Beyond Transcription Factors: Remodeling Chromatin in the Metanephric Mesenchyme

During development, cell fate determination is governed not only by the nuclear repertoire of lineage-specific transcription factors (TFs) but also by the access of TFs to their cognate cis-regulatory elements (CREs). Chromatin accessibility is a dynamic process and is constrained by the presence of repeating units of nucleosomes composed chiefly of histone proteins tightly packaged to form condensed chromatin structure. To overcome this constraint, several chromatin-based mechanisms work in concert through a process called chromatin remodeling, altering the nucleosome architecture exposing or hiding the regulatory elements.¹ Two major mechanisms directly regulate access to nucleosomal DNA, namely covalent histone modifications and ATP-dependent chromatin remodeling. Histone modifications (e.g., lysine acetylation and methylation) alter the binding affinity between DNA and histones and form platforms for additional interacting chromatin complexes, thereby loosening or tightening the condensed nucleosome structure. In comparison, several families of ATP-dependent chromatin remodeling complexes, all possessing a common ATPase domain that catalyzes energy from hydrolysis of ATP, reposition (slide), eject, or exchange histones to create nucleosome-free DNA-exposed regions.

Recent studies that examined chromatin accessibility in mouse nephron progenitors in bulk or at single-cell resolution have revealed dynamic reorganization of chromatin states (closed versus open) anteceding commitment to the podocyte, proximal, or distal tubular fates.² Although the basic mechanisms whereby these chromatin states are established and maintained are not well defined, there is evidence that the balance between Trithorax (activating, open chromatin) and Polycomb (repressive, closed chromatin) complexes is crucial in maintenance of cell identity and fate of nephron progenitors.^3–5 However, because epigenetic regulators do not bind DNA directly, their gene-specific function must depend on interaction and corecruitment by pioneer and lineage-specific TFs.

In this issue of JASN, Li et al.⁶ provide biochemical and genetic evidence for the interactions of the master transcriptional regulators, Eya1 and Six2, with the chromatin remodeling SWI/SNF complex cotargeting gene enhancers driving nephron fate induction and maintenance. Eye absent (Eya) proteins act as transcriptional coactivators. Heterozygous EYA1 mutations in humans cause branchio-oto-renal syndrome; patients with branchio-oto-renal syndrome lack kidneys or have severely dysplastic kidneys. Heterozygous SIX1/2 mutations also cause renal hypodysplasia. Genetic studies have shown that Eya1 is upstream of Six2 in the metanephric mesenchyme (MM). However, Six2 binds to the Eya1 regulatory elements in nephron progenitors,⁷ and Eya1 and Six2 proteins interact; however, it is unclear how these interactions regulate the chromatin landscape and gene expression in MM maintenance and differentiation (Figure 1).

Figure 1.

A schematic summary of the manuscript by Li et al.⁶ depicting the functions of the Six2-Eya1/Brg1 complex in mouse MM and nephron progenitor cells. E, Embryonic.

To address these questions, Li et al.⁶ engineered mice with a multitagged Eya1^HA-Flag knock-in allele allowing the pull down and purification of Eya1-interacting proteins. Mass spectrometry of purified multitagged Eya1 identified Six2 and the BAF155/BAF60 subunits of the ATP-dependent chromatin remodeling complex SWI/SNF. Importantly, Eya1 and Six2 were found to physically interact with Brm-related gene-1 (Brg1), a component of the mammalian BAF complex and an ATPase that uses energy from ATP hydrolysis to alter chromatin structure and accessibility. Using a temporal deletion strategy (Eya1^CreERXBrg1^fl/fl), the authors showed a dramatic effect of Brg1 deletion on the maintenance and fate of the MM. Deletion of Brg1 in the E9.5–E10.5 MM caused bilateral renal agenesis due to massive apoptosis associated with loss of markers of the early MM, including Eya1 itself. As a result, there was a failure of ureteric bud outgrowth in the mutant mice. Deletion of Brg1 at E10.5–E12.5 resulted in a severe hypodysplastic kidney phenotype. The mutant kidneys exhibited depletion of nephron progenitors secondary to loss of proliferation potential and activation of apoptosis. Simultaneously, Brg1-mutant nephron progenitors underwent premature mesenchyme-to-epithelium transition, which the authors attributed to loss of Six2-mediated repression of Wnt4. To determine how Six2 might cooperate with Brg1 in transcriptional regulation, the investigators carried out ChIP-seq experiments in E13.5 kidneys and identified a group of Six2/Brg1 cobound enhancers that are directly relevant to regulation of nephron progenitor growth and differentiation. Subsequent ChIP-seq on the Six2^−/− mesenchyme further revealed that Six2 is required for Brg1 recruitment to a subset of Six2-target genes. To prove the functional requirement of the Six2/Brg1 complex in gene regulation in vivo, the investigators generated transgenic reporter mice driven by the Six2/Brg1-bound CREs in the MycN and Pbx1 genes. These CREs were sufficient to drive tissue- and cell type-specific expression in the MM, and mutations of the Six2-binding sites eliminated reporter gene expression. A distant Brg1-bound CRE in the Eya1 gene was also sufficient to drive tissue-specific reporter, albeit the CRE did not contain a canonical Six-responsive element.

On the basis of the findings of this study, the authors propose that Brg1 is essential for Eya1 and Six2 expression in the MM as well as for Six2 and Eya1 downstream transcriptional responses. Using a similar strategy, the same group of investigators recently demonstrated the critical importance of the Brg1/Eya complex in fate induction of the otic ectodrem. Along the same lines, Basta et al.⁸ reported that Sall1 (a TF mutated in Townes–Brook syndrome) also forms a complex with Brg1 and Six2 and that Brg1 is required for Sall1 function in nephron progenitor maintenance. Other studies have shown that Six2 and Sall1 interact with the NURD/HDAC chromatin remodeling complex to regulate nephron progenitor fate.^9,10 Taken together, a common theme that emerges from these studies is that master transcriptional regulators partner with histone modifiers and/or chromatin remodelers to regulate chromatin accessibility.

In summary, this study illustrates the power of combining mouse genetics and epigenetics in deciphering the gene regulatory networks governing kidney organogenesis. The rigor of this study includes generation of a multitagged Eya1 protein in transgenic mice to aid in purifying the Eya1-containing multiprotein complex, the use of temporally induced deletion of Brg1, the meticulous analysis of the kidney phenotype, the combined use of RNA/ChIP-seq to identify direct Brg1/Six2 target genes, and the use of enhancer-linked reporter transgenic mice to prove the functionality of the identified CREs in vivo. Because Brg1 regulates chromatin accessibility directly, it remains to be determined how loss of Brg1 might affect accessibility of the regulatory genome.

Disclosures

S.S. El-Dahr reports scientific advisor or membership with American Journal of Kidney Diseases, Kidney360, and Louisiana Children's Museum.

Funding

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grant RO1 DK114050.