(C) PLOS One This story was originally published by PLOS One and is unaltered. . . . . . . . . . . Chromatin remodeler Dmp18 regulates apoptosis by controlling H2Av incorporation in Drosophila imaginal disc development [1] ['Ying Feng', 'State Key Laboratory Of Optometry', 'Ophthalmology', 'Vision Science', 'School Of Optometry', 'Eye Hospital', 'Wenzhou Medical University', 'Wenzhou', 'Zhejiang', 'Yan Zhang'] Date: 2022-11 Programmed Cell Death (PCD) or apoptosis is a highly conserved biological process and plays essential roles both in the development and stress context. In Drosophila, expression of pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death. Here, we demonstrate that the chromatin remodeler Dmp18, the homolog of mammalian Znhit1, plays a crucial role in regulating apoptosis in eye and wing development. We showed that loss of Dmp18 disrupted eye and wing development, up-regulated transcription of pro-apoptotic genes, and induced apoptosis. Inhibition of apoptosis suppressed the eye defects caused by Dmp18 deletion. Furthermore, loss of Dmp18 disrupted H2Av incorporation into chromatin, promoted H3K4me3, but reduced H3K27me3 modifications on the TSS regions of pro-apoptotic genes. These results indicate that Dmp18 negatively regulates apoptosis by mediating H2Av incorporation and histone H3 modifications at pro-apoptotic gene loci for transcriptional regulation. Our study uncovers the role of Dmp18 in regulating apoptosis in Drosophila eye and wing development and provides insights into chromatin remodeling regulating apoptosis at the epigenetic levels. Apoptosis is an important biological process in the development and stress context by removing unwanted or damaged cells. Dysregulation of apoptosis causes many diseases including cancers. Transcriptional induction of pro-apoptotic genes is sufficient to induce apoptosis in Drosophila. Znhit1 encodes a Zinc finger HIT-type containing protein and works as a component of the chromatin remodeling complex in yeast and mammals. In the current study, we identified that Dmp18, the homolog of Znhit1, regulates apoptosis by mediating the histone variant H2Av incorporation and H3K4me3 as well as H3K27me3 modifications around the transcription start site (TSS) regions of pro-apoptotic genes. Dmp18 deletion up-regulated the transcription of pro-apoptotic genes including rpr and hid, triggered massive cell death, and resulted in eye and wing defects. Importantly, loss of Dmp18 disrupted H2Av incorporation into chromatin, increased H3K4me3, but decreased H3K27me3 modifications on the TSS regions of pro-apoptotic genes for transcriptional activation. Together, this study reveals the mechanism by which Dmp18 regulates apoptosis in the eye and wing discs. Funding: This work was supported by the National Natural Science Foundation of China ( http://www.nsfc.gov.cn/ ) (31900529) to Y.F., National Natural Science Foundation of China ( http://www.nsfc.gov.cn/ ) (31730044 and 32192403) to X.L., Zhejiang Provincial Natural Science Foundation of China ( http://zjnsf.kjt.zj.gov.cn/portal/index.html ) (LQ17C120001) to Y.F. and Zhejiang Provincial Natural Science Foundation of China ( http://zjnsf.kjt.zj.gov.cn/portal/index.html ) (LQ18C070001) to X.Y. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability: All relevant data are within the paper and its Supporting Information files. The raw NGS data were deposited to the NCBI SRA database under Bioproject ID: PRJNA761186: accession number from SRR15734269 to SRR15734277 (RNA-seq) and from SRR15734939 to SRR15734950 (CUT&Tag-seq). Copyright: © 2022 Feng et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The dynamic exchange of histone H2A with histone variant H2A.Z on chromatin is catalyzed by the ATP-dependent chromatin remodeling complex SWR1/SRCAP [ 40 , 41 ]. Znhit1 encodes a Zinc finger HIT-type containing protein and is conserved from yeast to mammals [ 42 , 43 ]. Previous studies have shown that Znhit1 works as a component of the SWR1/SRCAP complex and is involved in regulating the exchange of H2A for H2A.Z [ 42 , 44 – 46 ]. Deletion of Znhit1 in mice disrupts organ development and homeostasis maintenance [ 45 – 51 ]. However, the physiological functions of Znhit1 in other organisms remain poorly understood. Here, we used Drosophila as a model system and generated the Drosophila Znhit1 (referred to as Dmp18) mutant fly, Dmp18 d1 , to investigate its role in eye and wing development. Our study showed that Dmp18 regulates apoptosis by controlling the transcription of pro-apoptotic genes in the eye and wing discs. We further demonstrated that Dmp18 mediates the incorporation of histone variant H2Av into chromatin and the modifications of both H3K4me3 and H3K27me3 on the TSS regions of pro-apoptotic genes for transcriptional regulation. Thus, our study reveals the role of Dmp18 in regulating apoptosis at the epigenetic levels in the eye and wing discs. Drosophila only has a single H2A variant, H2Av, which belongs to the H2A.Z family and is necessary for animal survival [ 38 , 39 ]. Its C-terminal contains an SQAY motif similar to H2A.X, suggesting that Drosophila H2Av may serve the functions of mammalian both H2A.Z and H2A.X in transcriptional regulation and DNA damage response [ 39 ]. Mammals have multiple H2A variants, including H2A.Z and H2A.X. H2A.Z is considered the most universal variant and is highly conserved in eukaryotes. Deletion of H2A.Z in mice leads to embryonic death [ 15 ], suggesting an essential role of H2A.Z for embryonic development. It has been reported that H2A.Z is specifically deposited around the transcription start site (TSS) of active promoters and positively or negatively regulates gene expression [ 16 – 18 ]. In addition to transcriptional regulation, H2A.Z is also important for multiple chromatin-based processes including heterochromatin formation, DNA replication, nuclear reassembly, chromosome segregation, meiotic recombination initiation, and formation of higher-ordered chromosomal structures [ 19 – 28 ]. Recent studies have shown that H2A.Z is involved in the regulation of fear memory in mice [ 29 – 32 ] and tumorigenesis [ 33 – 35 ]. In addition to H2A.Z, H2A.X is an important component in response to DNA damage and has been shown to play important functions in biological processes such as cell division, stem cell functions, and aging [ 36 , 37 ]. Maintaining the balance between cell death, proliferation, and differentiation is needed for organogenesis and histogenesis in multicellular organisms. Programmed Cell Death (PCD) or apoptosis plays essential roles both in the development and stress context by removing unwanted or damaged cells to keep the balance [ 1 , 2 ]. Dysregulation of apoptosis induces a variety of diseases, including cancers, autoimmune diseases, and neurodegenerative diseases [ 3 – 5 ]. Thus, understanding the regulatory mechanisms of apoptotic process can provide insights into disease treatment and prevention [ 3 , 6 , 7 ]. In Drosophila, expression of the pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death [ 8 – 10 ], which active apoptosis by inhibiting the activity of Death-associated inhibitor of apoptosis protein 1 (DIAP1) [ 11 – 14 ]. Thus, understanding the underlying regulatory mechanisms of pro-apoptotic gene expression is important. Results Dmp18 regulates apoptosis by mediating H2Av incorporation Since Dmp18 deletion and H2Av deletion generate similar phenotype, we hypothesized that Dmp18 regulates apoptosis by controlling H2Av deposition. To test our hypothesis, we examined the H2Av deposition on the polytene chromosome in the homozygous Dmp18d1. As expected, loss of Dmp18 disrupted H2Av deposition on chromatin (Fig 7A). Surprisingly, the protein level of H2Av was dramatically reduced in the Dmp18 mutant clones in the eye and wing discs (Fig 7B) and whole mutant larvae (Fig 7C), suggesting that Dmp18 not only controls H2Av deposition on chromatin but also regulates its protein level. To rule out the possibility that the apoptosis triggered by Dmp18 deletion is due to the degradation of H2Av, we generated the transgenic fly (UAS-H2Av-myc) and performed the rescue experiment with an over-expression of H2Av-myc in the Dmp18 mutant clones to examine the Cas3* activity. The result showed that over-expression of H2Av-myc alone did not activate Cas3* (S14B–S14B”’ Fig), and over-expression of H2Av-myc in the Dmp18 mutant clones failed to inhibit Cas3* activity (Fig 7E, compared to Fig 7D). The expression of Hid and rpr still increased when H2Av-myc was over-expressed in the Dmp18 mutant clones (Fig 7G) or homozygous Dmp18d1 (Fig 7H). We further examined the deposition of H2Av-myc on the polytene chromosome in the wild-type and homozygous Dmp18d1. H2Av-myc driven by tub-gal4 was incorporated into chromatin and co-localized with endogenous H2Av well in the wild-type, but it could not be deposited on chromatin in the homozygous Dmp18d1 (Fig 7J). Together, these data argue that Dmp18 regulates apoptosis by mediating H2Av incorporation. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 7. Dmp18 regulates apoptosis by mediating H2Av incorporation. (A) Loss of Dmp18 disrupted incorporation of H2Av into chromatin. (B) The protein level of H2Av was reduced in the Dmp18 mutant clones in the eye and wing discs. GFP-free region marked the Dmp18 mutant clones. (C) The whole larvae were used for immunoblotting with the H2Av antibody. The protein level of H2Av was reduced in the homozygous Dmp18d1. The GAPDH worked as the loading control. (D) Loss of Dmp18 activated Cas3* in the eye and wing discs. (E) Over-expression of H2Av-myc did not inhibit Cas3* activity in the Dmp18 mutant clones. (F) Loss of Dmp18 up-regulated Hid expression. (G) Over-expression of H2Av-myc did not reduce the Hid expression in the Dmp18 mutant clones. D-G: GFP marked the Dmp18 mutant clones or Dmp18 mutant clones with over-expressed H2Av-myc. (H) The RT-qPCR analysis showed the transcription level of rpr in the control, homozygous Dmp18d1 and homozygous Dmp18d1 with over-expressed H2Av-myc. The rpr still showed high expression when H2Av-myc was over-expressed in the homozygous Dmp18d1. (I) The RT-qPCR analysis showed the transcription of H2Av in control, H2Av-myc and homozygous Dmp18d1 with over-expressed H2Av-myc. (J) H2Av-myc driven by tub-gal4 was deposited on chromatin and co-localized with endogenous H2Av well in the wild-type, but it failed to be deposited on chromatin in the homozygous Dmp18d1. *p<0.05 and ****p<0.0001. Genotypes: A: w1118 and FRT40A-Dmp18d1/FRT40A-Dmp18d1; B: yw,hs-FLP/+; FRT40A-M(2L)-Ubi-GFP/FRT40A-Dmp18d1; D and F: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; E and G: yw, hs-FLP, tub-Gal4, UAS-nls-GFP/+; tub-Gal80, neoFRT40A/FRT40A-Dmp18d1; UAS-H2Av-myc/+; J: tub-gal4/UAS-H2Av-myc and FRT40A-Dmp18d1/FRT40A-Dmp18d1; tub-gal4/UAS-H2Av-myc. https://doi.org/10.1371/journal.pgen.1010395.g007 Previously, it has been shown that H2Av is particularly enriched downstream of TSS in Drosophila genes to regulate transcription [61]. To determine whether Dmp18 regulates pro-apoptotic gene transcription by controlling H2Av incorporation into the genome, we isolated the eye and wing discs from w1118, Dmp18d1, and H2Av810, and performed the Cleavage Under Targets and Tagmentation (CUT&Tag) assay and sequencing. The result showed that the H2Av-binding peaks around the TSS of rpr were lower than that of hid in wild-type (Fig 8A and 8B, row 1). The binding of H2Av at downstream of hid TSS region was almost lost in the homozygous Dmp18d1 and H2Av810 (Fig 8B, row 2 and 3), but its binding around the TSS of rpr was not obviously changed (Fig 8A, row 2 and 3). These data indicate that Dmp18 regulates H2Av deposition on the TSS region of hid, but not rpr. PPT PowerPoint slide PNG larger image TIFF original image Download: Fig 8. Dmp18 mediates H2Av incorporation and histone H3 modifications at the TSS regions of pro-apoptotic genes for transcriptional regulation. (A and B) The CUT&Tag assay and sequencing were performed to examine the binding of H2Av (row 1 to 3), H3K4me3 (row 4 to 6), H3K27me3 (row 7 to 9), and H3K27ac (row 10 to 12) at the TSS regions of rpr and hid in w1118, homozygous Dmp18d1 and homozygous H2Av810. The binding of H2Av at the TSS region of rpr (A) was not changed, but its binding at hid (B) was reduced in Dmp18d1 and H2Av810 (green asterisks marked). The binding of H3K4me3 at the TSS regions of hid and rpr was increased in Dmp18d1 and H2Av810 (red asterisks marked). The binding of H3K27me3 at the TSS regions of hid and rpr was slightly reduced in Dmp18d1 and H2Av810 (green asterisks marked). The binding of H3K27ac at the TSS regions of rpr and hid was not obviously changed (black asterisks marked). (C) Venn diagram showed the overlap of H2Av-binding reduced genes, H3K4me3-binding increased genes, and Dmp18 up-regulated genes. (D) GO analysis of the overlapped genes in C. (E) The working model of Dmp18 regulating apoptosis in Drosophila: Dmp18 regulates apoptosis by mediating H2Av incorporation and histone H3 modifications at the TSS regions of pro-apoptotic genes for transcriptional regulation. For hid transcription, Dmp18 mediates the H2Av incorporation and both H3K4me3 and H3K27me3 modifications for transcriptional regulation. For rpr transcription, Dmp18 directly or indirectly mediates H3K3me4 and H3K27me3 modifications for transcriptional regulation. https://doi.org/10.1371/journal.pgen.1010395.g008 [END] --- [1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010395 Published and (C) by PLOS One Content appears here under this condition or license: Creative Commons - Attribution BY 4.0. via Magical.Fish Gopher News Feeds: gopher://magical.fish/1/feeds/news/plosone/