k562 wildtype  (ATCC)

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) Epigenomic landscape of eNMU and its sub-elements e1 and e2 in K562 cells, showing DNase I hypersensitivity (DHS), H3K27ac, and GRO-cap–defined transcription start sites (TSSs). (B) NMU mRNA levels (RT-qPCR) in independent cultures of WT, ΔeNMU, Δe1 and Δe2 cell lines. Black dots = data from Tippens and Liang et al. ( GAPDH normalized); red dots = data from this study ( ACTB normalized). (C) PRO-seq signal at the NMU –eNMU locus in the same cell lines as (B). Tracks represent merged biological replicates (n = 2). (D) Relative NMU gene body read counts from PRO-seq in (C). (E) Workflow of the eNMU landing pad system to measure enhancer activity of a barcoded element library. (F) Rescue of NMU expression by inserting eNMU into the landing pad (LP); n = 2 independent LP clones. See also and .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Quantitative RT-PCR, Activity Assay, Expressing, Clone Assay

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: PRO-seq (3′-end) tracks of WT, ΔeNMU, Δe1, Δe2 and eNMU LP cell lines across a ∼1 Mb region around NMU ; insets show the full NMU gene and eNMU loci. Highlighted regions indicate NMU promoter (P) and eNMU (E). Note that at the eNMU locus in LP Clone E5, the prominent PRO-seq signal downstream (to the right) of the Bxb1 LP cassette originates from readthrough transcription driven by the strong EF1ɑ promoter within the selection cassette. However, transcriptional activity of the LP did not exhibit any enhancer function to activate the distal NMU gene. Tracks represent merged biological replicates (n = 2 independent cultures). WT K562 GRO-cap data shown as the TSS reference. Related to .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Selection, Activity Assay

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) NMU mRNA levels measured by RT-qPCR in single cell-derived recombinant clones; bars = median. Exact mutant sequences are listed in Table S1. (B–D) ChIP-qPCR of TF binding in select mutants: GATA1 (B) and RUNX1 (C) at e1; STAT5 (D) at e1 and e2 (n = 2 independent single cell clones per mutant). Statistical significance assessed using one-way ANOVA with Dunnett’s post hoc test vs. WT_eNMU (**, p < 0.01; ***, p < 0.001). Public ENCODE ChIP-seq tracks (fold change over control) shown as references: GATA1, ENCFF334KVR; RUNX1, ENCFF654QOE; STAT5A, ENCFF171KLX. (E) Left: p300 ChIP-seq profiles at the eNMU locus in the indicated mutants from (B–D); tracks represent merged biological replicates (n = 2). Track colors indicate specific motif disruptions: grey = WT_eNMU, green = e1_mRUNX1, red = e1_mGATA1, blue = e2_mSTAT5. Colored boxes below tracks indicate locations of disrupted TF motifs. Right: p300 signal at eNMU vs. NMU mRNA in matched single cell clones. (F) Schematic of regulatory factor interplay at eNMU. Note that, unlike normal physiological conditions where STAT5 proteins are activated in response to cytokine signaling, K562 cells express the constitutively active oncogenic BCR-ABL fusion protein that drives persistent STAT5 phosphorylation, dimerization and activation. , See also Methods.

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Quantitative RT-PCR, Derivative Assay, Recombinant, Clone Assay, Mutagenesis, ChIP-qPCR, Binding Assay, ChIP-sequencing, Control, Phospho-proteomics, Activation Assay

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A–D) ATAC-seq signal at eNMU (A), NMU promoter, and GAPDH control locus (B); PRO-seq signal at eNMU (C) and NMU promoter (D) in select eNMU mutants. In (B), black arrows highlight the proximal ATAC-seq peak only affected in the RUNX1 motif mutants. In (D), bar = NMU pausing index of merged replicates, dots = pausing index of individual replicates. Tracks represent merged biological replicates (n = 2 independent single cell clones). Colored boxes below tracks indicate locations of disrupted TF motifs. Fine vertical lines indicate positions of GRO-cap–defined TSSs (WT K562). See also .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Control, Clone Assay

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) ATAC-seq signal at eNMU, NMU promoter and GAPDH control locus for two independent single cell-derived clones of all analyzed eNMU mutants in this study. Colored boxes below tracks indicate locations of disrupted TF motifs. (B) PRO-seq tracks at the full NMU and GAPDH (control) genes in the same mutants as in (A). Tracks represent merged biological replicates (n = 2 independent single cell clones). Fine vertical lines indicate positions of GRO-cap–defined TSSs (WT K562). Related to .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Control, Derivative Assay, Clone Assay

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) Summarized information of selected K562 dTREs from previous studies. Effect sizes obtained from Fulco et al. (except for NMU e1, which is from Tippens and Liang et al. ). (B) Native genomic contexts of each dTRE; tested regions highlighted in light blue. Track scales are consistent across dTRE regions, except for GRO-cap, which uses an individually indicated scale. Detailed sources and accession information are provided in Table S4. (C) Correlation between GRO-cap read counts at dTREs and their intrinsic enhancer activity (−e2) at the eNMU locus. Pearson’s correlation coefficient (r) and corresponding p-value are shown. Related to .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) Workflow to test e2’s facilitator function on heterologous K562 dTREs using the eNMU landing pad. (B) Enhancer activity of dTREs in the absence or presence of e2, measured by RT-qPCR; e2 only serves as the baseline. n = 2 independent recombination experiments. (C) Correlation between intrinsic activity of elements (−e2) and the fold change with e2 fusion. Pearson’s correlation coefficient (r) and corresponding p-value are shown. Shaded region (blue) indicates the 95% confidence interval around the regression line. (D) NMU mRNA levels measured by RT-qPCR in single cell-derived recombinant clones (n ≥ 4) of e1_WT vs. e1_mGATA1 in the absence or presence of e2; e2 only serves as the baseline. Error bars = ± SEM. (E) PRO-seq signal at e1, NMU promoter and GAPDH control locus in e1_WT and e1_mGATA1 clones lacking e2. Tracks represent merged biological replicates (n = 2 independent single cell clones). Fine vertical lines indicate positions of GRO-cap–defined TSSs (WT K562). See also .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Activity Assay, Quantitative RT-PCR, Derivative Assay, Recombinant, Clone Assay, Control

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) ATAC-seq signal at the NMU –eNMU locus in WT, ΔeNMU, Δe1 and Δe2 cell lines, highlighting NMU promoter, eNMU, and facilitators (F1, F2, and F3 from Gasperini et al. ; F1 and F1′ from Reilly et al. ). Tracks represent merged biological replicates (n = 2 independent cultures). (B–C) Public intact Hi-C (B) and ChIP-seq (B and C) , at the NMU –eNMU locus in K562. Intact Hi-C is shown at 300-bp resolution, with black arrows indicating pairwise contacts between NMU promoter, facilitators, and eNMU; ChIP-seq tracks display signal p-values. Detailed sources and accession information are provided in Table S4. (D) Schematic model illustrating a 3D regulatory hub of enhancer–promoter–facilitator interactions at the NMU – eNMU locus. See also and .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Hi-C, ChIP-sequencing

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) Public Hi-C and ChIP-seq tracks of CTCF and RAD21 at the NMU –eNMU locus in K562. Hi-C is shown at 5-kb resolution, with dashed lines and open circles marking pairwise contacts between NMU promoter, facilitators, and eNMU. Note that some contact anchors may not align perfectly with the regulatory elements, possibly due to the limited resolution of this Hi-C dataset. (B) Expanded ChIP-seq tracks , displaying signal p-values. Grey box highlights the NMU promoter and its proximal region, with a zoomed-in view shown on the top right. A separate inset on the right zooms in at the eNMU region, shown at the same scale as the full locus, except where otherwise indicated. Detailed sources and accession information are provided in Table S4. Related to .

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Hi-C, ChIP-sequencing

Journal: bioRxiv

Article Title: Robust regulatory interplay of enhancers, facilitators, and promoters in a native chromatin context

doi: 10.1101/2025.07.07.663560

Figure Lengend Snippet: (A) Transcription-related sequence features of e1. (B) Sequence alignment between e1 and the MER72 LTR consensus. (C) NMU mRNA levels measured by RT-qPCR in single cell-derived recombinant clones; bars = median. (D–G) ATAC-seq signal at eNMU (D), NMU promoter, and GAPDH control locus (E); PRO-seq signal at eNMU (F) and NMU promoter (G) in select eNMU mutants. In (G), bar = NMU pausing index of merged replicates, dots = pausing index of individual replicates. Tracks represent merged biological replicates (n = 2 independent single cell clones). Colored boxes below tracks indicate locations of disrupted TF motifs. Fine vertical lines indicate positions of GRO-cap–defined TSSs (WT K562). (H) Proposed competition model between the LTR promoter and the NMU promoter. See also and Table S1.

Article Snippet: Parental wildtype K562 cells, an immortalized erythroleukemia cell line isolated from the bone marrow of a 53-year-old female patient with chronic myelogenous leukemia (CML), were obtained from the America Type Culture Collection (ATCC) (ATCC Number CCL-243) by the Yu lab and generously provided to us.

Techniques: Sequencing, Quantitative RT-PCR, Derivative Assay, Recombinant, Clone Assay, Control