Chromatin Remodelers Interact with Eya1 and Six2 to Target Enhancers to Control Nephron Progenitor Cell Maintenance

BackgroundEya1 is a critical regulator of nephron progenitor cell specification and interacts with Six2 to promote NPC self-renewal. Haploinsufficiency of these genes causes kidney hypoplasia. However, how the Eya1-centered network operates remains unknown.MethodsWe engineered a 2×HA-3×Flag-Eya1 knock-in mouse line and performed coimmunoprecipitation with anti-HA or -Flag to precipitate the multitagged-Eya1 and its associated proteins. Loss-of-function, transcriptome profiling, and genome-wide binding analyses for Eya1's interacting chromatin-remodeling ATPase Brg1 were carried out. We assayed the activity of the cis-regulatory elements co-occupied by Brg1/Six2 in vivo.ResultsEya1 and Six2 interact with the Brg1-based SWI/SNF complex during kidney development. Knockout of Brg1 results in failure of metanephric mesenchyme formation and depletion of nephron progenitors, which has been linked to loss of Eya1 expression. Transcriptional profiling shows conspicuous downregulation of important regulators for nephrogenesis in Brg1-deficient cells, including Lin28, Pbx1, and Dchs1-Fat4 signaling, but upregulation of podocyte lineage, oncogenic, and cell death–inducing genes, many of which Brg1 targets. Genome-wide binding analysis identifies Brg1 occupancy to a distal enhancer of Eya1 that drives nephron progenitor–specific expression. We demonstrate that Brg1 enrichment to two distal intronic enhancers of Pbx1 and a proximal promoter region of Mycn requires Six2 activity and that these Brg1/Six2-bound enhancers govern nephron progenitor–specific expression in response to Six2 activity.ConclusionsOur results reveal an essential role for Brg1, its downstream pathways, and its interaction with Eya1-Six2 in mediating the fine balance among the self-renewal, differentiation, and survival of nephron progenitors.

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Nephron progenitor commitment is a stochastic process influenced by cell migration

eLife ◽

10.7554/elife.41156 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 12

Author(s):

Kynan T Lawlor ◽

Luke Zappia ◽

James Lefevre ◽

Joo-Seop Park ◽

Nicholas A Hamilton ◽

...

Keyword(s):

Cell Migration ◽

Progenitor Cell ◽

Computational Modelling ◽

Rna Seq ◽

Self Renewal ◽

Tissue Microenvironment ◽

Nephron Progenitors ◽

Nephron Progenitor ◽

Robust Regulation ◽

Over Time

Progenitor self-renewal and differentiation is often regulated by spatially restricted cues within a tissue microenvironment. Here, we examine how progenitor cell migration impacts regionally induced commitment within the nephrogenic niche in mice. We identify a subset of cells that express Wnt4, an early marker of nephron commitment, but migrate back into the progenitor population where they accumulate over time. Single cell RNA-seq and computational modelling of returning cells reveals that nephron progenitors can traverse the transcriptional hierarchy between self-renewal and commitment in either direction. This plasticity may enable robust regulation of nephrogenesis as niches remodel and grow during organogenesis.

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Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment

eLife ◽

10.7554/elife.40392 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 11

Author(s):

Lori L O'Brien ◽

Alexander N Combes ◽

Kieran M Short ◽

Nils O Lindström ◽

Peter H Whitney ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cell ◽

Live Imaging ◽

Rna Seq ◽

Self Renewal ◽

Mammalian Kidney ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Cell Organization ◽

Nephron Progenitor

A normal endowment of nephrons in the mammalian kidney requires a balance of nephron progenitor self-renewal and differentiation throughout development. Here, we provide evidence for a novel action of ureteric branch tip-derived Wnt11 in progenitor cell organization and interactions within the nephrogenic niche, ultimately determining nephron endowment. In Wnt11 mutants, nephron progenitors dispersed from their restricted niche, intermixing with interstitial progenitors. Nephron progenitor differentiation was accelerated, kidneys were significantly smaller, and the nephron progenitor pool was prematurely exhausted, halving the final nephron count. Interestingly, RNA-seq revealed no significant differences in gene expression. Live imaging of nephron progenitors showed that in the absence of Wnt11 they lose stable attachments to the ureteric branch tips, continuously detaching and reattaching. Further, the polarized distribution of several markers within nephron progenitors is disrupted. Together these data highlight the importance of Wnt11 signaling in directing nephron progenitor behavior which determines a normal nephrogenic program.

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Chromatin accessibility and microRNA expression in nephron progenitor cells during kidney development

10.1101/2021.03.05.434138 ◽

2021 ◽

Author(s):

Andrew Clugston ◽

Andrew Bodnar ◽

Débora Malta Cerqueira ◽

Yu Leng Phua ◽

Alyssa Lawler ◽

...

Keyword(s):

Progenitor Cells ◽

Kidney Development ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Microrna Expression ◽

Genome Wide ◽

Extracellular Matrix Interactions ◽

Genomic Regions ◽

Nephron Progenitor ◽

Accessible Chromatin

AbstractMammalian nephrons originate from a population of nephron progenitor cells (NPCs), and it is known that NPCs’ transcriptomes change throughout nephrogenesis during healthy kidney development. To characterize chromatin accessibility and microRNA (miRNA) expression throughout this process, we collected NPCs from mouse kidneys at embryonic day 14.5 (E14.5) and postnatal day zero (P0) and assayed cells for transposase-accessible chromatin and small RNA expression. We observe 46,374 genomic regions of accessible chromatin, with 2,103 showing significant changes in accessibility between E14.5 and P0. In addition, we detect 1,104 known microRNAs, with 114 showing significant changes in expression. Genome-wide, changes in DNA accessibility and microRNA expression highlight biological processes like cellular differentiation, cell migration, extracellular matrix interactions, and developmental signaling pathways such as Notch. Furthermore, our data identify novel candidate cis-regulatory elements for Eya1 and Pax8, both genes with a role in NPC differentiation; we also associate expression-changing microRNAs, including let-7-5p, miR-125b-5p, miR-181a-2-3p, and miR-9-3p, with candidate cis-regulatory elements. Overall, our data characterize NPCs during kidney development and point out new candidate regulatory elements for genes and microRNA with key roles in nephrogenesis.

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Genome-Wide Regulatory Interactions in the Early Stages of Drosophila Speciation

10.36939/ir.202012151547 ◽

2020 ◽

Author(s):

◽

Alwyn Clark Go

Keyword(s):

Reproductive Isolation ◽

Expression Analysis ◽

Regulatory Elements ◽

Specific Expression ◽

Allele Specific Expression ◽

Regulatory Interactions ◽

Early Stages ◽

Genome Wide ◽

Allele Specific ◽

Incomplete Reproductive Isolation

Speciation occurs when reproductive barriers prevent the exchange of genetic information between individuals. A common form of reproductive barrier between species capable of interbreeding is hybrid sterility. Genomic incompatibilities between the divergent genomes of different species contribute to a reduction in hybrid fitness. These incompatibilities continue to accumulate after speciation, therefore, young divergent taxa with incomplete reproductive isolation are important in understating the genetics leading to speciation. Here, I use two Drosophila subspecies pairs. The first is D. willistoni consisting of D. w. willistoni and D. w. winge. The second subspecies pair is D. pseudoobscura, which is composed of D. p. pseudoobscura and D. p. bogotana. Both subspecies pairs are at the early stages of speciation and show incomplete reproductive isolation through unidirectional hybrid male sterility. In this thesis, I performed an exploratory survey of genome-wide expression analysis using RNA-sequencing on D. willistoni and determined the extent of regulatory divergence between the subspecies using allele-specific expression analysis. I found that misexpressed genes showed a degree of tissue specificity and that the sterile male hybrids had a higher proportion of misexpressed genes in the testes relative to the fertile hybrids. The analysis of regulatory divergence between this subspecies pair found a large (66-70%) proportion of genes with conserved regulatory elements. Of the genes showing evidence or regulatory divergence between subspecies, cis-regulatory divergence was more common than other types. In the D. pseudoobscura subspecies pair, I compared sequence and expression divergence and found no support for directional selection driving gene misexpression in their hybrids. Allele-specific expression analysis revealed that compensatory cis-trans mutations partly explained gene misexpression in the hybrids. The remaining hybrid misexpression occurs due to interacting gene networks or possible co-option of cis-regulatory elements by divergent transacting factors. Overall, the results of this thesis highlight the role of regulatory interactions in a hybrid genome and how these interactions could lead to hybrid breakdown by disrupting gene interaction networks.

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Genomic characterization of the adolescent idiopathic scoliosis associated transcriptome and regulome

10.1101/2020.03.02.973735 ◽

2020 ◽

Author(s):

Nadja Makki ◽

Jingjing Zhao ◽

Zhaoyang Liu ◽

Walter L. Eckalbar ◽

Aki Ushiki ◽

...

Keyword(s):

Adolescent Idiopathic Scoliosis ◽

Connective Tissue ◽

Idiopathic Scoliosis ◽

Association Studies ◽

Regulatory Elements ◽

Intervertebral Discs ◽

Genome Wide Association Studies ◽

Specific Expression ◽

Enhancer Activity ◽

Genome Wide

AbstractAdolescent idiopathic scoliosis (AIS), a sideways curvature of the spine, is the most common pediatric musculoskeletal disorder, affecting ∼3% of the population worldwide. However, its genetic bases and tissues of origin remain largely unknown. Several genome-wide association studies (GWAS) have implicated nucleotide variants in noncoding sequences that control genes with important roles in cartilage, muscle, bone, connective tissue and intervertebral discs (IVDs) as drivers of AIS susceptibility. Here, we set out to define the expression of AIS-associated genes and active regulatory elements by performing RNA-seq and ChIP-seq against H3K27ac in these tissues in mouse and human. Our study highlights genetic pathways involving AIS-associated loci that regulate chondrogenesis, IVD development and connective tissue maintenance and homeostasis. In addition, we identify thousands of putative AIS-associated regulatory elements which may orchestrate tissue-specific expression in musculoskeletal tissues of the spine. Quantification of enhancer activity of several candidate regulatory elements from our study identifies three functional enhancers carrying AIS-associated GWAS SNPs at the ADGRG6 and BNC2 loci. Our findings provide a novel genome-wide catalog of AIS-relevant genes and regulatory elements and aid in the identification of novel targets for AIS causality and treatment.

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Von Hippel-Lindau Acts as a Metabolic Switch Controlling Nephron Progenitor Differentiation

Journal of the American Society of Nephrology ◽

10.1681/asn.2018111170 ◽

2019 ◽

Vol 30 (7) ◽

pp. 1192-1205 ◽

Cited By ~ 3

Author(s):

Kasey Cargill ◽

Shelby L. Hemker ◽

Andrew Clugston ◽

Anjana Murali ◽

Elina Mukherjee ◽

...

Keyword(s):

Mitochondrial Respiration ◽

Metabolic Regulation ◽

Kidney Development ◽

Developmentally Delayed ◽

Metabolic Switch ◽

Von Hippel Lindau ◽

Conditional Deletion ◽

Nephron Progenitors ◽

Ubiquitin Ligase Complex ◽

Nephron Progenitor

BackgroundNephron progenitors, the cell population that give rise to the functional unit of the kidney, are metabolically active and self-renew under glycolytic conditions. A switch from glycolysis to mitochondrial respiration drives these cells toward differentiation, but the mechanisms that control this switch are poorly defined. Studies have demonstrated that kidney formation is highly dependent on oxygen concentration, which is largely regulated by von Hippel-Lindau (VHL; a protein component of a ubiquitin ligase complex) and hypoxia-inducible factors (a family of transcription factors activated by hypoxia).MethodsTo explore VHL as a regulator defining nephron progenitor self-renewal versus differentiation, we bred Six2-TGCtg mice with VHLlox/lox mice to generate mice with a conditional deletion of VHL from Six2+ nephron progenitors. We used histologic, immunofluorescence, RNA sequencing, and metabolic assays to characterize kidneys from these mice and controls during development and up to postnatal day 21.ResultsBy embryonic day 15.5, kidneys of nephron progenitor cell–specific VHL knockout mice begin to exhibit reduced maturation of nephron progenitors. Compared with controls, VHL knockout kidneys are smaller and developmentally delayed by postnatal day 1, and have about half the number of glomeruli at postnatal day 21. VHL knockout nephron progenitors also exhibit persistent Six2 and Wt1 expression, as well as decreased mitochondrial respiration and prolonged reliance on glycolysis.ConclusionsOur findings identify a novel role for VHL in mediating nephron progenitor differentiation through metabolic regulation, and suggest that VHL is required for normal kidney development.

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PLAG1 and USF2 Regulate Primitive Hematopoietic Expression of Musashi-2

Blood ◽

10.1182/blood.v126.23.3583.3583 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3583-3583

Author(s):

Muluken S Belew ◽

Stefan Rentas ◽

Laura de Rooij ◽

Kristin J Hope

Keyword(s):

Gene Expression ◽

Stem Cell ◽

Cell Lines ◽

Regulatory Elements ◽

Hematopoietic Cell ◽

Minimal Promoter ◽

Luciferase Reporter ◽

Specific Expression ◽

Self Renewal ◽

Model Cell

Abstract The Musashi-2 (MSI2) RNA binding protein is now recognized as a key regulator of hematopoietic stem cells (HSCs). Its expression is most elevated in the primitive HSC compartment and progressively decreases with differentiation. In mouse models of CML, ectopic expression of MSI2 drives progression from the chronic to the blast crisis state while in the human context its aberrantly high expression correlates with more aggressive CML disease states and is associated with poor prognosis in AML. These studies suggest that the precise molecular regulation of MSI2 gene expression may be among the critical mechanisms underlying balanced HSC self-renewal and differentiation and as a result, the prevention of leukemic transformation/progression. Despite the clear importance of understanding how Msi2 maintains an appropriate stem cell-specific expression level, very little is understood of the transcription factors (TFs) that mediate this. To define those factors that govern MSI2 expression and function specifically in the HSC compartment we undertook a systematic approach to map and define relevant regulatory elements of the MSI2 minimal promoter. We dissected a 3.5 kb region 5' upstream of MSI2's translational start site (TSS) shared between mouse and human and thus having the greatest potential of containing regulatory elements key to a conserved MSI2 stem-cell-specific gene expression program. Progressive 5'-terminal deletions of this region cloned upstream of a luciferase reporter gene and transfected into K562 and 293T model cell lines allowed us to define a minimal conserved promoter region from -588 to -203 bp upstream of the TSS that reports accurately on endogenous MSI2 expression. Coupled with in silico prediction of TF that bind this region, systematic TF binding site mutagenesis and luciferase reporter assays in model cell lines identified USF2 and PLAG1 as TFs whose direct binding to the MSI2 minimal promoter direct reporter activity. Loss and gain of function studies in K562 cells confirm that these factors co-regulate the transactivation of endogenous MSI2. Moreover we show in the most relevant primary human CD34+ hematopoietic cell context that these factors bind the MSI2 minimal promoter. While USF2 is a ubiquitously expressed TF across the hematopoietic hierarchy, the uniquely restricted expression of PLAG1 within only the most primitive of hematopoietic cells suggests that it specifically contributes to the heightened stem cell-specific expression of MSI2. Consistent with its role as a key driver of MSI2 and thus an enforcer of its pro-self-renewal functions, we found that overexpression of PLAG1 in human Lin-CD34+ cord blood cells enhanced MSI2 transcription and increased total Colony Forming Unit (CFU) output and re-plating efficiency of primitive CFU progenitors. PLAG1 overexpression also offered a pro-survival advantage to these cells as evidenced by a more than two-fold reduction in Annexin V positive cells compared to negative controls. We have thus described important transcriptional circuitry that governs stem-cell specific expression of MSI2 while at the same time functionally validated PLAG1 as a novel factor capable of modulating primitive hematopoietic cell self-renewal and survival. Disclosures No relevant conflicts of interest to declare.

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BET Proteins Regulate Expression of Osr1 in Early Kidney Development

Biomedicines ◽

10.3390/biomedicines9121878 ◽

2021 ◽

Vol 9 (12) ◽

pp. 1878

Author(s):

Janina Schreiber ◽

Nastassia Liaukouskaya ◽

Lars Fuhrmann ◽

Alexander-Thomas Hauser ◽

Manfred Jung ◽

...

Keyword(s):

Gene Expression ◽

Progenitor Cell ◽

Kidney Development ◽

Gene Activation ◽

Renal Development ◽

Prenatal Programming ◽

Bet Inhibitor ◽

Bet Inhibitors ◽

Nephron Progenitor

In utero renal development is subject to maternal metabolic and environmental influences affecting long-term renal function and the risk of developing chronic kidney failure and cardiovascular disease. Epigenetic processes have been implicated in the orchestration of renal development and prenatal programming of nephron number. However, the role of many epigenetic modifiers for kidney development is still unclear. Bromodomain and extra-terminal domain (BET) proteins act as histone acetylation reader molecules and promote gene transcription. BET family members Brd2, Brd3 and Brd4 are expressed in the nephrogenic zone during kidney development. Here, the effect of the BET inhibitor JQ1 on renal development is evaluated. Inhibition of BET proteins via JQ1 leads to reduced growth of metanephric kidney cultures, loss of the nephron progenitor cell population, and premature and disturbed nephron differentiation. Gene expression of key nephron progenitor transcription factor Osr1 is downregulated after 24 h BET inhibition, while Lhx1 and Pax8 expression is increased. Mining of BRD4 ChIP-seq and gene expression data identify Osr1 as a key factor regulated by BRD4-controlled gene activation. Inhibition of BRD4 by BET inhibitor JQ1 leads to downregulation of Osr1, thereby causing a disturbance in the balance of nephron progenitor cell self-renewal and premature differentiation of the nephron, which ultimately leads to kidney hypoplasia and disturbed nephron development. This raises questions about the potential teratogenic effects of BET inhibitors for embryonic development. In summary, our work highlights the role of BET proteins for prenatal programming of nephrogenesis and identifies Osr1 as a potential target of BET proteins.

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Age-Dependent Changes in the Progenitor Translatome Coordinated in part by Tsc1 Increase Perception of Signaling Inputs to End Nephrogenesis

10.1101/2020.03.12.989343 ◽

2020 ◽

Author(s):

Eric Brunskill ◽

Alison Jarmas ◽

Praneet Chaturvedi ◽

Raphael Kopan

Keyword(s):

Gradual Increase ◽

Tipping Point ◽

Ureteric Bud ◽

Self Renewal ◽

Nephron Endowment ◽

Nephron Progenitors ◽

Age Dependent ◽

Cellular Translation ◽

Nephron Progenitor

AbstractMammalian nephron endowment is determined by the coordinated cessation of nephrogenesis in independent niches. Here we report that in young niches, cellular Wnt agonists are poorly translated, Fgf20 levels are high and R-spondin levels are low, resulting in a pro self-renewal environment. By contrast, older niches are low in Fgf20 and high in R-spondin, with increased cellular translation of Wnt agonists, including the signalosome-promoting Tmem59. This suggests a hypothesis that the tipping point for nephron progenitor exit from the niche is controlled by the gradual increase in stability and clustering of Wnt/Fzd complexes in individual cells, enhancing the response to ureteric bud-derived Wnt9b inputs and driving differentiation. We show Tsc1 hemizygosity differentially promoted translation of Wnt antagonists over agonists, expanding a transitional (Six2+, Cited1+, Wnt4+) state and delaying the tipping point. As predicted by these findings, reducing Rspo3 dosage in nephron progenitors or Tmem59 globally increased nephron numbers in vivo.

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UNDERSTANDING DISTAL TRANSCRIPTIONAL REGULATION FROM SEQUENCE, EXPRESSION AND INTERACTOME PERSPECTIVES

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720010004756 ◽

2010 ◽

Vol 08 (02) ◽

pp. 219-246 ◽

Cited By ~ 1

Author(s):

ARVIND RAO ◽

DAVID J. STATES ◽

ALFRED O. HERO ◽

JAMES DOUGLAS ENGEL

Keyword(s):

Computational Biology ◽

De Novo ◽

Sequence Data ◽

Regulatory Elements ◽

Proximal Promoter ◽

Regulatory Sequences ◽

Sequence Motifs ◽

Specific Sequence ◽

Specific Expression ◽

Regulatory Regions

Gene regulation in eukaryotes involves a complex interplay between the proximal promoter and distal genomic elements (such as enhancers) which work in concert to drive precise spatio-temporal gene expression. The experimental localization and characterization of gene regulatory elements is a very complex and resource-intensive process. The computational identification of regulatory regions that confer spatiotemporally specific tissue-restricted expression of a gene is thus an important challenge for computational biology. One of the most popular strategies for enhancer localization from DNA sequence is the use of conservation-based prefiltering and more recently, the use of canonical (transcription factor motifs) or de novo tissue-specific sequence motifs. However, there is an ongoing effort in the computational biology community to further improve the fidelity of enhancer predictions from sequence data by integrating other, complementary genomic modalities. In this work, we propose a framework that complements existing methodologies for prospective enhancer identification. The methods in this work are derived from two key insights: (i) that chromatin modification signatures can discriminate proximal and distally located regulatory regions and (ii) the notion of promoter-enhancer cross-talk (as assayed in 3C/5C experiments) might have implications in the search for regulatory sequences that co-operate with the promoter to yield tissue-restricted, gene-specific expression.

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