2d projection of the metacell adjacency graph  (MetaCell Inc)

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: Meteor transcriptional elongation promotes Eomes expression in naive mESCs (A) Perturbations used to analyze the Meteor locus in this study. Shown are the locations of gRNAs used for deleting or inverting the Meteor promoter (pKO) or gene body (fKO) or for inhibiting transcription (dCas9-KD); the locations of insertion of ribozyme (Rz) or polyadenylation (pAS) sequences; and the scheme for knocking in the PGK promoter downstream of the Meteor promoter (pEX). The fKO and Rz-KD mESC lines are the same as in Tuck et al. (B) RNA-seq tracks showing Meteor expression in the various pKO and pInv lines (grown in serum-free 2i/LIF conditions) and dCas9-KD lines (serum/LIF conditions). All tracks are normalized to the same scale. Orange denotes transcription on the plus strand, and blue denotes transcription on the minus strand. (C) RNA-seq quantifications of Meteor and Eomes in Meteor pKO and pInv cell lines grown in serum/LIF (left) or serum-free 2i/LIF (right) conditions. Amounts normalized to WT1. Bars represent standard errors; n = 3. ∗ p < 0.05, ∗∗ p < 0.005. (D) qRT-PCR quantifications of Meteor and Eomes levels in Meteor fKO and Rz-KD mESCs grown in serum-free 2i/LIF conditions. Levels were normalized to WT4 and Ppib for internal control. Bars represent standard errors; n = 8. ∗ p < 0.05, ∗∗ p < 0.005. (E) Same as (D), for Meteor pKO and pInv mESCs grown in primed conditions, normalized to WT1. n = 4. (F) Same as (D), for pAS clones grown in serum-free 2i/LIF conditions. Levels were normalized to WT7. n = 3. (G) Same as (C), for Meteor dCas9-KD lines grown in serum/LIF conditions. Amounts normalized to Ctrl. n = 3. The dCas9-KD efficiencies of Meteor were 85% and 94% for KD1 and KD2, respectively. (H) Same as (D), for pEX clones grown in serum-free 2i/LIF conditions. Levels were normalized to WT9. n = 3. See also Figure S1 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Expressing, RNA Sequencing, Quantitative RT-PCR, Control, Clone Assay

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: Meteor depletion induces chromatin changes in mESCs (A) (Top) Genome browser image of the region surrounding the Meteor locus. Shown are representative transcript models; RNA-seq tracks where orange denotes transcription on the plus strand and blue denotes transcription on the minus strand; and ENCODE mESC ChIP-seq tracks. (Middle) 4C analysis in the indicated mESC lines using the Meteor or Eomes promoters as viewpoints. Domainograms showing mean contact per fragment end for a series of window sizes are placed below smoothed trend lines and raw counts of the contact profiles. (Bottom) ChIP-seq tracks of CTCF in the indicated mESC lines. All tracks are normalized to the same scale. (B) (Top) Genome browser image of the region surrounding the Meteor locus. RNA-seq track is the same as shown in (A); Cut&Run analysis of H3K27me3 levels in the indicated mESC lines grown in serum-free 2i/LIF conditions. (Bottom) Bar plot shows quantification of signal in the highlighted region, normalized to WT1 and to a H3K27me3-rich region near the Ppib gene (see ). Bars represent standard errors; n = 3. ∗ p < 0.05, one-sided t test. (C) Distribution of reads per kilobase per million (RPKM) of all transcripts identified in an EZH2 RNA immunoprecipitation (RIP) dataset taken from Zhao et al. RPKM of Meteor indicated by a red line. See also Figure S2 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: RNA Sequencing, ChIP-sequencing, RNA Immunoprecipitation

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph. Cells are shown as small dots, whose location indicates similarity to the adjacent cells and metacells (MCs). Color was assigned to each MC according to the ratio of WT and pKO cells that comprise it, with darker shades representing MCs comprised mostly of WT cells and lighter shades representing MCs comprised mostly of pKO cells. (B) Correlation between the log of the fold enrichment values (expression enrichment over the median MC) for the indicated gene pairs, separated by MCs. (C) Same as (A), separately for WT1 and pKO1 cells. (D) Scatterplot comparing gene expression between the indicated groups of MCs. Highlighted are the 20 most differentially expressed genes in each group. (E) Same as (D), for MCs 2 and 4. See also Figure S4 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Expressing, Gene Expression

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet: A distinct functional feature of the Meteor locus represses Eomes throughout neuronal differentiation (A) (Top) Genome browser image of the region surrounding the Meteor locus. Shown are representative transcript models, and RNA-seq tracks taken from Hezroni et al. where orange denotes transcription on the plus strand and blue denotes transcription on the minus strand. All tracks are normalized to the same scale. (Bottom) 4C analysis in the indicated cells using the Meteor or Eomes promoters as viewpoints. Domainograms showing mean contact per fragment end for a series of window sizes are placed below smoothed trend lines and raw counts of the contact profiles. (B) RSEM quantifications of Meteor and Eomes expression levels in the indicated cell types; RNA-seq data are the same as shown in (A). (C) ENCODE ChIP-seq tracks in mESCs (top), and H3K27ac ChIP-seq and H3K4me3 Cut&Run tracks in NPCs (bottom). Genomic coordinates are aligned to (A). (D) DESeq2 quantifications of Meteor and Eomes in NPCs derived from the indicated cell lines. Amounts normalized to WT1/Ctrl. Bars represent standard errors; n = 3. ∗∗ p adj < 0.005. (E) qRT-PCR quantifications of Meteor and Eomes in NPCs derived from the indicated cell lines. Levels were normalized to WT9 and Ppib for internal control. Bars represent standard errors; n = 3. (F) Model of Meteor function. In mESCs, the Meteor locus activates Eomes expression. Perturbing elongation through the locus is associated with increased H3K27me3 deposition and decreased Eomes expression, likely through decreasing the Eomes -expressing mESC subpopulation. As a function of the growth conditions of the cells, this might result in reduced efficiency of cardiac mesoderm formation. Following neuronal differentiation, the Meteor locus now represses Eomes levels, with the DNA element or transcription initiation serving as the functional feature. See also Figure S5 .

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Functional Assay, RNA Sequencing, Expressing, ChIP-sequencing, Derivative Assay, Quantitative RT-PCR, Control

Journal: Cell Reports

Article Title: Complex regulation of Eomes levels mediated through distinct functional features of the Meteor long non-coding RNA locus

doi: 10.1016/j.celrep.2023.112569

Figure Lengend Snippet:

Article Snippet: Figure 4 Distinct subpopulations of mESCs in Meteor WT and KO mESCs (A) 2D projection of the MetaCell adjacency graph.

Techniques: Transfection, Gene Expression, Control, Sequencing, Stable Transfection, Expressing, Recombinant, Plasmid Preparation, Software