Defining the dynamic chromatin landscape of nephron progenitors

Six2+ cap mesenchyme cells, also called nephrons progenitor cells (NPC), are precursors of all epithelial cell types of the nephron, the filtering unit of the kidney. Current evidence indicates that perinatal “old” NPC have a greater tendency to exit the progenitor niche and differentiate into nascent nephrons than their embryonic “young” counterpart. Understanding the underpinnings of NPC aging may offer insights to rejuvenate old NPC and expand the progenitor pool. Here, we compared the chromatin landscape of young and old NPC and found common features reflecting their shared lineage but also intrinsic differences in chromatin accessibility and enhancer landscape supporting the view that old NPC are epigenetically poised for differentiation. Annotation of open chromatin regions and active enhancers uncovered the transcription factor Bach2 as a potential link between the pro-renewal MAPK/AP1 and pro-differentiation Six2/b-catenin pathways that might be of critical importance in regulation of NPC fate. Our data provide the first glimpse of the dynamic chromatin landscape of NPC and serve as a platform for future studies of the impact of genetic or environmental perturbations on the epigenome of NPC. Summary statement Hilliard et al. investigated the chromatin landscape of native Six2+ nephron progenitors across their lifespan. They identified age-dependent changes in accessible chromatin and regulatory regions supporting the view that old nephron progenitors are epigenetically poised for differentiation.


INTRODUCTION
Reciprocal interactions between the ureteric bud and surrounding NPC of the cap mesenchyme govern nephron induction. The cap mesenchyme is composed of an early progenitor Cited1 + /Six2 + compartment and a transit Cited1 -/Six2 + compartment that subsequently differentiate into the pretubular aggregate, the precursor of the renal vesicle, the earliest epithelial precursor of the nephron (Brown et al., 2013;Mugford et al., 2009). Careful morphometric studies and cell cycle analyses have shown that the proportion of NPC progressing through the cell cycle decreases with NPC aging, whereas the contribution of cell death is minimal, suggesting that all NPC exit occurs via differentiation into early nephrons (Short et al., 2014). Using genetic and primary cell culture models, Fgf9 and Bmp7 were shown to stimulate the MAPK pathway activation of Fos and Jun in the cap mesenchyme leading to the formation of the AP-1 heterodimer which stimulates cell cycle and growth factor genes contributing to the maintenance of the NPC population (Muthukrishnan et al., 2015). Although Fgf9 levels do not fall appreciably during NPC aging, Fgf20 levels do (Barak et al., 2012). It is therefore possible that reduced growth factor availability/activity in the niche is partly responsible for the short lifespan of NPC. However, there are also intrinsic differences between young and old NPC. For example, Six2 (and other stemness factors such as Wt1, Osr1 and Sall1) levels decline in postnatal NPC suggesting that these low levels cannot sustain NPC stemness in the face of elevated canonical Wnt signaling. A decline in the glycolytic capacity has also been shown by RNA-seq on postnatal NPC (Chen et al., 2015) as well as in primary young and old NPC (Liu et al., 2017). These changes translate into differences in cell behavior as demonstrated in the heterochronic transplantation studies (Chen et al., 2015): whereas young NPC tend to remain in the progenitor niche, old NPC exit and differentiate. The biological underpinnings of NPC aging, i.e., the greater tendency of perinatal NPC to differentiate compared to their embryonic counterpart, are not well understood.
Here, we compared the chromatin landscape of young and old NPC and find that dynamic chromatin accessibility to developmental enhancers is an intrinsic property of aging NPC.
Genome-wide ATAC-seq and ChIP-seq uncovered common and differentially accessible chromatin regions in young vs. old NPC reflecting their shared identity but also their maturational differences. Relative gain and loss of enhancer accessibility correlated with NPC gene expression and identified the poised epigenetic state of differentiation genes. While the open chromatin of young NPC is enriched in binding sites for the core NPC transcription factors (Six2, Wt1, Hoxa/c/d, Tead, AP1), old NPC gain chromatin accessibility to the Bach2/Batf complex, a repressor of AP1mediated transcriptional activation. Importantly, Bach2 is a component of the transcription factor code of the renal vesicle, the earliest precursor of the nephron, and is a genomic target of the Six2/b-catenin complex, whereas MAPK/AP1 is required to maintain the progenitor state of NPC. In summary, our data support the notion that dynamic changes in the NPC epigenome over their lifespan balance NPC proliferation and differentiation. We propose Bach2 as a potential molecular link between the MAPK/AP1 and Six2/b-catenin pathways.

RESULTS
Experimental protocol (Fig. 1) To compare the open chromatin landscape of young (embryonic) and old (perinatal) NPC, we applied the assay for transposase-accessible chromatin by highthroughput sequencing (ATAC-seq) to fluorescence-activated cell sorted (FACS) GFP + cells isolated from embryonic (E13, E16) and perinatal (P0, P2) Six2 GC mice (Kobayashi et al., 2008), which specifically express GFP under the Six2 regulatory elements in the cap mesenchyme. By gating fluorescence and forward scatter, we also obtained enriched populations of GFP high and GFP low NPC from newborn mice (referred to as P0-H and P0-L), representing undifferentiated and differentiating NPC, respectively. To reduce background, remove mitochondria from the transposition reaction, and increase the complexity of the library, we used the Omni-ATAC protocol (Corces et al., 2017) on 50,000 FACS-GFP + (n=3-4 biological replicates per age group). In another set of experiments, we applied magnetic-activated cell sorting (MACS) (Brown et al., 2013) to isolate the NPC from E13 and P0 CD-1 mice. To enrich for the self-renewing NPC population, E13 and P0 MACS-NPC were expanded in nephron progenitor expansion medium (NPEM) (Brown et al., 2013;Brown et al., 2015) for two passages then subjected to chromatin immunoprecipitation.

Assessment of NPC diversity
We applied droplet-based single-cell RNA sequencing to 10,524 GFP + cells isolated from E16 Six2 GC mice. Clustering analysis identified 13 distinct cell clusters varying from as few as 150 cells to as many as 1616 cells per cluster ( Fig. 2A). Cluster 2 and Clusters 6-8 were enriched in NPC expressing centrosome duplication, cell cycle and DNA replication genes (Fig. 2B). Cluster 12 contained fewer numbers of NPC (343 cells, 3%) and expressed early differentiation genes such as Wnt4, Pax8, Wfdc2 and Kdm2b (Fig. 2C). This was not surprising since lower levels of Six2(GFP) continue to be present in early nascent elements such as the pretubular aggregate and renal vesicle.
As discussed later in the text (Fig 7A), chromatin profiling of E13 and P0 NPC confirmed that the promoters of these early differentiation genes are bivalent (H3K4me1/H3K27me3) and therefore in a poised transcriptional state expressing low transcript levels. Cluster 13 is composed of NPC co-expressing stromal mesenchymal markers such as Lgals1, Maged2, and Gpc3 (Fig. 2D). A previously published study using scRNA-seq showed that individual NPC exhibit stochastic expression of stroma markers (Brunskill et al., 2014). These findings confirm that Six2 + NPC are composed of diverse cell populations some which are actively engaged in self-renewal while others are poised to differentiate.

The open chromatin landscape of NPC
ATAC-seq results were highly reproducible between biological replicates and showed a clear enrichment at the regulatory elements. As an example (

NPC aging is associated with differential chromatin accessibility
We compared the open chromatin regions of E16 and P2 NPC as well as of P0-H and P0-L NPC using DiffBind R (http://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf).
The affinity analysis is a quantitative approach to assess for differential chromatin access at consensus peaks. This method takes read densities computed over consensus peak

NPC chromatin profiles in genes implicated in renal function
We examined the ATAC signature of genes found in genome-wide association studies of estimated glomerular filtration rate and demonstrate preferential mapping of variants to regulatory regions in kidney but not extra-renal tissues (Pattaro et al., 2016).  5C).

The histone modifications landscape of pro-renewal and differentiation pathways
Given that the niche microenvironment (growth factor availability) may not be similar in native young vs. old NPC, we examined the epigenetic states of MACS-isolated E13 and P0 NPC grown in NPEM for two passages. The MACS isolation, which is performed on wild-type tissue, also avoids the Six2GFPCre transgene, in case it had a non-specific effect on the chromatin landscape. ChIP-seq analysis was then performed to map active (H3K4me1/H3K27ac), repressed (H3K27me3) and poised (H3K4me1/H3K27me3) enhancers. Fig S1 A-D depicts that pro-renewal pathway genes (Akt signaling, cell cycle, RTK signaling, and epigenetic regulators) are all decorated with active histone marks, the exception being cell cycle inhibitors which are occupied by broad regions of the repressive mark H3K27me3 (Fig S1B). In comparison, Fig. 6A shows that genes required for mesenchyme-to-epithelium transition are occupied by bivalent chromatin regions (H3K4me1/K27me3) indicating they are silent but poised for transcription. Furthermore, poised differentiation genes showed a decrease in occupancy in the repressive H3K27me3 mark and a reciprocal gain of H3K27ac with NPC aging (Fig.  6 A,B). Finally, non-lineage genes, e.g., pro-neural developmental genes, are occupied by broad domains of H3K27me3 which serve to restrain their expression (Fig. 6C).
Collectively, these results indicate that young and old NPC exhibit intrinsic differences in chromatin accessibility and histone modifications at annotated and putative promoters and enhancers.

Chromatin profiling links the transcription factor Bach2 with NPC aging
We used ATAC-seq to identify transcription factor motifs within the accessible  Liu et al., 2018;McLaughlin et al., 2014).
While these studies were informative, additional work was needed to clarify whether the dynamic chromatin landscape observed in metanephric mesenchyme cell lines applies to native NPC.
In the present study, using native freshly isolated NPC across their lifespan, we demonstrate that as the NPC age in the niche, chromatin accessibility to the regulatory regions of poised differentiation genes increases, likely preparing these gene networks for activation of the epithelial nephrogenesis program. These changes are not simply the results of changing proportions of the self-renewing and differentiating NPC populations but likely intrinsic, since young and old NPC grown in the same growth factor expansion medium also show significant differences in their chromatin enhancer landscape. We speculate that these maturational changes in chromatin accessibility are likely orchestrated by concomitant changes in the epigenetic machinery such as the Polycomb complex, ATP-dependent chromatin remodelers, DNA methylation and the NuRD/HDAC complex, that govern the access of master regulators to their cis-acting elements. Indeed, there is genetic evidence that perturbations of the epigenetic machinery disrupt the balance between NPC proliferation and differentiation in vivo (Denner and Rauchman, 2013;El-Dahr and Saifudeen, 2018;Liu et al., 2018;Zhang et al., 2018).
A new finding of this study is that NPC aging is associated with enhanced accessibility at the Bach2/Batf occupancy sites. As mentioned earlier in the text, Bach2 was identified as part of the transcription factor signature of the distal renal vesicle, a compartment that receives high levels of Wnt9b/ß-catenin signaling (Brunskill et al., 2014). This leads us to the following working model (Fig. 8D). In young NPC, Six2/corepressor complexes inhibit Bach2 transcription. With further activation of Wnt/ß-catenin signaling and concomitant decline in Six2 levels, the Six2/TCF repressor is replaced by ß-catenin/TCF/co-activator complex leading to induction of Bach2 transcription. The Bach2/Batf complex subsequently displaces the AP-1 complex inhibiting expression of AP1 targets (e.g., cell cycle genes). It is also conceivable that Bach2 targets differentiation genes to activate them. To our knowledge, studies of renal development have not been reported in Bach2-or Bach1/Bach2-deficient mice. Future investigations of Bach2 function in nephrogenesis and delineation of Bach2-target genes will shed light on some of these gene regulatory networks. If indeed Bach2 links the proliferation and differentiation pathways in the NPC, targeting Bach2 may be a useful tool to manipulate the fate and lifespan of the NPC in renal regenerative medicine.

Isolation of NPC
NPC were isolated from E13.5, E16.5, P0 and P2 CD1 mice or Six2GFPCre mice by magnetic-activated cell sorting (MACS) (Brown et al., 2015) or fluorescent-activated cell sorting (FACS). Animal protocols utilized in this study were approved by and in strict adherence to guidelines established by the Tulane University Institutional Animal Care and Use Committee.

ATAC-seq
For sample library preparation we followed the Omni-ATAC method outlined by (Buenrostro et al., 2015;Corces et al., 2017). Briefly 50,000 nuclei from FACS-sorted cells were processed for Tn5 transposase-mediated tagmentation and adaptor incorporation at sites of accessible chromatin. This reaction was carried out using the  Table S2. Peaks sets generated with peak calling analysis were analyzed using DiffBind R/Bioconductor package.

Acknowledgments
The Authors are grateful to Kejing Song (Tulane Genomic and epigenomic and sequencing cores), and the laboratory of Dr. Mazhar Adli (University of Virginia) for help in the early phase of the study.

Competing interests
No competing interests declared.

Funding
This work was supported by NIH grant DK114500 (SED) and the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program (JKK).

Data availability
The files have been deposited in NCBI GSE124804.                   Suppl. Fig. 2