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Rapid clearance of epigenetic protein reporters from wound edge cells in Drosophila larvae does not depend on the JNK or PDGFR/VEGFR signaling pathways.

Anderson AE, Galko MJ - Regeneration (Oxf) (2014)

Bottom Line: Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin.In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance.Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

ABSTRACT
The drastic cellular changes required for epidermal cells to dedifferentiate and become motile during wound closure are accompanied by changes in gene transcription, suggesting corresponding alterations in chromatin. However, the epigenetic changes that underlie wound-induced transcriptional programs remain poorly understood partly because a comprehensive study of epigenetic factor expression during wound healing has not been practical. To determine which chromatin modifying factors might contribute to wound healing, we screened publicly available fluorescently-tagged reporter lines in Drosophila for altered expression at the wound periphery during healing. Thirteen reporters tagging seven different proteins showed strongly diminished expression at the wound edge. Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin. In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance. Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

No MeSH data available.


Related in: MedlinePlus

Reporters for seven proteins are downregulated in the vicinity of the wound. (A)−(G′′′) Dissected epidermal whole mounts of larvae heterozygous for e22c‐Gal4, UAS‐DsRed2‐Nuc and the indicated reporter transgene were immunostained for Fasciclin III 4 h post‐wounding. For all genotypes, DsRed panels (A)−(G), (A′′)−(G′′), (A′′′)−(G′′′) show the location of epidermal nuclei while reporter panels (A′)−(G′), (A′′)−(G′′), (A′′′)−(G′′′) highlight all cells expressing the reporter. Fasciclin III staining (magenta, A′′′−G′′′) highlights epidermal cell membranes and the wound edge. Arrows indicate epidermal nuclei that have lost detectable reporter expression. Scale bar 200 μm.
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reg212-fig-0001: Reporters for seven proteins are downregulated in the vicinity of the wound. (A)−(G′′′) Dissected epidermal whole mounts of larvae heterozygous for e22c‐Gal4, UAS‐DsRed2‐Nuc and the indicated reporter transgene were immunostained for Fasciclin III 4 h post‐wounding. For all genotypes, DsRed panels (A)−(G), (A′′)−(G′′), (A′′′)−(G′′′) show the location of epidermal nuclei while reporter panels (A′)−(G′), (A′′)−(G′′), (A′′′)−(G′′′) highlight all cells expressing the reporter. Fasciclin III staining (magenta, A′′′−G′′′) highlights epidermal cell membranes and the wound edge. Arrows indicate epidermal nuclei that have lost detectable reporter expression. Scale bar 200 μm.

Mentions: We were interested in identifying epigenetic proteins that might regulate wound healing. We made use of existing protein trap resources to identify any reporter corresponding to a protein involved in epigenetic regulation that showed differential expression at the wound edge. We screened 54 independent GFP‐ or YFP‐ tagged protein trap lines from the Flytrap and Cambridge Protein Trap Insertion (CPTI) collections (Morin et al. 2001; Kelso et al. 2004; Buszczak et al. 2007; Quinones‐Coello et al. 2007; Ryder et al. 2009). The selected lines tag known or suspected proteins involved in epigenetic regulation, including proteins that directly interact with histones, proteins found in chromatin modifying complexes, and those that contain critical domains common to known epigenetic factors. Most of the reporters examined were expressed in multiple cell types, including the underlying muscles (for an example see Fig. 1D−D′′′). Therefore, to specifically identify epidermal nuclei, we crossed each reporter to an epidermal Gal4 (e22c‐Gal4) driving UAS‐DsRed2nuc (Lesch et al. 2010) and considered only nuclei expressing DsRed when evaluating reporter expression at the wound edge. For the initial screen we examined wounded larvae (Galko and Krasnow 2004; Lesch et al. 2010) 4 h post‐wounding as this is a time‐point when the wound edge cells are actively migrating (Wu et al. 2009) and transcriptional changes have been observed at the wound edge (Galko and Krasnow 2004; Lesch et al. 2010; Baek et al. 2012; Brock et al. 2012; Stevens and Page‐McCaw 2012). In the majority of reporter lines, the GFP or YFP signal in the epidermis was too weak for live microscopy. We therefore visualized reporter expression by immunostaining dissected whole mounts with an anti‐GFP antibody that recognizes both GFP and YFP. To label wound edge membranes, the epidermis was also stained with anti‐Fasciclin III. The majority of the reporters showed no obvious difference between distal and proximal nuclei, and none of the reporters screened showed increased expression at the wound edge (see Table S1 for a list of reporters without wound‐edge‐specific staining patterns, and Fig. S1 for a representative example). However, we observed a striking decrease in the expression of 13 reporters in wound‐proximal cells (see arrows in Figs. 1 and S2). These 13 reporters tag seven different proteins involved in epigenetic regulation: Osa, Sin3A, Sap130, Kismet, Mi‐2, Mip120, and Spt6 (Table 1). Two of the lines tag Osa, two tag Sin3A, and five tag Mi‐2. Interestingly, these reporters corresponded to chromatin remodeling proteins that are thought to be both activators (Osa, Spt6, Kismet) and repressors (Mi‐2, Sin3A, Sap130, and Mip120). Two additional lines with decreased expression were also identified (asterisks in Table S1), but because it is unclear whether they tag Mi‐2 or the overlapping Su(Tpl) protein, these lines were not included in subsequent analyses.


Rapid clearance of epigenetic protein reporters from wound edge cells in Drosophila larvae does not depend on the JNK or PDGFR/VEGFR signaling pathways.

Anderson AE, Galko MJ - Regeneration (Oxf) (2014)

Reporters for seven proteins are downregulated in the vicinity of the wound. (A)−(G′′′) Dissected epidermal whole mounts of larvae heterozygous for e22c‐Gal4, UAS‐DsRed2‐Nuc and the indicated reporter transgene were immunostained for Fasciclin III 4 h post‐wounding. For all genotypes, DsRed panels (A)−(G), (A′′)−(G′′), (A′′′)−(G′′′) show the location of epidermal nuclei while reporter panels (A′)−(G′), (A′′)−(G′′), (A′′′)−(G′′′) highlight all cells expressing the reporter. Fasciclin III staining (magenta, A′′′−G′′′) highlights epidermal cell membranes and the wound edge. Arrows indicate epidermal nuclei that have lost detectable reporter expression. Scale bar 200 μm.
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reg212-fig-0001: Reporters for seven proteins are downregulated in the vicinity of the wound. (A)−(G′′′) Dissected epidermal whole mounts of larvae heterozygous for e22c‐Gal4, UAS‐DsRed2‐Nuc and the indicated reporter transgene were immunostained for Fasciclin III 4 h post‐wounding. For all genotypes, DsRed panels (A)−(G), (A′′)−(G′′), (A′′′)−(G′′′) show the location of epidermal nuclei while reporter panels (A′)−(G′), (A′′)−(G′′), (A′′′)−(G′′′) highlight all cells expressing the reporter. Fasciclin III staining (magenta, A′′′−G′′′) highlights epidermal cell membranes and the wound edge. Arrows indicate epidermal nuclei that have lost detectable reporter expression. Scale bar 200 μm.
Mentions: We were interested in identifying epigenetic proteins that might regulate wound healing. We made use of existing protein trap resources to identify any reporter corresponding to a protein involved in epigenetic regulation that showed differential expression at the wound edge. We screened 54 independent GFP‐ or YFP‐ tagged protein trap lines from the Flytrap and Cambridge Protein Trap Insertion (CPTI) collections (Morin et al. 2001; Kelso et al. 2004; Buszczak et al. 2007; Quinones‐Coello et al. 2007; Ryder et al. 2009). The selected lines tag known or suspected proteins involved in epigenetic regulation, including proteins that directly interact with histones, proteins found in chromatin modifying complexes, and those that contain critical domains common to known epigenetic factors. Most of the reporters examined were expressed in multiple cell types, including the underlying muscles (for an example see Fig. 1D−D′′′). Therefore, to specifically identify epidermal nuclei, we crossed each reporter to an epidermal Gal4 (e22c‐Gal4) driving UAS‐DsRed2nuc (Lesch et al. 2010) and considered only nuclei expressing DsRed when evaluating reporter expression at the wound edge. For the initial screen we examined wounded larvae (Galko and Krasnow 2004; Lesch et al. 2010) 4 h post‐wounding as this is a time‐point when the wound edge cells are actively migrating (Wu et al. 2009) and transcriptional changes have been observed at the wound edge (Galko and Krasnow 2004; Lesch et al. 2010; Baek et al. 2012; Brock et al. 2012; Stevens and Page‐McCaw 2012). In the majority of reporter lines, the GFP or YFP signal in the epidermis was too weak for live microscopy. We therefore visualized reporter expression by immunostaining dissected whole mounts with an anti‐GFP antibody that recognizes both GFP and YFP. To label wound edge membranes, the epidermis was also stained with anti‐Fasciclin III. The majority of the reporters showed no obvious difference between distal and proximal nuclei, and none of the reporters screened showed increased expression at the wound edge (see Table S1 for a list of reporters without wound‐edge‐specific staining patterns, and Fig. S1 for a representative example). However, we observed a striking decrease in the expression of 13 reporters in wound‐proximal cells (see arrows in Figs. 1 and S2). These 13 reporters tag seven different proteins involved in epigenetic regulation: Osa, Sin3A, Sap130, Kismet, Mi‐2, Mip120, and Spt6 (Table 1). Two of the lines tag Osa, two tag Sin3A, and five tag Mi‐2. Interestingly, these reporters corresponded to chromatin remodeling proteins that are thought to be both activators (Osa, Spt6, Kismet) and repressors (Mi‐2, Sin3A, Sap130, and Mip120). Two additional lines with decreased expression were also identified (asterisks in Table S1), but because it is unclear whether they tag Mi‐2 or the overlapping Su(Tpl) protein, these lines were not included in subsequent analyses.

Bottom Line: Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin.In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance.Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The University of Texas MD Anderson Cancer Center, Unit 1000, 1515 Holcombe Boulevard, Houston, TX 77030, USA.

ABSTRACT
The drastic cellular changes required for epidermal cells to dedifferentiate and become motile during wound closure are accompanied by changes in gene transcription, suggesting corresponding alterations in chromatin. However, the epigenetic changes that underlie wound-induced transcriptional programs remain poorly understood partly because a comprehensive study of epigenetic factor expression during wound healing has not been practical. To determine which chromatin modifying factors might contribute to wound healing, we screened publicly available fluorescently-tagged reporter lines in Drosophila for altered expression at the wound periphery during healing. Thirteen reporters tagging seven different proteins showed strongly diminished expression at the wound edge. Three downregulated proteins, Osa, Kismet, and Spt6, are generally associated with active chromatin, while four others, Sin3A, Sap130, Mi-2, and Mip120, are associated with repressed chromatin. In all cases reporter down regulation was independent of the Jun N-terminal Kinase and Pvr pathways, suggesting that novel signals control reporter clearance. Taken together, our results suggest that clearance of chromatin modifying factors may enable wound edge cells to rapidly and comprehensively change their transcriptional state following tissue damage.

No MeSH data available.


Related in: MedlinePlus