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Methylomic correlates of autophagy activity in cystic fibrosis.

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society (2019-02-10)
Kyle Caution, Alexander Pan, Kathrin Krause, Asmaa Badr, Kaitlin Hamilton, Anup Vaidya, Hawin Gosu, Kylene Daily, Shady Estfanous, Mikhail A Gavrilin, Mark E Drew, Estelle Cormet-Boyaka, Xi Chen, David E Frankhouser, Ralf Bundschuh, Pearlly Yan, Duaa Dakhlallah, Amal O Amer
ABSTRACT

Autophagy is a highly regulated, biological process that provides energy during periods of stress and starvation. This conserved process also acts as a defense mechanism and clears microbes from the host cell. Autophagy is impaired in Cystic Fibrosis (CF) patients and CF mice, as their cells exhibit low expression levels of essential autophagy molecules. The genetic disorder in CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for a chloride channel. CF patients are particularly prone to infection by pathogens that are otherwise cleared by autophagy in healthy immune cells including Burkholderia cenocepacia (B. cenocepacia). The objective of this study is to determine the mechanism underlying weak autophagic activity in CF macrophages and find therapeutic targets to correct it. Using reduced representation bisulfite sequencing (RRBS) to determine DNA methylation profile, we found that the promoter regions of Atg12 in CF macrophages are significantly more methylated than in the wild-type (WT) immune cells, accompanied by low protein expression. The natural product epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. Accordingly, EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy and preventing dissemination. In addition, EGCG improved the function of CFTR protein. Altogether, utilizing RRBS for the first time in the CF field revealed a previously unrecognized mechanism for reduced autophagic activity in CF. Our data also offers a mechanism by which EGCG exerts its positive effects in CF.

MATERIALS
Product Number
Brand
Product Description

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5-Aza-2′-deoxycytidine, ≥97%
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(−)-Epigallocatechin gallate, ≥95%
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Rapamycin from Streptomyces hygroscopicus, ≥95% (HPLC), powder
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(−)-Epigallocatechin, ≥95% (HPLC), from green tea
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(−)-Epicatechin gallate, ≥98% (HPLC), from green tea
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3-Methyladenine, autophagy inhibitor
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Anti-ATG5 (N-terminal) antibody produced in rabbit, affinity isolated antibody, PBS solution