Journal: Nucleic Acids Research

Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence

doi: 10.1093/nar/gkag642

Figure Lengend Snippet: H3K9me3 heterochromatin domains undergo global reorganization during ME differentiation and EMT. ( A ) Schematic overview of the differentiation protocols utilized to generate NE, ME, and DE from hPSCs. ( B ) Immunostaining of CDH1 (epithelial marker) and CDH2 (mesenchymal marker), along with nuclear staining (DAPI), in hPSC and differentiated cells. Scale bars, 50 μm. ( C ) Spearman correlation analysis of H3K9me3 ChIP-seq data across individual replicates of hPSC and differentiated cells ( n = 2 replicates from two independent differentiations). ( D ) Averaged profiles of H3K9me3 ChIP-seq at de novo H3K9me3 domains in DE ( n = 2). ( E ) Intersection between genes associated with de novo H3K9me3 domains and genes downregulated during hPSC differentiation into ME and DE. GO terms associated with these downregulated genes are shown, along with representative genes by EnrichR. Adjusted P -values by the Benjamini–Hochberg (BH) method. ( F ) Genome browser tracks displaying H3K9me3 ChIP-seq and RNA-seq data for hPSC and differentiated cells at representative loci of hPSC, NE, and neural marker genes.

Article Snippet: Total RNA was extracted using AurumTM Total RNA Mini Kit (7326 820, Bio-Rad) and sent to Plasmidsaurus (3' messenger RNA sequencing [3′ mRNA-seq], ∼90 bp sequencing) or Novogene (whole transcriptome, paired-end 150 bp sequencing) for library preparation and sequencing on an Illumina NovaSeq 6000 or NovaSeq X Plus.

Techniques: Immunostaining, Marker, Staining, ChIP-sequencing, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence

doi: 10.1093/nar/gkag642

Figure Lengend Snippet: MAPK/ERK signaling regulates H3K9me3 domain reorganization within a critical temporal window of ME differentiation. ( A ) RNA-seq heatmaps of representative FGF genes across cell types are shown in log 2 (fragments per kilobase of sequence per million mapped reads [FPKM] + 1; n = 3 replicates from three independent differentiations; top). Schematic overview of MEK inhibition using PD0325901 (MEKi) during differentiation (bottom). ( B ) Immunostaining of the NE markers (SOX1 and SOX2), ME marker (TBXT), and DE marker (SOX17), epithelial marker (CDH1), along with nuclear staining (DAPI), in control and MEKi-treated ME and DE. Scale bars, 100 or 50 μm. ( C ) Differential H3K9me3 domain analysis comparing control and MEKi-treated cells ( n = 2 replicates from two independent differentiations; FC > 2.0 and P < 0.01 by DESeq2). ( D ) Genome-wide Spearman correlation analysis of H3K9me3 ChIP-seq data across individual replicates of control and MEKi-treated cells ( n = 2). ( E ) Western blot quantification of relative H3K9me3 signal intensity, normalized to total H3 ( n = 3 replicates from three independent differentiations, means ± standard error of the mean).

Article Snippet: Total RNA was extracted using AurumTM Total RNA Mini Kit (7326 820, Bio-Rad) and sent to Plasmidsaurus (3' messenger RNA sequencing [3′ mRNA-seq], ∼90 bp sequencing) or Novogene (whole transcriptome, paired-end 150 bp sequencing) for library preparation and sequencing on an Illumina NovaSeq 6000 or NovaSeq X Plus.

Techniques: RNA Sequencing, Sequencing, Inhibition, Immunostaining, Marker, Staining, Control, Genome Wide, ChIP-sequencing, Western Blot

Journal: Nucleic Acids Research

Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence

doi: 10.1093/nar/gkag642

Figure Lengend Snippet: MAPK/ERK signaling regulates EMT and ME gene expression program in association with H3K9me3 reorganization. ( A ) PCA of RNA-seq data from hPSC, NE, ME, DE, and MEKi-treated ME and DE, based on the top 5000 expressed genes ( n = 3 replicates from three independent differentiations for control cells, n = 2 replicates from two independent differentiations for MEKi-treated cells). ( B ) RNA-seq heatmaps of representative marker gene expression across control and MEKi-treated ME and DE in log 2 (FPKM + 1). Mesenchyme/epithelial, M/E. ( C ) Intersection between genes associated with gained H3K9me3 domains and genes downregulated in MEKi-treated ME and DE. GO terms associated with these downregulated genes are shown, along with representative genes by EnrichR. Adjusted P -values by the BH method.

Article Snippet: Total RNA was extracted using AurumTM Total RNA Mini Kit (7326 820, Bio-Rad) and sent to Plasmidsaurus (3' messenger RNA sequencing [3′ mRNA-seq], ∼90 bp sequencing) or Novogene (whole transcriptome, paired-end 150 bp sequencing) for library preparation and sequencing on an Illumina NovaSeq 6000 or NovaSeq X Plus.

Techniques: Gene Expression, RNA Sequencing, Control, Marker

Journal: Nucleic Acids Research

Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence

doi: 10.1093/nar/gkag642

Figure Lengend Snippet: Phosphorylated ERK is enriched at chromatin domains and contributes to restricting ectopic H3K9me3 accumulation at key developmental gene loci. ( A ) Genome browser tracks displaying RNA-seq, P-ERK ChIP-seq, and H3K9me3 ChIP-seq data in control and MEKi-treated ME (MEKi-1d) and DE cells (MEKi-2d) at representative loci. ( B ) Differential P-ERK peak analysis comparing control and MEKi-treated ME cells ( n = 2 replicates from two independent differentiations; FC > 1.5 and P < 0.01 by DESeq2). Representative genes associated with Lost P-ERK peaks by GREAT. ( C ) Averaged profiles of P-ERK and H3K9me3 ChIP-seq in control and MEKi-treated ME cells at Lost P-ERK-associated gene loci ( n = 2). Transcriptional start site (TSS).

Article Snippet: Total RNA was extracted using AurumTM Total RNA Mini Kit (7326 820, Bio-Rad) and sent to Plasmidsaurus (3' messenger RNA sequencing [3′ mRNA-seq], ∼90 bp sequencing) or Novogene (whole transcriptome, paired-end 150 bp sequencing) for library preparation and sequencing on an Illumina NovaSeq 6000 or NovaSeq X Plus.

Techniques: RNA Sequencing, ChIP-sequencing, Control

Journal: Nucleic Acids Research

Article Title: MAPK/ERK signaling regulates H3K9me3 heterochromatin reorganization to confer mesendoderm developmental competence

doi: 10.1093/nar/gkag642

Figure Lengend Snippet: Proper establishment of H3K9me3 domains plays a dual role in repressing off-target genes and supporting robust activation of lineage-specific programs. ( A ) Schematic overview of the strategy for generating Dox-inducible CRISPRi hPSC lines targeting three H3K9me3 MTases, SUV39H1, SUV39H2 , and SETDB1 (MTase-TKD). ( B ) Western blot quantification of relative H3K9me3 signal intensity, normalized to total H3 in DE ( n = 2 replicates from two independent differentiations, means ± standard deviation). ( C ) Differential gene expression analysis of RNA-seq comparing MTase-TKD versus control ( n = 2 replicates; FC > 1.5 and FDR < 0.05). ( D ) RNA-seq heatmaps showing log 2 FC of representative marker gene expression in MTase-TKD relative to control cells. ( E ) Scatter plots comparing gene expression changes in MTase-TKD versus control ( x -axis) and MTase-TKD + MEKi versus control ( y -axis) in ME and DE. Differentially expressed genes (FC > 1.5 and FDR < 0.05) are highlighted. The top 20 differentially expressed genes are labeled.

Article Snippet: Total RNA was extracted using AurumTM Total RNA Mini Kit (7326 820, Bio-Rad) and sent to Plasmidsaurus (3' messenger RNA sequencing [3′ mRNA-seq], ∼90 bp sequencing) or Novogene (whole transcriptome, paired-end 150 bp sequencing) for library preparation and sequencing on an Illumina NovaSeq 6000 or NovaSeq X Plus.

Techniques: Activation Assay, Western Blot, Standard Deviation, Gene Expression, RNA Sequencing, Control, Marker, Labeling