Journal: bioRxiv

Article Title: Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation

doi: 10.1101/2024.06.06.597604

Figure Lengend Snippet: ERH depletion reverts fibroblast H3K9me3 patterns to an hESC like state (A) Schematic of siRNA treatments and timing for H3K9me3 CUT&RUN experiments shown in and SOX2 CUT&RUN and RNA-seq experiments shown in . (B) Browser track of H3K9me3 levels in siControl and siERH treated human fibroblasts and H1 hESCs (GSM1003585) at the multi-domain level, domain level and gene level. Example H3K9me3 peaks common between siERH fibroblasts and H1 hESCs are indicated by red triangles. (C) Heatmap of fibroblast siControl and siERH H3K9me3, H1 hESC H3K9me3 (GSM1003585) and H1 hESC HP1α (GSE123229) levels centered at siERH retained H3K9me3 peaks located within fibroblast H3K9me3 domains +/-10kb. (D) Browser track comparing siControl and siERH fibroblast H3K9me3 with H1 hESC H3K9me3 patterns. Peak locations called by MACS2 analysis on siERH H3K9me3 data are shown. (E) Correlation of H3K9me3 from BJ fibroblasts after siControl or siERH with H1 hESC H3K9me3. See also and Table S7.

Article Snippet: Human BJ foreskin fibroblasts were obtained from ATCC (CRL-2522) and cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM Glutamax (ThermoFisher 35050061) at 37°C and 5% CO 2 .

Techniques: RNA Sequencing Assay

Journal: bioRxiv

Article Title: Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation

doi: 10.1101/2024.06.06.597604

Figure Lengend Snippet: ERH depletion reverts fibroblast H3K9me3 patterns to an hESC like state (A) Immunofluorescence of ERH (red), H3K9me3 (red) and DAPI (blue) in siControl and siERH human fibroblasts. Scale bars, 50µm. (B) Occurrence of repeats from RepEnrich database, and repeat occurrence at sites of siERH fibroblasts H3K9me3 peaks. (C) Example browser track of siERH H3K9me3 peak at 3’ region of a ZNF gene. (D) Browser tracks showing H3K9me3 (GSM1003585) and HP1a (GSM1701825) in H1 hESCs and H3K9me3 in siControl and siERH human fibroblasts. (E) Top 10 statistically enriched transcription factor binding motifs at H3K9me3 peaks in siERH treated human fibroblasts (homer findMotifs).

Article Snippet: Human BJ foreskin fibroblasts were obtained from ATCC (CRL-2522) and cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM Glutamax (ThermoFisher 35050061) at 37°C and 5% CO 2 .

Techniques: Immunofluorescence, Binding Assay

Journal: bioRxiv

Article Title: Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation

doi: 10.1101/2024.06.06.597604

Figure Lengend Snippet: ERH depletion enhances gene activation of pluripotency (A) Expression of genes activated during fibroblasts to iPSC reprogramming timepoints that are located in unmarked, H3K27me3 or H3K9me3 marked heterochromatin as annotated by . (B) Venn diagram showing overlap between SOX2 peak calls in siControl and siERH fibroblasts+OSKM. (C) SOX2 CUT&RUN average profile in siControl and siERH fibroblasts+OSKM for genome regions identified as being differentially bound by reprogramming transcription factors . (D) Rank ordered GIGGLE enrichment scores. Each circle indicates similarity between siERH SOX2-gained sites and a ChIP-seq peak set from cistromeDB. Blue circles are peak sets profiled in hESCs, white circles are peaks from a non-embryonic origin. (E) Browser track of example hESC SOX2 and NANOG peak coincident with fibroblast siERH H3K9me3 loss and SOX2 peak gain. (F) Volcano plot of expression changes (DESeq2) in siERH vs siControl fibroblasts+OSKM of iPSC activated genes located in fibroblast H3K9me3 domains. (G) Browser track of H3K9me3 in siControl and siERH fibroblasts and RNA in siControl and siERH fibroblasts+OSKM at DPPA4. (H) Example 8 cell genes activated in siERH+OSKM but not activated during eight time points of iPSC reprogramming time course . (I) Plot of expression change of specific L1 element loci vs expression change of corresponding nearest gene within 25kb in fibroblasts+OSKM. R, Pearson correlation. (J) Browser tracks of H3K9me3 in siControl and siERH fibroblasts and RNA in siControl and siERH fibroblasts+OSKM, untreated H1, 1 day DE and 2 day EC at a locus of an LTR7 repeat. (K) Profile plots of H3K9me3 and RNA levels from LTR7 and HERVH repeats in siERH vs siControl fibroblasts+OSKM. (L) Browser track of H3K9me3 in siControl and siERH fibroblasts and RNA in siControl and siERH fibroblasts+OSKM, untreated H1 and 1 day DE at an LTR7Y-HERVH transposable element and nearby gene. (M) Volcano plot showing differential repeat expression in siERH vs siControl fibroblast+OSKM for repeats annotated in as repressed by H3K9me3 in human blastocysts. (TETranscripts, DESeq2, padj<=0.05, log2fc>0.5).

Article Snippet: Human BJ foreskin fibroblasts were obtained from ATCC (CRL-2522) and cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM Glutamax (ThermoFisher 35050061) at 37°C and 5% CO 2 .

Techniques: Activation Assay, Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation

doi: 10.1101/2024.06.06.597604

Figure Lengend Snippet: ERH depletion enhances gene activation of pluripotency and alternative lineage genes during iPSC reprogramming (A) Heatmap of H3K9me3 CUT&RUN from siControl and siERH human fibroblasts and SOX2 CUT&RUN from siControl and siERH fibroblasts+OSKM at siERH unique SOX2 peak sites. (B) Browser track of example hESC SOX2 and NANOG peak coincident with fibroblast siERH H3K9me3 loss and SOX2 peak gain. (C) Expression of ERH and example naïve pluripotency genes activated in 48 hours OSKM by ERH depletion, transcripts per million (TPM) values from RNA-seq replicates shown (DESeq2 adjusted p-value). (D) Expression change and significance, TEtranscripts analysis, of L1 LTR transposable elements in by ERH depletion in fibroblasts+OSKM listed in order of evolutionary age as determined in . (E) Profile plots of H3K9me3 and RNA levels from LTR7 and HERVH repeats in siERH vs siControl fibroblasts+OSKM. (F) Browser track showing H3K9me3 in siControl and siERH fibroblasts and RNA levels in siControl+OSKM and siERH+OSKM for gene and adjacent LTR7Y naïve pluripotency repeat. (G) Radar plot showing cumulative expression profile across indicated human tissues, data downloaded from BBI Descartes database, for genes upregulated (padj<=0.05, log2 fold change >= 0.5) in siERH vs siControl fibroblasts+OSKM and fibroblasts+NoTF . See also , and Tables S8,S9.

Article Snippet: Human BJ foreskin fibroblasts were obtained from ATCC (CRL-2522) and cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM Glutamax (ThermoFisher 35050061) at 37°C and 5% CO 2 .

Techniques: Activation Assay, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Heterochromatin protein ERH represses alternative cell fates during early mammalian differentiation

doi: 10.1101/2024.06.06.597604

Figure Lengend Snippet: ERH depletion activates alternative lineage genes and repeats during iPSC reprogramming (A) Representative phase image showing cell confluency of siControl and siERH fibroblasts+OSKM cells 2 weeks after initial OSKM infection with titrated siRNA concentration. All treatments were plated at identical cell numbers 1 day prior to infection. (B) Plot of iPSC colony count from siControl and siERH fibroblasts+OSKM cells 3 weeks after initial OSKM infection with titrated siRNA concentration. Each value represents the number of colonies generated in 1 well of a 24 well plate. (C) Expression change and significance, TEtranscripts analysis, of transposable elements by ERH depletion in fibroblasts+OSKM grouped by lineage/stage at which they are typically expressed as determined in . Negative log2 fold changes are represented as unfilled circles. (D) Browser track of H3K9me3 in siControl and siERH fibroblasts and RNA in siControl and siERH fibroblasts+OSKM at an L1PA4 repeat element and nearby neuronal gene.

Article Snippet: Human BJ foreskin fibroblasts were obtained from ATCC (CRL-2522) and cultured in Eagle’s Minimum Essential Medium (EMEM, Sigma-Aldrich M2279) supplemented with 10% fetal bovine serum (FBS, Hyclone SH30071) and 2mM Glutamax (ThermoFisher 35050061) at 37°C and 5% CO 2 .

Techniques: Infection, Concentration Assay, Generated, Expressing