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

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

Genomics ◽

10.1016/j.ygeno.2021.12.017 ◽

2021 ◽

Author(s):

Andrew Clugston ◽

Andrew Bodnar ◽

Débora Malta Cerqueira ◽

Yu Leng Phua ◽

Alyssa Lawler ◽

...

Keyword(s):

Progenitor Cells ◽

Kidney Development ◽

Chromatin Accessibility ◽

Microrna Expression ◽

Nephron Progenitor

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Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes

10.1101/2021.03.10.434810 ◽

2021 ◽

Author(s):

Jaclyn M Noshay ◽

Zhikai Liang ◽

Peng Zhou ◽

Peter A Crisp ◽

Alexandre P Marand ◽

...

Keyword(s):

Dna Methylation ◽

Sequence Variation ◽

Reference Genome ◽

De Novo ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Genome Context ◽

Genomic Regions ◽

Genome Assemblies ◽

Accessible Chromatin

AbstractAccessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43 and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. The number of UMRs and ACRs that can be identified is more accurate when chromatin data is aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are substantially enriched within shared regions, as determined by chromosomal alignments. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only a small number being polymorphic between genotypes. However, the majority of UMRs between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.Article summaryRegions of the genome that have accessible chromatin or unmethylated DNA are often associated with cis-regulatory elements. We assessed chromatin accessibility and DNA methylation in four structurally diverse maize genomes. There are accessible or unmethylated regions within the non-shared portions of the genomes but these features are depleted within these regions. Evaluating the dynamics of methylation and accessibility between genotypes reveals conservation of features, albeit with variable boundaries suggesting some instability of the precise edges of unmethylated regions.

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

Journal of the American Society of Nephrology ◽

10.1681/asn.2021040525 ◽

2021 ◽

Vol 32 (11) ◽

pp. 2815-2833

Author(s):

Jun Li ◽

Jinshu Xu ◽

Huihui Jiang ◽

Ting Zhang ◽

Aarthi Ramakrishnan ◽

...

Keyword(s):

Progenitor Cell ◽

Kidney Development ◽

Regulatory Elements ◽

Proximal Promoter ◽

Specific Expression ◽

Self Renewal ◽

Content Type ◽

Nephron Progenitors ◽

Genome Wide ◽

Nephron Progenitor

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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Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab190 ◽

2021 ◽

Author(s):

Jaclyn M Noshay ◽

Zhikai Liang ◽

Peng Zhou ◽

Peter A Crisp ◽

Alexandre P Marand ◽

...

Keyword(s):

Dna Methylation ◽

Sequence Variation ◽

Reference Genome ◽

De Novo ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Genome Context ◽

Genomic Regions ◽

Genome Assemblies ◽

Accessible Chromatin

Abstract Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43 and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. A more complete set of UMRs and ACRs can be identified when chromatin data is aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are 6-12 fold enriched within regions between genomes. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only ∼5% being polymorphic between genotypes. However, the majority (71%) of UMRs that are shared between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.

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Genome-wide cancer-specific chromatin accessibility patterns derived from archival processed xenograft tumors

Genome Research ◽

10.1101/gr.275219.121 ◽

2021 ◽

Author(s):

Shelsa S. Marcel ◽

Austin L. Quimby ◽

Melodie P. Noel ◽

Oscar C. Jaimes ◽

Marjan Mehrab-Mohseni ◽

...

Keyword(s):

Human Tumor ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Primary Tumors ◽

Genome Wide ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Cellular Components ◽

Accessible Chromatin

Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.

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ATAC-STARR-seq v2

10.17504/protocols.io.b2nuqdew ◽

2021 ◽

Author(s):

Tyler Hansen ◽

Emily Hodges

Keyword(s):

Regulatory Region ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

High Signal ◽

Regulatory Pathways ◽

Information Accessibility ◽

Genome Wide ◽

A Genome ◽

Wide Scale ◽

Accessible Chromatin

Massively parallel reporter assays test the capacity of putative cis-regulatory elements (CREs) to drive transcription on a genome-wide scale. In nearly all cases, chromatin accessibility is necessary to drive activity, so most CREs are inactive due to chromatin context rather than intrinsic DNA sequence properties. Here, we combined assay for transposase-accessible chromatin (ATAC-seq) with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of nucleosome-free DNA genome-wide. Our approach enabled high-resolution testing of ~50 million unique DNA fragments tiling ~101,000 accessible chromatin regions in human lymphoblastoid cells. To illustrate the application of our approach, we show that 30% of all accessible regions contain an activator, a silencer or both. Benchmarking against standard ATAC-seq, our approach faithfully captures chromatin accessibility and transcription factor (TF) footprints with high signal-to-noise. Integrating three layers of genomic information (accessibility, TF occupancy, and activity) provided by ATAC-STARR-seq, we stratified active and silent CREs by the presence of several TF footprints and show that CREs with specific TF combinations are associated with distinct gene regulatory pathways. Altogether, these data highlight the power of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome from a single DNA source.

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Assessing the regulatory potential of transposable elements using chromatin accessibility profiles of maize transposons

Genetics ◽

10.1093/genetics/iyaa003 ◽

2020 ◽

Vol 217 (1) ◽

Author(s):

Jaclyn M Noshay ◽

Alexandre P Marand ◽

Sarah N Anderson ◽

Peng Zhou ◽

Maria Katherine Mejia Guerra ◽

...

Keyword(s):

Transposable Elements ◽

Allelic Variation ◽

Regulatory Elements ◽

Chromatin Accessibility ◽

Transcriptional Responses ◽

Maize Genotypes ◽

Show Evidence ◽

Regulatory Potential ◽

Dna Regulatory Elements ◽

Accessible Chromatin

Abstract Transposable elements (TEs) have the potential to create regulatory variation both through the disruption of existing DNA regulatory elements and through the creation of novel DNA regulatory elements. In a species with a large genome, such as maize, many TEs interspersed with genes create opportunities for significant allelic variation due to TE presence/absence polymorphisms among individuals. We used information on putative regulatory elements in combination with knowledge about TE polymorphisms in maize to identify TE insertions that interrupt existing accessible chromatin regions (ACRs) in B73 as well as examples of polymorphic TEs that contain ACRs among four inbred lines of maize including B73, Mo17, W22, and PH207. The TE insertions in three other assembled maize genomes (Mo17, W22, or PH207) that interrupt ACRs that are present in the B73 genome can trigger changes to the chromatin, suggesting the potential for both genetic and epigenetic influences of these insertions. Nearly 20% of the ACRs located over 2 kb from the nearest gene are located within an annotated TE. These are regions of unmethylated DNA that show evidence for functional importance similar to ACRs that are not present within TEs. Using a large panel of maize genotypes, we tested if there is an association between the presence of TE insertions that interrupt, or carry, an ACR and the expression of nearby genes. While most TE polymorphisms are not associated with expression for nearby genes, the TEs that carry ACRs exhibit enrichment for being associated with higher expression of nearby genes, suggesting that these TEs may contribute novel regulatory elements. These analyses highlight the potential for a subset of TEs to rewire transcriptional responses in eukaryotic genomes.

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Identification and classification of cis-regulatory elements in the amphipod crustacean Parhyale hawaiensis

10.1101/2021.09.16.460328 ◽

2021 ◽

Author(s):

Dennis A Sun ◽

Nipam H Patel

Keyword(s):

Gene Expression ◽

Genome Annotation ◽

Time Course ◽

Regulatory Elements ◽

Rna Seq ◽

Genome Wide ◽

Long Read ◽

Amphipod Crustacean ◽

Accessible Chromatin

AbstractEmerging research organisms enable the study of biology that cannot be addressed using classical “model” organisms. The development of novel data resources can accelerate research in such animals. Here, we present new functional genomic resources for the amphipod crustacean Parhyale hawaiensis, facilitating the exploration of gene regulatory evolution using this emerging research organism. We use Omni-ATAC-Seq, an improved form of the Assay for Transposase-Accessible Chromatin coupled with next-generation sequencing (ATAC-Seq), to identify accessible chromatin genome-wide across a broad time course of Parhyale embryonic development. This time course encompasses many major morphological events, including segmentation, body regionalization, gut morphogenesis, and limb development. In addition, we use short- and long-read RNA-Seq to generate an improved Parhyale genome annotation, enabling deeper classification of identified regulatory elements. We leverage a variety of bioinformatic tools to discover differential accessibility, predict nucleosome positioning, infer transcription factor binding, cluster peaks based on accessibility dynamics, classify biological functions, and correlate gene expression with accessibility. Using a Minos transposase reporter system, we demonstrate the potential to identify novel regulatory elements using this approach, including distal regulatory elements. This work provides a platform for the identification of novel developmental regulatory elements in Parhyale, and offers a framework for performing such experiments in other emerging research organisms.Primary Findings-Omni-ATAC-Seq identifies cis-regulatory elements genome-wide during crustacean embryogenesis-Combined short- and long-read RNA-Seq improves the Parhyale genome annotation-ImpulseDE2 analysis identifies dynamically regulated candidate regulatory elements-NucleoATAC and HINT-ATAC enable inference of nucleosome occupancy and transcription factor binding-Fuzzy clustering reveals peaks with distinct accessibility and chromatin dynamics-Integration of accessibility and gene expression reveals possible enhancers and repressors-Omni-ATAC can identify known and novel regulatory elements

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Premature differentiation of nephron progenitor cell and dysregulation of gene pathways critical to kidney development in a model of preterm birth

Scientific Reports ◽

10.1038/s41598-021-00489-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aleksandra Cwiek ◽

Masako Suzuki ◽

Kimberly deRonde ◽

Mark Conaway ◽

Kevin M. Bennett ◽

...

Keyword(s):

Preterm Birth ◽

Progenitor Cells ◽

Kidney Development ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Neonatal Morbidity ◽

Expression Of Genes ◽

Nephron Progenitor ◽

Lower Expression ◽

Nephrogenic Zone

AbstractPreterm birth is a leading cause of neonatal morbidity. Survivors have a greater risk for kidney dysfunction and hypertension. Little is known about the molecular changes that occur in the kidney of individuals born preterm. Here, we demonstrate that mice delivered two days prior to full term gestation undergo premature cessation of nephrogenesis, resulting in a lower glomerular density. Kidneys from preterm and term groups exhibited differences in gene expression profiles at 20- and 27-days post-conception, including significant differences in the expression of fat-soluble vitamin-related genes. Kidneys of the preterm mice exhibited decreased proportions of endothelial cells and a lower expression of genes promoting angiogenesis compared to the term group. Kidneys from the preterm mice also had altered nephron progenitor subpopulations, early Six2 depletion, and altered Jag1 expression in the nephrogenic zone, consistent with premature differentiation of nephron progenitor cells. In conclusion, preterm birth alone was sufficient to shorten the duration of nephrogenesis and cause premature differentiation of nephron progenitor cells. These candidate genes and pathways may provide targets to improve kidney health in preterm infants.

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