Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury.

In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in reparative responses to injury are not well understood. We have studied the mechanisms of transcriptional reprogramming in disease using murine kidney podocytes as a model for tissue injury. Podocytes are a crucial component of glomeruli, the filtration units of each nephron. Podocyte injury is the initial event in many processes that lead to end-stage kidney disease. Wilms tumor-1 (WT1) is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Using murine models and human kidney organoids, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Reprogramming the transcriptome involved highly dynamic changes in the binding of WT1 to target genes during a reparative injury response, affecting chromatin state and expression levels of target genes.

EED, a member of the polycomb group, is required for nephron differentiation and the maintenance of nephron progenitor cells.

Epigenetic regulation of gene expression has a crucial role allowing for the self-renewal and differentiation of stem and progenitor populations during organogenesis. The mammalian kidney maintains a population of self-renewing stem cells that differentiate to give rise to thousands of nephrons, which are the functional units that carry out filtration to maintain physiological homeostasis. The polycomb repressive complex 2 (PRC2) epigenetically represses gene expression during development by placing the H3K27me3 mark on histone H3 at promoter and enhancer sites, resulting in gene silencing. To understand the role of PRC2 in nephron differentiation, we conditionally inactivated the Eed gene, which encodes a nonredundant component of the PRC2 complex, in nephron progenitor cells. Resultant kidneys were smaller and showed premature loss of progenitor cells. The progenitors in Eed mutant mice that were induced to differentiate did not develop into properly formed nephrons. Lhx1, normally expressed in the renal vesicle, was overexpressed in kidneys of Eed mutant mice. Thus, PRC2 has a crucial role in suppressing the expression of genes that maintain the progenitor state, allowing nephron differentiation to proceed.

Genome-Wide Analysis of Wilms' Tumor 1-Controlled Gene Expression in Podocytes Reveals Key Regulatory Mechanisms.

The transcription factor Wilms' tumor suppressor 1 (WT1) is key to podocyte development and viability; however, WT1 transcriptional networks in podocytes remain elusive. We provide a comprehensive analysis of the genome-wide WT1 transcriptional network in podocytes in vivo using chromatin immunoprecipitation followed by sequencing (ChIPseq) and RNA sequencing techniques. Our data show a specific role for WT1 in regulating the podocyte-specific transcriptome through binding to both promoters and enhancers of target genes. Furthermore, we inferred a podocyte transcription factor network consisting of WT1, LMX1B, TCF21, Fox-class and TEAD family transcription factors, and MAFB that uses tissue-specific enhancers to control podocyte gene expression. In addition to previously described WT1-dependent target genes, ChIPseq identified novel WT1-dependent signaling systems. These targets included components of the Hippo signaling system, underscoring the power of genome-wide transcriptional-network analyses. Together, our data elucidate a comprehensive gene regulatory network in podocytes suggesting that WT1 gene regulatory function and podocyte cell-type specification can best be understood in the context of transcription factor-regulatory element network interplay.

Epigenetic control of NF-κB-dependent FAS gene transcription during progression of myelodysplastic syndromes.

The death domain containing TNF receptor 6 (CD95/Fas) is a direct target for the NF-κB transcription factor and is repressed in solid tumors such as colon carcinomas. Previously, we reported that the Fas death receptor, while overexpressed in low-risk myelodysplastic syndromes (MDS), becomes undetectable on CD34(+) progenitors when the disease progresses to secondary acute myeloid leukemia (AML). This study determined the interplay between NF-κB and Fas during MDS progression. We first observed that Fas was induced by TNF-α in the HL60 cell line. In these cells, p65 (RELA) was associated with the FAS promoter, and inhibition of the NF-κB pathway by an IKKα inhibitor (BAY11-7082) or lentiviral expression of a nondegradable mutant of IκBα (IκSR) blocked Fas expression. In contrast, TNF-α failed to induce Fas expression in the colon carcinoma cell line SW480, due to hypermethylation of the FAS promoter. Azacitidine rescued p65 binding on FAS promoter in vitro, and subsequently Fas expression in SW480 cells. Furthermore, inhibition of the NF-κB pathway decreased the expression of Fas in MDS CD45(lo)CD34(+) bone marrow cells. However, despite the nuclear expression of p65, Fas was often low on CD45(lo)CD34(+) AML cells. TNF-α failed to stimulate its expression, while azacitidine efficiently rescued p65 binding and Fas reexpression. Overall, these data suggest that DNA methylation at NF-κB sites is responsible for FAS gene silencing.