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H4K20 di-methylation and multiciliogenesis. how a ubiquitous epigenetic mark affects a specific cell organelle
H4K20 di-methylation and multiciliogenesis. how a ubiquitous epigenetic mark affects a specific cell organelle
Epigentic information is preserved in different states of chromatin structure and accessibility. Covalent modifications of histones affect DNA compaction and can recruit further histone modifying enzymes. Among these post-translational modifications are different methylation states (mono-, di-, tri-methylation) on the lysine (K) residue at position 20 of histone 4 (H4K20). Previous studies from our laboratory in the larval model organism Xenopus laevis have shown that over-representation of H4K20 mono-methylation resulted in the embryos’ failure to create a directional fluid flow across the morphant epidermis, which normally is induced by metachronal beating of interspersed motile cilia tufts. A strong reduction in ciliary axonemes within multiciliated cells was found to be at the basis of this. Therefore, the intriguing question arose how H4K20me2, being an abundant and ubiquitous chromatin mark, could be crucial to the development of specific organelles such as motile cilia in a relatively selective manner. The present study further characterised the underlying ciliogenic defect in loss-offunction experiments by means of antisense morpholino oligonucleotide - mediated translational blockage of the respective histone methyl-transferases Suv4-20h1/-h2. This did not primarily affect lineage commitment and differentiation, but above all elicited altered epithelial morphology and cell-to-cell interaction. The shape and the capacity of MCCs to perform radial intercalation were compromised. On a sub-cellular level, the apical actin cytoskeleton was not properly established and while amplification of the centriole-like basal bodies still occured, their disengagement from perinuclear deuterosomes as well as their apical transport and docking were impeded. This phenotype could neither be rescued by overexpression of the ciliogenic master regulator multicilin, nor by overexpression of the transcription factor foxj1 which is known to promote axonemal outgrowth - suggesting an epistatic role of the Suv4-20h1/h2 enzymes affecting the deployment of cytoskeletal components. Complementary transcriptomic studies from our laboratory on morphant ectodermal explants confirmed the downregulation of a large-subset of cilia- and cytoskeleton-associated genes at neurula stage due to over-representation of H4K20 mono-methylation. Subsequently, partial reversibility of the ciliogenic phenotype by means of concomitant mRNA injections of the histone de-methylase Phf8, removing H4K20 mono-methyl, could be demonstrated. Taken together, the findings of the present study suggest that the cellcycle dependent placement of H4K20 di-methylation in G1/ G0 neutralizes the repressive role of the residue’s mono-methylation, thereby allowing the expression of the necessary cytoskeletal genes to perform basal body disengagement, apical transport and axonemal outgrowth.
Cilia, H4K20 methylation, epigenetics, Xenopus laevis
Berges, Julian
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Berges, Julian (2021): H4K20 di-methylation and multiciliogenesis: how a ubiquitous epigenetic mark affects a specific cell organelle. Dissertation, LMU München: Faculty of Medicine
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Abstract

Epigentic information is preserved in different states of chromatin structure and accessibility. Covalent modifications of histones affect DNA compaction and can recruit further histone modifying enzymes. Among these post-translational modifications are different methylation states (mono-, di-, tri-methylation) on the lysine (K) residue at position 20 of histone 4 (H4K20). Previous studies from our laboratory in the larval model organism Xenopus laevis have shown that over-representation of H4K20 mono-methylation resulted in the embryos’ failure to create a directional fluid flow across the morphant epidermis, which normally is induced by metachronal beating of interspersed motile cilia tufts. A strong reduction in ciliary axonemes within multiciliated cells was found to be at the basis of this. Therefore, the intriguing question arose how H4K20me2, being an abundant and ubiquitous chromatin mark, could be crucial to the development of specific organelles such as motile cilia in a relatively selective manner. The present study further characterised the underlying ciliogenic defect in loss-offunction experiments by means of antisense morpholino oligonucleotide - mediated translational blockage of the respective histone methyl-transferases Suv4-20h1/-h2. This did not primarily affect lineage commitment and differentiation, but above all elicited altered epithelial morphology and cell-to-cell interaction. The shape and the capacity of MCCs to perform radial intercalation were compromised. On a sub-cellular level, the apical actin cytoskeleton was not properly established and while amplification of the centriole-like basal bodies still occured, their disengagement from perinuclear deuterosomes as well as their apical transport and docking were impeded. This phenotype could neither be rescued by overexpression of the ciliogenic master regulator multicilin, nor by overexpression of the transcription factor foxj1 which is known to promote axonemal outgrowth - suggesting an epistatic role of the Suv4-20h1/h2 enzymes affecting the deployment of cytoskeletal components. Complementary transcriptomic studies from our laboratory on morphant ectodermal explants confirmed the downregulation of a large-subset of cilia- and cytoskeleton-associated genes at neurula stage due to over-representation of H4K20 mono-methylation. Subsequently, partial reversibility of the ciliogenic phenotype by means of concomitant mRNA injections of the histone de-methylase Phf8, removing H4K20 mono-methyl, could be demonstrated. Taken together, the findings of the present study suggest that the cellcycle dependent placement of H4K20 di-methylation in G1/ G0 neutralizes the repressive role of the residue’s mono-methylation, thereby allowing the expression of the necessary cytoskeletal genes to perform basal body disengagement, apical transport and axonemal outgrowth.