Requirement of Split ends for epigenetic regulation of Notch signal-dependent genes during infection-induced hemocyte differentiation

Abstract

Drosophila producing a mutant form of the putative transcription coregulator, Split ends (Spen), originally identified in the analysis of neuronal development, display diverse immune defects. In order to understand the role of Spen in the innate immune response, we analyzed the transcriptional defects associated with spen mutant hemocytes and their relationship to the Notch signaling pathways. Spen is regulated by the Notch pathway in the lymph glands and is required for Notch-dependent activation of a large number of genes involved in energy metabolism and differentiation. Analysis of the epigenetic marks associated with Spen-dependent genes indicates that Spen performs its function as a coactivator by regulating chromatin modification. Intriguingly, expression of the Spen-dependent genes was transiently downregulated in a Notch-dependent manner by the Dif activated upon recognition of pathogen-associated molecules, demonstrating the existence of cross talk between hematopoietic regulation and the innate immune response. Our observations reveal a novel connection between the Notch and Toll/IMD signaling pathways and demonstrate a coactivating role for Spen in activating Notch-dependent genes in differentiating cells.

FIG. 1.

Spen mutant flies are highly susceptible to fungal infection. (A) The survival rates (%) of wild-type (wt) flies, flies carrying several alleles of spen, and spen^rev2 adult flies, were measured after subjecting them to septic injury using B. bassiana. Infections were performed in at least four replicates. spen^rev2, a P-element excision derivative of spen^GE10359. (B) Determination of the protein level of Spen in circulating plasmatocytes. Immunostaining was performed in wild-type (wt), spen^GE10359, and spen^rev2 hemocytes with rat anti-spen antibody. Scale bars, 10 μm. Experiments were repeated four times with similar results.

FIG. 2.

Transcriptome analysis of the circulating hemocytes in spen and Notch pathway mutants. (A) Bivariate scatter plots of log₂-transformed microarray data comparing E(spl) versus Su(H) and spen versus Su(H). Those genes downregulated in spen hemocytes are denoted by blue dots. The Pearson correlation coefficient (r) is shown for each plot. (B) Microarray analysis of hemocytes in E(spl), Su(H), and spen mutant flies. Columns correspond to the different mutants, and rows to different genes. The expression levels of genes up (red)- and down (green)-regulated more than threefold (at least one observation with that absolute value) compared to that of wild-type hemocytes are shown (left). Representative GO groups belonging groups I and II are shown on the right, together with functional annotations and gene symbols.

FIG. 3.

Notch-dependent expression of Spen in lymph glands. (A) Immunohistochemical analysis of the lymph glands of third-instar larvae of the indicated genotypes with anti-N^icd (red) and anti-Spen (green) antibodies, along with DAPI (blue) staining. Scale bar, 200 μm. (B) In situ hybridization analysis of sut4 and CG32064 transcripts in lymph glands. The genotypes of the lymph glands are indicated on the top. The cortical zone (CZ) and medullary zone of the lymph glands are marked with gray and yellow lines, respectively. Scale bar, 100 μm. The N^ts1 mutant was raised at 18°C. To obtain the Notch mutant phenotype, we incubated third-instar larvae at 29°C for 12 h and analyzed the lymph glands after further incubation of the larvae for 18 h at 18°C.

FIG. 4.

Epigenetic regulation of Spen-dependent genes. The statistical status of the Spen-dependent genes (S) and Spen-independent genes (I) for expression levels (A) and epigenetic patterns (B) are shown. (A) The expression levels of Spen-dependent genes are significantly downregulated compared to other genes in Kc cells. The statistical significance of the downregulation is shown at the bottom. (B) Epigenetic status of Spen-dependent genes compared to Spen-independent genes. Of 921 genes that were repressed more than threefold in our full genome Drosophila Affymetrix microarray analysis, only 198 are matched to specific probes of the ChIP-chip analysis, which includes probes for 5,186 Drosophila genes. This cohort of genes was compared to the rest of the genes, and box plots comparing the two groups with respect to activating histone modifications (H3K4me3, H3K79me2, H3Ac, and H4Ac), the repressing epigenetic mark (H3K27me3), and the binding strength of polycomb group proteins (Pc, Esc, and Sce) are shown. The green boxes indicate the levels of activating histone marks in the Spen-dependent genes; the red boxes indicate the levels of repressing histone marks or binding strength of polycomb group proteins in the Spen-dependent genes; the white boxes represent the status of the Spen-independent genes. (C) ChIP assays of wild-type (wt) and spen mutant hemocytes. Immunoprecipitation was performed with IgG, anti-Spen, or anti-trimethylated H3K4 antibodies. Coimmunoprecipitated DNA fragments were amplified with specific primers for the promoter regions of the genes indicated on the left.

FIG. 5.

Requirement of Spen for lamellocyte differentiation. (A) Counts of circulating lamellocytes in third-instar larvae after fungal infection. The numbers of lamellocytes in the circulation of third-instar larvae with (+) or without (−) spore infection are shown. The means and standard deviations of five independent experiments are shown. In each experiment, at least six larvae of each genotype were counted, and the results were averaged. (B) Immunohistochemical analysis of the lymph glands of wild-type (wt) and spen mutant third-instar larvae with lamellocyte-specific anti-L1 antibody (red) at the indicated times after infection with live B. bassiana spores. DAPI staining patterns are shown. The cortical zones (CZ) are marked by white lines. Scale bar, 50 μm. (C) Immunohistochemical analysis of wild-type lymph glands with anti-L1 (red) and anti-Spen (green) antibodies, along with the DAPI (blue) staining 18 h after infection with live B. bassiana spores. Scale bar, 10 μm. The data are representative of three independent experiments. (D) Microarray analysis of wild-type hemocytes 1 h after fungal infection. Bivariate scatter plots of log₂-transformed microarray data compare fungal infection of wild-type versus spen mutant hemocytes. Genes whose expression decreased in the spen hemocytes relative to wild-type (wt) hemocytes are indicated by blue dots. The Pearson correlation coefficient (r) is shown. (E) In situ hybridization analysis of sut4 transcripts in lymph glands after spore infection. Wild-type larvae were subjected to septic injury with B. bassiana spores, and lymph glands were dissected from third-instar larvae at 6-h intervals. Scale bar, 100 μm. The results are representative of three independent experiments.

FIG. 6.

Dif-dependent downregulation of Notch signaling. Immunohistochemical analysis of lymph glands with antibodies against Notch (red) and Spen (green). Third-instar larvae of the indicated genotype were infected with live B. bassiana spores (A) or E. coli (B), and lymph glands were dissected at 6-h intervals as indicated on the left. Tl^r3 flies were obtained from the embryo collection, grown at 25°C for 30 h and shifted to 29°C for 72 h. Scale bar, 200 μm. The results are representative of three independent experiments.

FIG. 7.

Novel connection between the Notch and Toll signal pathways. Genes (signals) with active or inactive status are marked with solid lines and boxes with colors or dashed lines and boxes without colors, respectively. (A) Chromatin regulation by Spen mediating Notch signaling in hematopoiesis in the absence of infection was determined. Spen expression in the Drosophila lymph glands was completely dependent on Notch activity and appeared to be regulated downstream of E(spl). Spen functions as a coactivator by regulating the chromatin status of a large group of genes mostly involved in energy metabolism and development. Notch inhibits the expression of genes involved in cell proliferation. The expression pattern may favor the maintenance of stem cell phenotypes. (B) Cross talk between the Toll and Notch pathways during infection-induced hematopoiesis. In Drosophila lymph glands, activated Dif is required for transient downregulation of Notch levels, and activated Dif may stimulate the degradation pathways mediated by ubiquitination or endocytosis. Notch downregulation inactivates genes involved in energy metabolism while releasing the inhibition of genes involved in proliferation. This expression pattern may favor the differentiation of hemocytes.