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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

RNAi knockdown reduces expression of the Sap130, Mip120, and Kis reporters. (A)−(F′) Unwounded segments of dissected epidermal whole mounts of larvae heterozygous for an epidermal Gal4 driver (see Table 1), UAS‐DsRed2nuc, the indicated reporter, and the indicated UAS‐RNAi. Larvae were raised at 30°C. (A)−(A′) Sap130 reporter with UAS‐Sap130RNAi1. (B)−(B′) Sap130 reporter with w1118. (C)−(C′) Mip120 reporter with UAS‐Mip120RNAi. (D)−(D′) Mip120 reporter with w1118. (E)−(E′) Kis reporter with UAS‐KisRNAi. (F)−(F′) Kis reporter with w1118. Arrowheads, muscle nuclei that do not express dsRed2Nuc; arrows, epidermal nuclei; note the lack of GFP staining in (A)−(A′), (C)−(C′), and (E)−(E′) compared with controls. (G), (I) Quantification of reporter intensity in epidermal nuclei of indicated genotypes. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0005 (Student's t test). For the remaining reporter/RNAi combinations tested no statistically significant reduction in signal was observed. (J)−(J′′′′) Kis antibody staining (J, J′′, J′′′′) is reduced at the wound edge 4 h post‐wounding (J′′), and overlaps with Kis reporter staining (J). Scale bar 200 μm.
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reg212-fig-0002: RNAi knockdown reduces expression of the Sap130, Mip120, and Kis reporters. (A)−(F′) Unwounded segments of dissected epidermal whole mounts of larvae heterozygous for an epidermal Gal4 driver (see Table 1), UAS‐DsRed2nuc, the indicated reporter, and the indicated UAS‐RNAi. Larvae were raised at 30°C. (A)−(A′) Sap130 reporter with UAS‐Sap130RNAi1. (B)−(B′) Sap130 reporter with w1118. (C)−(C′) Mip120 reporter with UAS‐Mip120RNAi. (D)−(D′) Mip120 reporter with w1118. (E)−(E′) Kis reporter with UAS‐KisRNAi. (F)−(F′) Kis reporter with w1118. Arrowheads, muscle nuclei that do not express dsRed2Nuc; arrows, epidermal nuclei; note the lack of GFP staining in (A)−(A′), (C)−(C′), and (E)−(E′) compared with controls. (G), (I) Quantification of reporter intensity in epidermal nuclei of indicated genotypes. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0005 (Student's t test). For the remaining reporter/RNAi combinations tested no statistically significant reduction in signal was observed. (J)−(J′′′′) Kis antibody staining (J, J′′, J′′′′) is reduced at the wound edge 4 h post‐wounding (J′′), and overlaps with Kis reporter staining (J). Scale bar 200 μm.

Mentions: Next, we tested if the tagged proteins accurately reflect the expression of the native proteins. We raised larvae bearing a reporter, its corresponding RNAi transgene, and an epidermal Gal4 driver and UAS‐dsRed2Nuc, at 29°C to maximize epidermal RNAi expression. We then compared the expression level of each reporter in epidermal nuclei when its cognate gene was targeted (Fig. 2A−I). RNAi lines for Sap130 (Fig. 2A−C), Mip120 (Fig. 2D,F), and Kis (Fig. 2G,I) yielded a statistically significant reduction in reporter signal compared with controls. For the RNAi transgenes targeting the remaining genes, we did not see a statistically significant reduction in antibody signal. In these cases, we cannot distinguish between insufficient knockdown from the RNAi transgenes or inaccurate tagging of unintended proteins. Because of this, the localization and RNAi wound closure results for Mi‐2, Spt6, and Sin3A should be interpreted with caution pending development of additional reagents for verification of knockdown and/or localization.


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)

RNAi knockdown reduces expression of the Sap130, Mip120, and Kis reporters. (A)−(F′) Unwounded segments of dissected epidermal whole mounts of larvae heterozygous for an epidermal Gal4 driver (see Table 1), UAS‐DsRed2nuc, the indicated reporter, and the indicated UAS‐RNAi. Larvae were raised at 30°C. (A)−(A′) Sap130 reporter with UAS‐Sap130RNAi1. (B)−(B′) Sap130 reporter with w1118. (C)−(C′) Mip120 reporter with UAS‐Mip120RNAi. (D)−(D′) Mip120 reporter with w1118. (E)−(E′) Kis reporter with UAS‐KisRNAi. (F)−(F′) Kis reporter with w1118. Arrowheads, muscle nuclei that do not express dsRed2Nuc; arrows, epidermal nuclei; note the lack of GFP staining in (A)−(A′), (C)−(C′), and (E)−(E′) compared with controls. (G), (I) Quantification of reporter intensity in epidermal nuclei of indicated genotypes. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0005 (Student's t test). For the remaining reporter/RNAi combinations tested no statistically significant reduction in signal was observed. (J)−(J′′′′) Kis antibody staining (J, J′′, J′′′′) is reduced at the wound edge 4 h post‐wounding (J′′), and overlaps with Kis reporter staining (J). Scale bar 200 μm.
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reg212-fig-0002: RNAi knockdown reduces expression of the Sap130, Mip120, and Kis reporters. (A)−(F′) Unwounded segments of dissected epidermal whole mounts of larvae heterozygous for an epidermal Gal4 driver (see Table 1), UAS‐DsRed2nuc, the indicated reporter, and the indicated UAS‐RNAi. Larvae were raised at 30°C. (A)−(A′) Sap130 reporter with UAS‐Sap130RNAi1. (B)−(B′) Sap130 reporter with w1118. (C)−(C′) Mip120 reporter with UAS‐Mip120RNAi. (D)−(D′) Mip120 reporter with w1118. (E)−(E′) Kis reporter with UAS‐KisRNAi. (F)−(F′) Kis reporter with w1118. Arrowheads, muscle nuclei that do not express dsRed2Nuc; arrows, epidermal nuclei; note the lack of GFP staining in (A)−(A′), (C)−(C′), and (E)−(E′) compared with controls. (G), (I) Quantification of reporter intensity in epidermal nuclei of indicated genotypes. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0005 (Student's t test). For the remaining reporter/RNAi combinations tested no statistically significant reduction in signal was observed. (J)−(J′′′′) Kis antibody staining (J, J′′, J′′′′) is reduced at the wound edge 4 h post‐wounding (J′′), and overlaps with Kis reporter staining (J). Scale bar 200 μm.
Mentions: Next, we tested if the tagged proteins accurately reflect the expression of the native proteins. We raised larvae bearing a reporter, its corresponding RNAi transgene, and an epidermal Gal4 driver and UAS‐dsRed2Nuc, at 29°C to maximize epidermal RNAi expression. We then compared the expression level of each reporter in epidermal nuclei when its cognate gene was targeted (Fig. 2A−I). RNAi lines for Sap130 (Fig. 2A−C), Mip120 (Fig. 2D,F), and Kis (Fig. 2G,I) yielded a statistically significant reduction in reporter signal compared with controls. For the RNAi transgenes targeting the remaining genes, we did not see a statistically significant reduction in antibody signal. In these cases, we cannot distinguish between insufficient knockdown from the RNAi transgenes or inaccurate tagging of unintended proteins. Because of this, the localization and RNAi wound closure results for Mi‐2, Spt6, and Sin3A should be interpreted with caution pending development of additional reagents for verification of knockdown and/or localization.

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