Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease
Syamantak Majumder, … , Ferhan S. Siddiqi, Andrew Advani
Syamantak Majumder, … , Ferhan S. Siddiqi, Andrew Advani
Published December 11, 2017
Citation Information: J Clin Invest. 2018;128(1):483-499. https://doi.org/10.1172/JCI95946.
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Research Article Endocrinology Nephrology

Shifts in podocyte histone H3K27me3 regulate mouse and human glomerular disease

Abstract

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain–containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways.

Authors

Syamantak Majumder, Karina Thieme, Sri N. Batchu, Tamadher A. Alghamdi, Bridgit B. Bowskill, M. Golam Kabir, Youan Liu, Suzanne L. Advani, Kathryn E. White, Laurette Geldenhuys, Karthik K. Tennankore, Penelope Poyah, Ferhan S. Siddiqi, Andrew Advani

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Figure 2

Decreasing H3K27me3 levels promotes Notch-dependent podocyte dedifferentiation.

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Decreasing H3K27me3 levels promotes Notch-dependent podocyte dedifferent...
(A and B) Immunoblotting for H3K27me3 (n = 3) (A) and N1-ICD (n = 3) (B) in mouse podocytes exposed to 10 μM EPZ-6438 for 48 hours. (C) Immunofluorescence to detect N1-ICD in mouse podocytes treated with EPZ-6438, showing increased nuclear N1-ICD expression following EPZ-6438 treatment (n = 3). Original magnification, ×630. (D) mRNA levels of the Notch target gene Hey1 following EPZ-6438 treatment (n = 3). (E) Immunoblotting of cultured mouse podocytes for the cleaved NTM of Notch1 after knockdown of Notch1 with siRNA (n = 5). (F and G) mRNA levels of the mesenchymal marker α-SMA (F) and the podocyte marker podocin (G) in podocytes treated with EPZ-6438 for 72 hours (n = 12), with or without pretreatment with the γ secretase inhibitor DAPT (10 μM, n = 6) or knockdown of Notch1 with siRNA (n = 6). (H) Urine albumin/creatinine ratio in EZH2podKO (n = 6) mice injected with adriamycin (n = 4) and treated with DAPT (20 mg/kg i.p. every 3 days) (n = 3) for 10 days. (I) Jagged-1 mRNA levels in mouse podocytes treated with EPZ-6438 (n = 3). (J) ChIP for the Jag1 promoter following enrichment with an H3K27me3 antibody (n = 3). (K) Immunoblotting for Jagged-1 in mouse podocytes treated with EPZ-6438 (n = 3). (L) Immunofluorescence to detect the podocyte marker nephrin and Jagged-1 in glomeruli from a BALB/c mouse and from a BALB/c mouse treated with EPZ-6438 (100 mg/kg by daily gavage for 10 days) (vehicle, n = 5; EPZ-6438, n = 6). DAPI staining is shown in blue. Zoomed images are enlargements of the outlined areas. Original magnification, ×630. Values represent the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001, by 2-tailed Student’s t test (A, B, D, E, I, J, and K) and 1-way ANOVA followed by Fisher’s LSD post-hoc test (F–H).