The Drosophila histone variant H2Av facilitates Notch signaling activity in a two-tier regulatory fashion

Abstract

Background: H2Av is an evolutionarily conserved H2A variant protein involved in the regulation of transcription. The Tip60 complex is recruited by different transcription factors to facilitate the incorporation and acetylation of H2Av, thereby influencing target gene expression. The Tip60-H2Av axis is involved in various developmental processes, though its precise roles are not yet fully understood.

Methods: RNA interference and gene mutation technology were used to screen essential genes in regulating Notch signaling pathway. Immunostaining was used to detect the protein level of H2Av, Tip60 complex as well as Notch signaling pathway components. Chromatin immunoprecipitation assays were performed to detect the specific binding site of H2Av in E(spl)-Complex and Su(H) genes.

Result: Here we report that H2Av is required for Notch signaling activation during Drosophila wing development. H2Av depletion disrupts the expression of Notch target genes, resulting in wing marginal defects. Unexpectedly, we find that H2Av regulates the expression of the Su(H) gene which encodes for the transcription factor of the Notch signaling cascade. We further demonstrate that the Tip60 complex modulates the transcription of both Notch targets and Su(H) likely through H2Av. Based on these observations, we propose a model that the Tip60-H2Av axis facilitates Notch pathway activation by simultaneously promoting the expression of both the target genes and the transcription factor.

Conclusion: This study offers insights into the diverse roles of the Tip60-H2Av axis in Notch pathway activation by identifying a novel two-tier regulatory mechanism which may also be utilized by other chromatin remodeling factors.

Keywords: Drosophila; H2Av; Notch; Su(H); Tip60.

Fig. 1

H2Av positively regulates Notch signaling activity in the Drosophila wing. A-C: Adult wings from flies harboring H2Av^LL00733 (A) and H2Av^KO (B). homozygous clones, and en > H2Av RNAi flies (C). Wing nicking is marked by black arrows (A-C). Scale bars = 100 µm. D: Representative image of wing discs stained for Cut from flies harboring H2Av^KO minute clones. The clones are marked by the absence of GFP, with a representative clone outlined by dashed lines. E: Representative image of wing discs stained for Cut from en > H2Av RNAi larvae. The expression region of en-Gal4 is marked by GFP and outlined by dashed lines. F-G: Representative images of adult wings and wing discs stained for Cut and Wg from wild-type flies (F-F’’) and flies expressing H2Av RNAi (G-G’’). H: Representative image of wing discs expressing Notch signaling reporter NRE-GFP from hh > H2Av RNAi larvae. The expression region of hh-Gal4 is marked by GFP and outlined by dashed lines. I: Representative image of wing discs stained for Cut from flies harboring H2Av^KO mutant clones with a H2Av^WT transgene expressed in all cells. Mutant clones are marked by the absence of RFP, with a representative clone outlined by dashed lines. J: Wing disc bearing H2Av^KO MARCM clones stained for Cut. In these MARCM clones, UAS-H2Av^WT was expressed under the control of tub-Gal4. The MARCM clones are marked by GFP, with a representative clone outlined by dashed lines. Scale bars = 50 μm

Fig. 2

H2Av is involved in activating the expression of Notch target genes. A-H: Wing discs bearing MARCM clones stained for Cut (A, B and E-H) or Wg (C, D). In the control group, no transgene was expressed in the H2Av^KO clones (A). In the experimental groups, UAS- H2Av^S137A (B), UAS-Ser (C-D), UAS-N^FL (E-F) or UAS-NICD (G-H) was expressed under the control of tub-Gal4 in the clones. The MARCM clones are marked by GFP and representative clones are circled by dished lines. Scale bars = 50 μm. I: Percentage of Cut positive clones in UAS-NICD; WT and UAS-NICD; H2Av^KO MARCM clones. Three biological replicates were performed, and more than 20 wing discs were examined for each genotype. Data are presented as mean ± SEM. P-values from unpaired two-tailed Student’s t-test are indicated, *P < 0.05

Fig. 3

H2Av modulates the transcription of Notch target genes. A: The mRNA level of H2Av, E(spl)-C family genes and Cut were examined by qRT-PCR in wing discs collected from C765-Gal4 and C765 > H2Av RNAi larvae. Data are presented as mean ± SEM. P-values from unpaired two-tailed Student’s t-test are indicated. B-C: The occupancy of H2Av (B) and H2Av^ac (C) at regulatory regions in the E(spl)-C family genes and Cut as examined by ChIP-qPCR. The results are presented as means ± SE and asterisks indicate significant differences (independent t-test, *P < 0.05, **P < 0.01 and ***P < 0.001). Abbreviations are as follows: “igr”, intergenic region; “tr”, transcribed region; and “enh”, enhancer

Fig. 4

H2Av regulates Su(H) transcription. A-C: Representative image of wing discs stained for H2Av (A), Dl (B) and NICD (C) from Dpp > H2Av RNAi larvae. The expression region of Dpp-Gal4 is marked by GFP and outlined by dashed lines. D-E: Representative image of wing discs stained for H2Av (D) and Su(H) (E) from en > H2Av RNAi larvae. The expression region of en-Gal4 is marked by GFP and outlined by dashed lines. F: Representative image of wing discs expressing Su(H)-GFP and stained for H2Av from Ci > H2Av RNAi larvae. The expression region of Ci-Gal4 is marked by RFP and outlined by dashed lines. Scale bars = 50 μm. G: The mRNA level of Su(H) was examined by qRT-PCR in wing discs collected from C765-Gal4 and C765 > H2Av RNAi larvae. Two pairs of primers which amplify non-overlapping regions were used. The results are presented as means ± SE and asterisks indicate significant differences (independent t-test, ***P < 0.001). H: The enrichment of H2Av on Su (H) is represented by the ChIP-seq signal as the average log2 ratio of IP/Input in the 10 kb window of the entire genome. I: The occupancy of H2Av at Su(H) regulatory regions as examined by ChIP-qPCR

Fig. 5

The Tip60 complex regulates Notch signaling activity. A-B: Representative images of adult wings from Dpp > Tip60 RNAi (A) and Dpp > E(Pc) RNAi (B) flies. Scale bars = 50 μm. C-E: Representative image of wing discs stained for Cut and H2Av^ac from Dpp > Tip60 RNAi (C), Dpp > dom RNAi (D) and Dpp > E(Pc) RNAi (E) larvae. The expression region of Dpp-Gal4 is marked by GFP and outlined by dashed lines. Scale bars = 50 μm

Fig. 6

The Tip60 complex controls Notch signaling activity by acetylating H2Av. A-B: Representative image of wing discs stained for Cut from larvae overexpressing UAS-H2Av^5KR (A) and UAS-H2Av^5KQ (B). C-D: Representative image of wing discs stained for Cut from larvae expressing Tip60 RNAi along with UAS-H2Av^5KR (C) and UAS-H2Av^5KQ (D). E-F: Representative images of wing discs stained for Cut from larvae expressing dom RNAi along with UAS-H2Av^5KR (E) and UAS-H2Av^5KQ (F). The expression region of Dpp-Gal4 is marked by GFP and outlined by dashed lines. Scale bars = 50 μm

Fig. 7

The Tip60 complex regulates the expression of Su(H). A-B: Representative image of wing discs stained for Delta (A) and NICD (B) from Dpp > Tip60 RNAi larvae. C: Representative image of wing discs stained for NICD from Dpp > dom RNAi larvae. The expression region of Dpp-Gal4 is marked by GFP and outlined by dashed lines. D-E: Wing discs stained for Su(H) from en > Tip60 RNAi (D) and en > dom RNAi (E) larvae. The en-Gal4 expression region is marked by GFP and outlined by dashed lines. Scale bars = 50 μm

Fig. 8

The Tip60-H2Av module regulates Notch signaling pathway in a two-tier fashion. The Tip60-H2Av module plays a dual role in the Notch pathway: (i) In the Notch signal receiving cells, the Tip60 complex acetylates H2Av (H2Av^ac) and facilitates its integration into nucleosomes within the enhancer region of Notch target genes. H2Av^ac promotes an open chromatin state on the enhancer region of Notch targets, leading to increased transcription of target genes such as Cut, Wg, and E (spl)-complex. (ii) The Tip60-H2Av module promotes the expression of transcription factor Su(H), thereby further enhancing Notch signaling pathway activity