right), H4K16ac (bottom left), and H3K9me2 (bottom right). X chromosomal genes (n = 1214) are represented by a solid line and autosomal genes (chromosomes 2L, 2R, 3L, and 3R, n = 5785) by a dashed line.

Fig. 5

JIL-1 and JASPer depletion in cells modulates the transcriptional output of genes, especially on the male X chromosome. a MA-plot showing mean log₂ fold-change of RNA-seq counts upon jasper RNAi versus control (upper panel, n = 4) and jil-1 RNAi versus controls (lower panel, n = 5) against mean RNA-seq counts for robustly detected genes at autosomes (left, chromosomes 2L, 2R, 3L, and 3R n = 6833) and X chromosome (right, n = 1441) in male S2 cells (left site). Statistically significant differentially expressed genes between RNAi and control conditions (fdr < 0.05) are marked in red and the number of significant genes is indicated on the plot. On the right, mean log₂ fold-change of RNA-seq counts upon jasper RNAi versus control (upper panel, n = 4) and jil-1 RNAi versus controls (lower panel, n = 4) against mean RNA-seq counts for autosomal genes (left, chromosomes 2L, 2R, 3L, and 3R n = 7144) and X chromosomal genes (right, n = 1509) in female Kc cells (left site). b Density plot showing mean log₂ fold-change of RNA-seq counts upon jasper RNAi versus controls (n = 4) and jil-1 RNAi versus controls (n = 5) at genes in male S2 cells, in left panel, as in a. X chromosomal genes (n = 1441) are marked with solid line and autosomal genes (chromosomes 2L, 2R, 3L, and 3R, n = 6833) with dashed line and jasper RNAi additionally in orange. Right panel, mean log₂ fold-change of RNA-seq counts upon jasper RNAi and jil-1 RNAi versus controls (n = 4 each) at genes in female Kc cells. X chromosomal genes (n = 1509) and autosomal genes (chromosomes 2L, 2R, 3L, and 3R, n = 7144).

Fig. 6

JIL-1 and JASPer depletion in S2 cells decrease the transcript level of transposons of the telomeric transposons of the HTT arrays. a Heatmap showing mean normalized log₂ enrichment in H3K36me3 (n = 4), JASPer (n = 4), JIL-1 (n = 5), and MSL3 (n = 3) MNase ChIP-seq at transposons (n = 124) in male S2 cells. Transposons of the HTT array are marked in red. b Scatter plot showing mean log₂ fold-change of RNA-seq counts upon jasper RNAi versus control and jil-1 RNAi versus control against mean normalized log₂ enrichment in JASPer (n = 4, upper panel) and JIL-1 (n = 5, lower panel) MNase ChIP-seq, respectively, at robustly detected transposons in male S2 cells (n = 111). Statistically significant differentially expressed transposons between RNAi and control conditions (fdr < 0.05) are marked in red and the number of significant genes is indicated on the plot. TEs of the HTT arrays, gypsy5, and 3S18 are labeled. c Bar plot of difference of mean H3K9me2 (n = 3 each) spike-in ChIP-seq normalized coverage after jil-1 RNAi treatment and control male S2 cells at transposons (n = 124) in male S2 cells. Error bars represent standard error of the mean. TEs of the HTT arrays are marked in red.

Fig. 7

The JASPer interaction network and other H3K36me3 binding proteins and complexes in Drosophila melanogaster. a Volcano plot of IP-MS showing –log₁₀(p-values) against mean log₂ fold-change in α-JASPer IP (n = 6) versus control IP (n = 5). Significantly enriched (p-value < 0.05 and log₂ fold-change > 4) proteins (n = 69) are highlighted in dark gray. JIL-1 and JASPer are marked in red, Set1/COMPASS complex members in blue, PBAP/Brm complex members in purple, other proteins involved in chromatin remodeling in orange, NSL complex members in pink, Su(var)3-7, Su(var)205, woc and pzg in green, Chro and Rpd3 in yellow and NDF in brown. b Bar plot showing GO term enrichment of significantly enriched proteins shown in a. The five statistically significantly (fdr < 0.01) most enriched GO terms are shown. c Model of JJ-complex binding at H3K36me3 marked gene bodies and interactions with other complexes. Interactions presented here are indicated by arrows. Other known H3K36me3 binding proteins (NDF and Mrg15) are drawn at the lower side.  We propose that phosphorylation by JIL-1 kinase is tightly regulated in space and time in part by its partner JASPer which stabilizes and anchores the kinase to active genes and telomeric transposons by binding to H3K36me3 nucleosomes via its PWWP domain. Regulation by JASPer affects any potential phosphorylation by JIL-1, in particular H3Ser10 phosphorylation which is involved in inhibition of heterochromatinisation.