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Gene & Protein in Disease BCL11A targets and chromatin binding patterns

of the membrane-associated AXL receptor kinase. In that 3. Results
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regard, B-pDCs phenotypically and functionally resemble
previously characterized human AXL DCs. 9-12 The transcriptional dependency of BCL11A in both pDCs
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and B cells, along with their documented overlapping
To further investigate the presence of a B-pDC subset expression, led us to hypothesize that B-pDCs and B
in human neoplasia, we performed transcriptional analyses lymphocytes share common overlapping transcriptional
on several human malignant hematopoietic lines. Our programs. To test this hypothesis, we performed ChIP-
findings revealed a conserved dependency on the B-cell seq analyses to identify BCL11A target genes in human
lymphoma/leukemia 11A (BCL11A) transcription factor leukemias and compared these findings to BCL11A targets
for their specification, with a partial overlap of BCL11A in the human pDC CAL-1 cell line. We analyzed data
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targets seen in pDCs and B cells in murine hematopoietic from the NALM6 pre-B leukemia, the Raji B-cell Burkitt’s
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stem cells. 8 lymphoma, and the GM12878 pre-B-cell leukemia and
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The development of both classical pDCs and B compared them to the data from the CAL-1 human pDC
lymphocytes is regulated by BCL11A. 8,13-15 Both lineages share cell line. An important feature of CAL-1, in addition to its
several BCL11A-regulated genes. BCL11A overexpression classical pDC expression, was its upregulation of the AXL
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has been observed in multiple malignancies, 16-18 including receptor kinase. 25,26 Furthermore, CAL-1 was derived from
murine leukemia. 19-21 To determine if a B-pDC lineage exists malignant pDCs that express high levels of BCL11A. 23,27,28
in humans, we utilized chromatin immunoprecipitation The ChIP-seq analysis for BCL11A was executed
sequencing (ChIP-seq) to identify direct BCL11A and plotted as previously detailed. Figure 1A illustrates
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transcription targets and chromatin binding patterns that the overlap of genes bound by BCL11A among the four
are shared between human pDCs and B-cell leukemias. leukemias. Peak scores ≥10 are shown for NALM6

2. Materials and methods (green), Raji (black), CAL-1 (blue), and GM12878 (red).
Numbers within the outer circle indicate statistically
2.1. Data deposition significant targets for each cell line, whereas the inner
Data in this study were deposited and can be accessed circle numbers denote the overlap of these targets among
through the following accession numbers: current ChIP- the four leukemias. Figure 1B presents BCL11A binding
seq (GSE99019) as well as previously published data, within critical target genes as defined as chromatin targets
including pre-B ChIP-seq (GSE52868) and CAL-1 ChIP- within 50 kb up- or downstream of transcriptional start
20
seq (GSE55043) in the gene expression omnibus database, sites. Benjamini–Hochberg statistics were performed to
and BCL11A ChIP-seq (ENCODE GM12878) in the determine q-values and associated false discovery rates.
Encyclopedia of DNA Elements repository. As shown in Figure 1A, the expression of several
prototypic B-cell transcripts (e.g., IGLL1, IGLL5, and SPIB)
2.2. Chromatin immunoprecipitation followed by in CAL-1 indicated a close relationship to both pDCs
chromatin immunoprecipitation sequencing
and AXL transitional DCs. However, the distribution of
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We previously described the details of the BCL11A BCL11A occupancy in CAL-1 closely resembled that seen
ChIP assays, analyzing the ChIP-seq data using Illumina in three human leukemias, with approximately one-quarter
technology. The human lines employed for BCL11A ChIP- of CAL-1 targets being shared (Figure 1A). Notably, we
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seq included NALM6 (human pre-B leukemia) and Raji observed that in all leukemias, BCL11A is bound to its own
(human Burkitt’s lymphoma line). These were compared promoter.
to the ChIP-seq of the human CAL-1 pDC cell line and
the ChIP-seq of GM12878 (human B lymphoblastoid In addition to the array of BCL11A leukemia-related
leukemia), acquired from the Encyclopedia of DNA targets shared by B cells and pDCs, we observed context-
Elements consortium. We analyzed all peak scores ≥10, dependent binding in others. While BCL11A bound
employing the previously established BCL11A consensus promoter regions of genes, such as PAX5, TCF3, and
binding site GGAAgcTGAAA. 8 ID3, exclusively in B cells, it occupied distinct sites in
CAL-1 cells (e.g., AXL, SIGLEC1, and IGLL1). Notably,
2.3. Statistical analysis some target genes exhibited binding at different promoter
positions, as evidenced by non-overlapping peaks in TCF4
We defined a target gene as any gene with binding sites and IRF4 or in SPIB and ID2 (Figure 1B).
that occur within 50 kb upstream to the 5’ end, including
any occurring introns. Benjamini–Hochberg association Moreover, BCL11A did not bind directly to the CD19
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and associated q-values were performed to eliminate false locus in CAL-1 pDCs, but promoter-associated CD19
discovery rates. peaks were present in all B-cell lines. Conversely, CD86

Volume 4 Issue 2 (2025) 2 doi: 10.36922/gpd.8131