Journal: Blood

Article Title: Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation ∗

doi: 10.1182/blood.2025030211

Figure Lengend Snippet: BCL11A is developmentally regulated by lineage-specific enhancers and epigenetic silencing. (A) Chromatin accessibility profiles based on publicly available ATAC-seq data sets across human hematopoietic lineages are shown for the BCL11A locus at chr2:60,319,000-60,600,000 (hg38). The dashed lines indicate the BCL11A TSS and erythroid-specific enhancers. The right panel displays BCL11A mRNA expression levels (TPM), with each dot representing an independent replicate. (B) Chromatin immunoprecipitation sequencing (ChIP-seq) profiles of H3K27ac and H3K27me3 in primary human hematopoietic stem cell (HSCs) (blue), erythroblasts (red), and T lymphocytes (black). H3K27me3 deposition is observed at the BCL11A promoter and gene body in T lymphocytes. (C) ChIP-seq profiles of H3K27ac and H3K27me3 in primary mouse HSCs, erythroblasts, and T lymphocytes, revealing conserved patterns of Bcl11a repression in T lymphocytes via H3K27me3-mediated silencing. The dashed line indicates the Bcl11a TSS. The sources of the public data sets are listed in . (D) Schematic representation of BCL11A gene expression and enhancer activity in HSCs, erythroblasts, and T lymphocytes, illustrating a shared CTCF binding profile but distinct epigenetic landscapes that correlate with lineage-specific gene expression. Erythroid-specific enhancers (E+55, E+58, and E+62) are highlighted in red, whereas the regions of active and repressive chromatin are indicated in green and gray, respectively. BasoE, basophilic erythroblast; BFU-E, burst-forming unit–erythroid; CFU-E, colony-forming unit–erythroid; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; DC, dendritic cell; GMP, granulocyte-monocyte progenitor; MEP, megakaryocyte-erythroid progenitor; MPP, multipotent progenitor; MK, megakaryocyte; NK, natural killer; orthoE, orthochromatic erythroblast; PolyE, polychromatic erythroblast; ProE, proerythroblast; TPM, transcripts per million.

Article Snippet: Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed using Zymo-Seq ATAC Library Kit (Zymo, catalog no. D5458).

Techniques: Expressing, ChIP-sequencing, Gene Expression, Activity Assay, Binding Assay

Journal: Blood

Article Title: Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation ∗

doi: 10.1182/blood.2025030211

Figure Lengend Snippet: BCL11A enhancers are required for epigenetic insulation. (A) Epigenetic landscape of the BCL11A locus in human primary erythroblasts and HUDEP-2 cells, showing chromatin accessibility (ATAC-seq) and the occupancy of active histone marks (H3K27ac and H3K4me3), the repressive histone mark (H3K27me3), and CTCF. The dashed lines indicate the BCL11A TSS and erythroid-specific enhancers. (B) BCL11A and HBG mRNA expression levels, along with the percentage of HBG and the levels of HbF by high-performance liquid chromatography and HbF + cells, in WT and CRISPR-edited single-cell HUDEP-2 clones. Each dot represents an independent single-cell–derived clone. Data are presented as mean ± standard deviation (SD) and analyzed by 1-way analysis of variance (ANOVA). ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. (C) Chromatin accessibility, CUT&Tag (H3K27ac), and CUT&RUN (H3K27me3) profiles in WT HUDEP-2 cells and cells with BCL11A exon 2 mutation, enhancer KO, or sg1617-mediated enhancer editing (sg1617). The right panel shows a representative genomic region with no detectable chromatin state changes. (D) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of CD34 + HSPCs from healthy donors and patients with SCD edited with Cas9-RNP targeting E+58 (sg1617) or a control locus (sgAAVS). Samples were collected at differentiation day 8. Results are shown as mean ± SD (n ≥ 3 replicates) and analyzed by 1-way ANOVA. ∗∗∗ P < .001; ∗∗∗∗ P < .0001. (E) Genome browser tracks of ATAC-seq, H3K27ac, and H3K27me3 in primary erythroblasts derived from CD34 + HSPCs from healthy donors or patients with SCD, showing H3K27me3 deposition at the BCL11A promoter and gene body following Cas9-RNP-mediated E+58 enhancer editing (sg1617) or a control locus (sgAAVS), as indicated by the log 2 fold change of H3K27me3 CUT&RUN signals. CD34 + HSPCs were induced for erythroid differentiation for 8 days (healthy donors) or 12 days (SCD). (F) A model depicting the critical role of the erythroid enhancer E+58 in BCL11A gene regulation and the maintenance of epigenetic insulation. Enh KO, enhancer knockout; Exon2 Mut, BCL11A exon 2 mutation; NS, not significant; sgAAVS, sgRNA targeting the adenovirus-associated virus integration site.

Article Snippet: Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed using Zymo-Seq ATAC Library Kit (Zymo, catalog no. D5458).

Techniques: Insulation, Expressing, High Performance Liquid Chromatography, CRISPR, Single Cell, Clone Assay, Derivative Assay, Standard Deviation, Mutagenesis, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Knock-Out, Virus

Journal: Blood

Article Title: Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation ∗

doi: 10.1182/blood.2025030211

Figure Lengend Snippet: BCL11A locus is organized into enhancer-dependent chromatin structures. (A) Schematic representation of the CPC workflow. Cells were crosslinked with formaldehyde, followed by in situ digestion with a restriction enzyme and chromatin fragment ligation. The chromatin was then de-crosslinked, and proteins and peptides were removed. Long DNA concatemers underwent 2 rounds of hybridization capture using biotinylated probes, followed by PCR amplification and size selection. Libraries were prepared and sequenced on the Oxford Nanopore PromethION platform. CPC data were analyzed to identify pairwise and multiway chromatin interactions. (B) Pairwise chromatin interactions at the BCL11A locus in WT, enhancer KO, and sg1617-edited HUDEP-2 cells. The upper panel shows genome browser tracks for ATAC-seq, H3K27ac, H3K27me3, and CTCF binding at the BCL11A locus, with the dashed lines indicating CBS serving as CPC baits. The bottom panel displays the significant chromatin interactions ( P < .05) identified using CHiCANE for all the CTCF and promoter baits within chr2:60,319,000-60,600,000. Specifically, the first panel shows the combined interactions from all the baits, whereas the subsequent panels display the interactions for each bait. Within each panel, the virtual 4C track anchored at the bait region and the interaction arcs are shown, with a color scale indicating the interaction frequency. (C) CPC analysis of the pairwise interactions ( P < .05) in WT and enhancer-edited (sg1617) cells, with enhancers E+55, E+58, and E+62 serving as baits (dashed lines). The results show a reduction in enhancer-mediated pairwise interactions across all 3 enhancers following editing. (D) A model illustrating chromatin interactions at the BCL11A locus in WT and enhancer-edited cells. CPC analysis revealed both E-P interactions and CTCF-mediated chromatin loops, both of which were weakened upon enhancer editing, leading to altered chromatin organization and epigenetic silencing of BCL11A . E-P, enhancer-promoter.

Article Snippet: Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed using Zymo-Seq ATAC Library Kit (Zymo, catalog no. D5458).

Techniques: In Situ, Ligation, Hybridization, Amplification, Size Selection, Binding Assay

Journal: Blood

Article Title: Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation ∗

doi: 10.1182/blood.2025030211

Figure Lengend Snippet: Enhancer-dependent chromatin configuration is required for epigenetic insulation. (A) Multiway chromatin interactions at the BCL11A locus in WT, enhancer KO, and sg1617-edited HUDEP-2 cells. The upper tracks show chromatin accessibility (ATAC-seq), H3K27ac, H3K27me3 signals, and CTCF binding at the BCL11A locus. In the lower graphs, each line represents an independent Nanopore sequencing read, with the dots indicating bait regions used in CPC. The number of multiway interactions is reduced following enhancer KO or sg1617 editing. (B) The distribution of promoter-associated multiway interactions with individual CBS in WT, enhancer KO, and sg1617-edited HUDEP-2 cells based on the total number of paired-end tags (PETs) for each condition. The pie charts illustrate the changes in the number and distribution of promoter-CTCF contacts following enhancer KO or editing, as indicated by the sizes of the pie charts. (C) Top-ranked multiway interactions affected by enhancer editing relative to WT. Interaction frequencies were normalized to the total PETs per sample. The bar graph highlights the most significantly altered interactions, with the dots and lines below each column indicating the involved genomic regions. (D) CPC analysis of multiway interactions using BCL11A enhancers as baits. Multiway interactions associated with enhancers E+55, E+58, and E+62 are significantly diminished upon sg1617 editing, illustrating the role of enhancers in maintaining higher-order chromatin architecture. (E) Pie chart analysis of enhancer-associated multiway interactions with other enhancers, the promoter, or CBS. The upper panels illustrate global changes in enhancer connectivity, whereas the lower panels depict enhancer-CBS interactions at individual CBS across different conditions. Enhancer editing leads to a marked reduction in interaction frequency and complexity. (F) Top-ranked multiway interactions altered upon enhancer editing relative to WT. The bar graph represents the most significant changes normalized to the total PET numbers. The dots and lines below each column indicate the involved genomic regions. (G) A model depicting the 3D chromatin organization of the BCL11A locus in WT and enhancer-edited erythroid cells. In normal erythroid cells, CREs and CBS within the BCL11A locus form clusters, termed BCL11A enhancer-dependent chromatin rosette, which facilitate multiway chromatin looping, enhancer-promoter interactions, and epigenetic insulation to sustain BCL11A expression. Following enhancer editing via CRISPR-Cas9, epigenetic insulation and enhancer-promoter interactions are disrupted, accompanied by the loss of CTCF-mediated looping, leading to the epigenetic silencing of BCL11A . Enh, enhancer.

Article Snippet: Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed using Zymo-Seq ATAC Library Kit (Zymo, catalog no. D5458).

Techniques: Insulation, Binding Assay, Nanopore Sequencing, Expressing, CRISPR

Journal: Blood

Article Title: Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation ∗

doi: 10.1182/blood.2025030211

Figure Lengend Snippet: BCL11A eRNAs orchestrate NIPBL and cohesin binding for epigenetic insulation. (A) Genome browser tracks showing chromatin accessibility (ATAC-seq) and the occupancy of SMC1, NIPBL, and CTCF at the BCL11A locus in WT HUDEP-2 cells and single-cell clones following sg1617-mediated enhancer editing. The dashed lines indicate the BCL11A TSS and erythroid-specific enhancers. The right panel shows a representative genomic region with no detectable changes in chromatin state or NIPBL/cohesin binding. (B) CLIP-qPCR analysis of NIPBL and eRNA interactions in HUDEP-2 cells. A schematic of the workflow is shown on the left. Results are presented for 2 independent CLIP experiments using NIPBL or IgG (negative control) antibodies. The known NIPBL-interacting small nuclear RNA, RNU-1 , was analyzed as a positive control, whereas PAPOLG , a transcribed genomic region upstream of the BCL11A locus, served as a negative control. Two independent PCR primers were used to detect E+58 and E+55 eRNA transcripts. Results are mean ± SD (n ≥ 3 replicates) and analyzed by 1-way ANOVA for each primer. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001. (C) qRT-PCR and F-cell analysis of HUDEP-2 cells transduced with nontargeting control (control ASO) or E+58 eRNA-targeting ASO (ASO12 and ASO23). ASO treatment resulted in a significant reduction in E+58 eRNA and BCL11A mRNA expression, accompanied by increased HBG expression and F cells. Results are shown as mean ± SD (n ≥ 3 replicates) and analyzed by 1-way ANOVA. ∗∗ P < .01; ∗∗∗ P < .001. (D) E+58 eRNA-targeting ASO reduced NIPBL binding at the BCL11A enhancers and increased H3K27me3-associated repressive chromatin at the BCL11A promoter in HUDEP-2 cells, as indicated by the log 2 fold change of H3K27me3 CUT&RUN signals. The right panel shows a representative genomic region with no detectable changes in chromatin state. (E) A model illustrating the 3D chromatin organization of the BCL11A locus in WT and enhancer-edited erythroid cells. In normal erythroid cells, the E+58 enhancer maintains chromatin insulation and prevents epigenetic silencing by serving as a TSS for eRNA production and as a NIPBL/cohesin landing site for chromatin loop formation. CRISPR-mediated enhancer editing disrupts both functions, leading to destabilized chromatin architecture, impaired chromatin insulation, and epigenetic silencing of BCL11A . CLIP, crosslinking immunoprecipitation; Ctrl ASO, control antisense oligonucleotide; IgG, immunoglobulin G; NS, not significant.

Article Snippet: Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) was performed using Zymo-Seq ATAC Library Kit (Zymo, catalog no. D5458).

Techniques: Binding Assay, Insulation, Single Cell, Clone Assay, Negative Control, Positive Control, Quantitative RT-PCR, Cell Analysis, Transduction, Control, Expressing, CRISPR, Cross-linking Immunoprecipitation

Journal: bioRxiv

Article Title: Mega-frequency mutagenesis: generation of non-random precise mutations with extremely high frequency upon adaptation of cancer cells to drugs and stress

doi: 10.64898/2026.02.20.707073

Figure Lengend Snippet: Development of recurrent mutations after drug treatments. A. The number of mutations in three parallel subclones following development of resistance to Dox. The number of mutations common to all three subclones (red), common to two out of three subclones (green) and mutations unique for each subclone (blue). B. A scheme showing that generation of mutations common between clones cannot occur during the propagation of the parental clone. See explanation in the text. C.Example of the lack of enrichment of mutations in open chromatin. Open chromatin assessed by ATACseq is shown in the lane with blue peaks, positions of mutations are shown in the lane with red bars. Data analysis in this experiment is shown in Table S2. C. Overlap of mutations between GDR and Lorlatinib. D. Overlap of mutations between osimertinib and Doxorubicin. E. Overlap of mutations between two independent lorlatinib-treated samples and one crizotinib-treated sample. F. Computer simulation of random generation of osimertinib-induced mutations. Observed number of common mutations is shown as red line. Random sampling of mutations and their overlap (100,000 pairs) is shown as blue lines. G. Overlap of mutations between osimertinib day 21 (0SM-3T) and osimertinib subclone 1 samples (OSM-1CL). H. Overlap of mutations between osimertinib day 21 (0SM-3T) and osimertinib mutations common between three subclones (OSM-TRPL).

Article Snippet: Samples for ATAC sequencing (ATACseq) were prepared using Zymo-Seq ATAC Library Kit (Cat#D5458) following the manufacturer protocol.

Techniques: Clone Assay, Sampling