Homozygous Loss of a Critical CTCF Recognition Sequence in the HOXA gene Cluster Eliminates CTCF Binding but Does Not Appreciably Reduce HOXA9 expression in NPM1 Mutated AML Cells

HOX genes encode homeodomain transcription factors that play a central role in hematopoietic stem/progenitor cell (HSPCs) self-renewal and the development of acute myeloid leukemia (AML). HOXA and HOXB genes display canonical expression patterns in primary AML samples that correlate with specific recurrent mutations, including expression of only HOXA genes in AMLs with MLL rearrangements and both HOXA and HOXB gene expression in normal karyotype AMLs with mutations in NPM1 (i.e., the NPMc mutation). Although regulation of HOXA genes in MLL-rearranged AML cells has been studied extensively, the regulatory mechanisms that govern HOXA expression in other AML types remains unclear. The chromatin organizing factor CTCF contributes to HOXA gene regulation by controlling chromatin looping and three-dimensional structure in a variety of cell types, including AML cells, and a recent study demonstrated that expression of HOXA9 in MLL-rearranged AML cell lines is dependent on a critical CTCF-binding site between HOXA7 and HOXA9 (CBS7/9). However, whether this site is required for HOXA gene expression in AMLs with NPMc remains unknown.

The AML cell line OCI-AML3 contains the NPMc mutation (in addition to a DNMT3AR882C mutant allele) and expresses both HOXA and HOXB genes. We therefore used this cell line to investigate the role of CTCF in regulating HOXA expression in AML cells with this canonical stem cell expression pattern. ChIP-seq for CTCF via 'ChIPmentation' identified eight CTCF binding sites across the HOXA cluster in OCI-AML3 cells, including CBS7/9, which is consistent with previous data in AML cells. These sites were identical to those identified in MLL-rearranged AML cell lines, suggesting that CTCF binding is conserved between these AML types. Similar to previous work, ChIP-seq for histone methylation marks associated with active (H3K4 trimethylation) and repressed (H3K27 trimeylation) chromatin demonstrated distinct chromatin domains at the HOXA locus on either side of CBS7/9, indicating that CTCF acts as a barrier between these domains. Using a computational prediction tool for CTCF binding, we identified two potential CTCF-binding sequences at the CBS7/9 locus, including one bidirectional major site (CBS7/9-A; 20 bp) with a high binding score, separated by only 6 bp from a secondary unidirectional site (CBS7/9-B; 9 bp).

To define the precise sequence important for CTCF binding in OCI-AML3 cells, we used CRISPR-Cas9 to generate a library of mutant cells with deletions of different sizes at the CBS7/9 locus, and then performed ChIP-seq on the mutant cells to measure CTCF binding. We found that mutants with deletions at CBS7/9-A were depleted relative to wild-type cells. Further analysis demonstrated that deletions as small as 10 bp affecting the CBS7/9-A site were sufficient to reduce CTCF binding to background levels. CRISPR-Cas9 generated deletions in the MLL-rearranged THP-1 cell line that spanned the same sequence also eliminated CTCF ChIP-seq signal, indicating that it is equally important for CTCF binding in MLL-rearranged and NPM1-mutant AML cells.

Our collection of mutant OCI-AML3 cell lines included five with biallelic deletions of the CBS7/9-A site, which completely eliminated all CTCF binding at this locus, but did not affect cellular proliferation or survival. Surprisingly, preliminary analysis of expression in the five homozygous mutant OCI-AML3 cells demonstrated that HOXA9 gene expression by RT-qPCR remained largely intact compared to wild-type OCI-AML3 cells, and was not statistically different from three OCI-AML3 cell lines with a heterozygous deletion of the CBS7/9 site.

In summary, we have mapped the precise recognition sequence for a critical CTCF binding site in the HOXA locus in human AML cells. Deletion of both alleles of this regulatory sequence in NPM1-mutated OCI-AML3 cells does not affect cell viability and appears to have only modest effects on expression of the adjacent HOXA9 gene. This is in contrast to a recent report suggesting that homozygous deletion of this CTCF binding site is critical for maintaining HOXA gene expression and may be required for cell viability in MLL-rearranged AML cell lines. These differences suggest that maintenance of HOXA expression in NPM1-mutated vs. MLL-rearranged AML cells may occur through distinct regulatory mechanisms.