MAFB drives differentiation by permitting WT1 binding to podocyte specific promoters

Reviewer #1 (Public Review):

Summary:
In their manuscript, Massa and colleagues provide a map of the epigenetic landscape in podocytes and analyze the role of the transcription factor MafB in podocyte gene expression. They initially map the histone profile in adult podocytes of the mouse by assaying three different histone methylation marks, namely H3K4me3, H3K4me1, and H3K27me3 for active, primed, and repressed states. They then perform Wt1- and MafB-ChIP-Seq analysis to identify respective direct targets of those transcription factors. Subsequently, they employ an inducible MafB knockout model and show that homozygous knockout mice show proteinuria and FSGS, suggesting an important role for MafB in podocyte homeostasis. RNA-Seq analysis in mice two days after tamoxifen application identified direct and indirect MafB target genes. Finally, the authors turn to a constitutive MafB knockout model, carry out anti-H3K4me3 and anti-Wt1 ChIP experiments, and examine selected promoters. One main conclusion from this work is that MafB opens chromatin and thus facilitates the binding of other transcription factors like Wt1 to podocyte-specific genes.

Strengths and weaknesses:
The authors have performed an impressive number of experiments and generated very valuable data. They use state-of the-art technology and the data are presented well and are sound. This being said the manuscript contains significant novel data, but also experiments that are already available in some sort. The histone profile in adult mouse podocytes is novel and provides an interesting map of epigenetic marks in this particular cell type. It is maybe not too surprising that podocyte-differentiation genes have different chromatin accessibility than genes associated with general development. The Wt1-ChIP has been done before by several labs but is certainly an important control in this work. The MafB-ChIP is new. The inducible MafB knockout model including the identification of Tcf21 as a target gene has been published by others in 2020 (and is acknowledged by the authors). The experiments addressing the potential role of MafB in chromatin opening are new. I find that the data are certainly compatible with the model put forward by the authors, but they are not compelling.

Reviewer #2 (Public Review):

Summary:
The authors investigate the role of MafB in regulating podocyte genes. Mafb is required for podocyte differentiation and maintenance. Mutations of this gene cause FSGS in mice and humans. They profiled MafB binding genome-wide in isolated glomeruli and defined overlap with Wt1. They provide evidence that Mafb is required for Wt1 binding and H3K4me3 methylation at the promoters of two essential podocyte genes, Nphs1 and Nphs2. Understanding how the action of different transcription factors is coordinated to control gene expression - the main goal of this paper - is an important line of investigation.

While the main conclusion of the paper is supported by their data, the scope is limited. Additional ChIP-seq experiments and data analysis are needed to solidify and extend their conclusions.

Strengths:

1. Performing ChIP-seq for histone modifications on isolated podocytes provides valuable cell-type-specific information. Similarly, profiling Mafb and Wt1 in isolated glomeruli provides podocyte-specific binding patterns because these transcription factors (TFs) are not expressed in other cell types in glomeruli. The significant overlap of their Wt1 binding genome-wide with that of prior published work is reassuring. RNA-seq on isolated podocytes provides the appropriate cell-type specific gene expression data to integrate with ChIP-seq data. Together, the RNA-seq and ChIP-seq data are valuable resources for other investigators examining gene regulation in mouse podocytes.

2. The phenotype analysis of their FSGS model is convincing and well done.

3. Testing how Wt1 binding is affected by loss of Mafb provides insight into how these key podocyte TFs may cooperate to regulate genes.

Weaknesses:

1. The conclusion that Mafb is required for Wt1 binding and H3K4me3 methylation is based solely on ChIP-PCR at two gene promoters (Nphs1, Nphs2). This result should be validated and extended by ChIP-seq. Mafb and Wt1 binding overlap at more than 200 sites. If their model is correct, it is likely that Wt1 binding would be affected at other genomic sites. This result would add strong support to their model of how Wt1 and Mafb cooperate to regulate genes in podocytes. Moreover, ChIP-seq would define whether the dependence of Wt1 on Mafb is also evident at distal regulatory regions (defined H3K4me1, which is typically found at predicted enhancers).

2. The FSGS model generated by the authors involved conditional deletion of Mafb in podocytes at 8 weeks of age. They found that this resulted in reduced expression of Nphs1 and Nphs2 within 48 hours post-deletion. However, they investigated Wt1 binding and H3K4me3 genomic binding in Mafb homozygous null embryos. While this result provides information about podocyte differentiation, it does not address the maintenance of expression of these essential podocyte genes in the adult kidney. Because post-natal deletion of Mafb led to FSGS and reduced expression of Nphs1/2, ChIP-seq should be performed on the adult conditional mutants in order to provide mechanistic information about the disease.

3. H3K4me1 binds enhancer regions. The authors performed ChIP-seq to profile H3K4me1 in isolated podocytes. However, there was no analysis reported of these results. It would be valuable to determine if Wt1 and Mafb co-localize at predicted enhancers in podocytes and if Wt1 binding is lost at these regions in Mafb mutant glomeruli.

Author Response

eLife assessment

This study presents valuable insights into the epigenetic landscape in adult kidney podocytes. A series of solid experiments demonstrate that genes that are regulated by a key kidney transcription factor, Mafb, are essential for H3K4me3 methylation and recruitment of Wt1 to Nphs1 and Nphs2. This new information provides insights into the potential relationship and coordination of transcription factors in regulating target genes in podocytes in glomerular diseases, although the conclusion that MafB is generally required for Wt1 to bind to podocyte-specific promoters is incomplete and should be extended beyond two or three genes.

We thank the reviewers and editors for critically reading our manuscript and their insightful comments. We will strive to revise

Reviewer #1 (Public Review):

Summary:

In their manuscript, Massa and colleagues provide a map of the epigenetic landscape in podocytes and analyze the role of the transcription factor MafB in podocyte gene expression. They initially map the histone profile in adult podocytes of the mouse by assaying three different histone methylation marks, namely H3K4me3, H3K4me1, and H3K27me3 for active, primed, and repressed states. They then perform Wt1- and MafB-ChIP-Seq analysis to identify respective direct targets of those transcription factors. Subsequently, they employ an inducible MafB knockout model and show that homozygous knockout mice show proteinuria and FSGS, suggesting an important role for MafB in podocyte homeostasis. RNA-Seq analysis in mice two daysafter tamoxifen application identified direct and indirect MafB target genes. Finally, the authors turn to a constitutive MafB knockout model, carry out anti-H3K4me3 and anti-Wt1 ChIP experiments, and examine selected promoters. One main conclusion from this work is that MafB opens chromatin and thus facilitates the binding of other transcription factors like Wt1 to podocyte-specific genes.

Strengths and weaknesses:

The authors have performed an impressive number of experiments and generated very valuable data. They use state-of the-art technology and the data are presented well and are sound. This being said the manuscript contains significant novel data, but also experiments that are already available in some sort. The histone profile in adult mouse podocytes is novel and provides an interesting map of epigenetic marks in this particular cell type. It is maybe not too surprising that podocyte-differentiation genes have different chromatin accessibility than genes associated with general development. The Wt1-ChIP has been done before by several labs but is certainly an important control in this work. The MafB-ChIP is new. The inducible MafB knockout model including the identification of Tcf21 as a target gene has been published by others in 2020 (and is acknowledged by the authors). The experiments addressing the potential role of MafB in chromatin opening are new. I find that the data are certainly compatible with the model put forward by the authors, but they are not compelling.

We agree that additional data on changes in chromatin accessibility in the absence of Mafb would help to support our model and we will be working towards this data for a revised version of the manuscript.

Reviewer #2 (Public Review):

Summary:

The authors investigate the role of MafB in regulating podocyte genes. Mafb is required for podocyte differentiation and maintenance. Mutations of this gene cause FSGS in mice and humans. They profiled MafB binding genome-wide in isolated glomeruli and defined overlap with Wt1. They provide evidence that Mafb is required for Wt1 binding and H3K4me3 methylation at the promoters of two essential podocyte genes, Nphs1 and Nphs2 Understanding how the action of different transcription factors is coordinated to control gene expression - the main goal of this paper - is an important line of investigation.

While the main conclusion of the paper is supported by their data, the scope is limited. Additional ChIP-seq experiments and data analysis are needed to solidify and extend their conclusions.

Strengths:

1. Performing ChIP-seq for histone modifications on isolated podocytes provides valuable cell-type-specific information. Similarly, profiling Mafb and Wt1 in isolated glomeruli provides podocyte-specific binding patterns because these transcription factors (TFs) are not expressed in other cell types in glomeruli. The significant overlap of their Wt1 binding genome-wide withthat of prior published work is reassuring. RNA-seq on isolated podocytes provides the appropriate cell-type specific gene expression data to integrate with ChIP-seq data. Together, the RNA-seq and ChIP-seq data are valuable resources for other investigators examining gene regulation in mouse podocytes.

1. The phenotype analysis of their FSGS model is convincing and well done.

1. Testing how Wt1 binding is affected by loss of Mafb provides insight into how these key podocyte TFs may cooperate to regulate genes.

Weaknesses:

1. The conclusion that Mafb is required for Wt1 binding and H3K4me3 methylation is based solely on ChIP-PCR at two gene promoters (Nphs1, Nphs2). This result should be validated and extended by ChIP-seq. Mafb and Wt1 binding overlap at more than 200 sites. If their model is correct, it is likely that Wt1 binding would be affected at other genomic sites. This result would add strong support to their model of how Wt1 and Mafb cooperate to regulate genes in podocytes. Moreover, ChIP-seq would define whether the dependence of Wt1 on Mafb is also evident at distal regulatory regions (defined H3K4me1, which is typically found at predicted enhancers).

We agree that a genome wide analysis of chromatin accessibility would help corroborating our model and will work towards this data for a revised version.

1. The FSGS model generated by the authors involved conditional deletion of Mafb in podocytes at 8 weeks of age. They found that this resulted in reduced expression of Nphs1 and Nphs2 within 48 hours post-deletion. However, they investigated Wt1 binding and H3K4me3 genomic binding in Mafb homozygous null embryos. While this result provides information about podocyte differentiation, it does not address the maintenance of expression of these essential podocyte genes in the adult kidney. Because post-natal deletion of Mafb led to FSGS and reduced expression of Nphs1/2, ChIP-seq should be performed on the adult conditional mutants in order to provide mechanistic information about the disease.

The fact that the phenotype in Mafb conditional mutant animals is progressive means that epigenetic changes are also likely to be quantitative. Indeed, Nphs1/Nphs2 are still expressed 6 weeks after Mafb deletion, albeit at lower levels. Since ChIP-seq experiments are not necessarily quantitative, we believe it may be difficult to detect statistically significant changes in this model. We will discuss this limitation of our study in a revised version of our manuscript.

1. H3K4me1 binds enhancer regions. The authors performed ChIP-seq to profile H3K4me1 in isolated podocytes. However, there was no analysis reported of these results. It would be valuable to determine if Wt1 and Mafb co-localize at predicted enhancers in podocytes and if Wt1 binding is lost at these regions in Mafb mutant glomeruli.

We well reanalyse the data taking the reviewer’s comments into account.