Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.

To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1^C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.

To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis.