CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia

Abstract

gene dysregulation is a common feature of acute myeloid leukemia (AML). The molecular mechanisms underlying aberrant gene expression and associated AML pathogenesis remain unclear. The nuclear protein CCCTC-binding factor (CTCF), when bound to insulator sequences, constrains temporal gene-expression patterns within confined chromatin domains for normal development. Here, we used targeted pooled CRISPR-Cas9-knockout library screening to interrogate the function of CTCF boundaries in the gene loci. We discovered that the CTCF binding site located between and genes (CBS7/9) is critical for establishing and maintaining aberrant gene expression in AML. Disruption of the CBS7/9 boundary resulted in spreading of repressive H3K27me3 into the posterior active chromatin domain that subsequently impaired enhancer/promoter chromatin accessibility and disrupted ectopic long-range interactions among the posterior genes. Consistent with the role of the CBS7/9 boundary in locus chromatin organization, attenuation of the CBS7/9 boundary function reduced posterior gene expression and altered myeloid-specific transcriptome profiles important for pathogenesis of myeloid malignancies. Furthermore, heterozygous deletion of the CBS7/9 chromatin boundary in the locus reduced human leukemic blast burden and enhanced survival of transplanted AML cell xenograft and patient-derived xenograft mouse models. Thus, the CTCF boundary constrains the normal gene-expression program, as well as plays a role in maintaining the oncogenic transcription program for leukemic transformation. The CTCF boundaries may serve as novel therapeutic targets for the treatment of myeloid malignancies.

Functional Interrogation ofHOXA9Regulome in MLLr Leukemia via Reporter-based CRISPR/Cas9 screen

“…Additionally, siRNA-mediated knock-down of CTCF in SEM cells did not change the transcription level of HOXA7 or HOXA9 (Figures 4E-4G). However, suppressing CTCF in human colorectal cancer cell line HCT116 notably reduced HOXA7 and HOXA9 expression (Figures S5A-S5C), consistent with the finding in MLLr AML cell line MOLM13 (24). Collectively, these data further confirmed the results of our CRISPR screening that CTCF is not a key regulator of HOXA9 in MLLr B-ALL SEM and likely plays a role in regulating HOXA9 transcription in a cell-type-specific manner.…”

“…Interestingly, the most-characterized looping factors, CTCF and YY1, were not enriched in the HOXA9 P2A-mCherry reporter screen (Figure 3B). CTCF was reported to be essential for HOXA9 expression by occupying the boundary sequence between HOXA7 and HOXA9 (CBS7/9) in MLLr AML cell line MOLM13 (24). CRISPR-mediated deletion of the core sequence CTCF binding motif in CBS7/9 significantly decreased HOXA9 expression and tumor progression (24, 43).…”

“…CTCF was reported to be essential for HOXA9 expression by occupying the boundary sequence between HOXA7 and HOXA9 (CBS7/9) in MLLr AML cell line MOLM13 (24). CRISPR-mediated deletion of the core sequence CTCF binding motif in CBS7/9 significantly decreased HOXA9 expression and tumor progression (24, 43). Given that CTCF is generally essential for cell survival, it is possible that cells targeted by CTCF sgRNAs in the HOXA9 P2A-mCherry reporter and TF screen quickly dropped out of the population and were unable to be enriched as a regulator of HOXA9 .…”

“…Previous studies have also advocated that the organization of chromatin domains at the HOXA gene cluster contributes to high HOXA9 expression in cancer cells (24, 25). Specifically, CCCTC-binding factor CTCF may potentiate HOXA9 expression through direct binding at the conserved motif between HOXA7 and HOXA9 (CBS7/9) to establish necessary chromatin looping interaction networks in AML (24).…”

“…Previous studies have also advocated that the organization of chromatin domains at the HOXA gene cluster contributes to high HOXA9 expression in cancer cells (24, 25). Specifically, CCCTC-binding factor CTCF may potentiate HOXA9 expression through direct binding at the conserved motif between HOXA7 and HOXA9 (CBS7/9) to establish necessary chromatin looping interaction networks in AML (24). In contrast, Ghasemi et al (BioRxiv, https://doi.org/10.1101/2020.02.17.952390) reported that HOXA gene expression was maintained in the CTCF binding site deletion mutants, suggesting that transcriptional activity at the HOXA locus in NPM1-mutant AML cells does not require long-range CTCF-mediated chromatin interactions.…”