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1 Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-
2 mutant AML cells
3
4 Reza Ghasemi1, Heidi Struthers1, Elisabeth R. Wilson1, and David H. Spencer1,2*
5
6 1 Department of Medicine, Division of Oncology, Section of Stem Cell Biology, Washington
7 University School of Medicine, St. Louis, MO, USA
8 2 McDonnell Genome Institute, Washington University, St. Louis, MO, USA
9
10 Running title: HOXA CTCF binding in NPM1-mutant AML
11
12 The authors have no conflicts of interest relating to the work described in this manuscript.
13
14 * Corresponding author
15 David H. Spencer, MD, PhD 16 Assistant Professor 17 Division of Oncology 18 Department of Medicine 19 Washington University School of Medicine 20 Campus Box 8007 21 660 South Euclid Avenue 22 St. Louis, MO. 63110 23 24
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25 Abstract
26 Transcriptional regulation of the HOXA genes is thought to involve CTCF-mediated chromatin
27 loops and the opposing actions of the COMPASS and Polycomb epigenetic complexes. We
28 investigated the role of these mechanisms at the HOXA cluster in AML cells with the common
29 NPM1c mutation, which express both HOXA and HOXB genes. CTCF binding at the HOXA
30 locus is conserved across primary AML samples, regardless of HOXA gene expression, and
31 defines a continuous chromatin domain marked by COMPASS-associated histone H3
32 trimethylation in NPM1-mutant primary AML samples. Profiling of the three-dimensional
33 chromatin architecture of NPM1-mutant OCI-AML3 cells identified chromatin loops between the
34 active HOXA9-HOXA11 genes and loci in the SNX10 gene and an intergenic region located
35 1.4Mbp upstream of the HOXA locus. Deletion of CTCF binding sites in OCI-AML3 cells
36 reduced these interactions, but resulted in new, CTCF-independent loops with regions in the
37 SKAP2 gene that were marked by enhancer-associated histone modifications in primary AML
38 samples. HOXA gene expression was maintained in the CTCF deletion mutants, indicating that
39 transcriptional activity at the HOXA locus in NPM1-mutant AML cells does not require long-
40 range CTCF-mediated chromatin interactions, and instead may be driven by intrinsic factors
41 within the HOXA gene cluster.
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42 Introduction
43 The HOX genes encode developmentally regulated transcription factors that are highly
44 expressed in acute myeloid leukemia (AML) and are important drivers of malignant self-renewal
45 in this disease. Understanding how HOX genes are regulated in AML may therefore provide
46 valuable information about the mechanisms that initiate and promote leukemogenesis. Previous
47 studies have shown that expression of HOX family members in AML is nearly always restricted
48 to specific genes in the HOXA and/or HOXB gene clusters (HOXC and HOXD genes are rarely
49 expressed), and that expression patterns correlate with recurrent AML mutations (1). HOX
50 expression is most closely associated with AMLs with MLL rearrangements, which exclusively
51 express HOXA genes, and AMLs with the recurrent NPM1c mutation, which nearly always
52 express both HOXA and HOXB cluster genes. The high prevalence of NPM1 mutations make
53 the combined HOXA/HOXB expression pattern the most common HOX phenotype in AML
54 patients. However, the regulatory mechanisms that drive this expression pattern are poorly
55 understood.
56
57 Studies of HOX gene regulation in model organisms have established that colinear expression
58 of each HOX cluster is mediated by COMPASS/Trithorax and Polycomb group proteins (PcG),
59 which promote gene activation and repression and perform methylation of histone H3 at lysine 4
60 (H3K4me3) and 27 (H3K27me3), respectively (2,3). These regulatory pathways are also
61 involved in HOX gene regulation in AML cells, and are best understood for the HOXA cluster in
62 AMLs with MLL rearrangements. MLL1 (KMT2A) is a component of the COMPASS complex,
63 and MLL fusion proteins bind to the HOXA locus in AML cells and recruit the non-COMPASS
64 histone H3 methyltransferase DOT1L, which is required for HOXA activation and AML
65 development in MLL-rearranged leukemia models (4–7). Regulatory DNA elements that control
66 three-dimensional chromatin architecture also play a role in HOXA gene regulation in AML cells.
67 Specifically, the HOXA and HOXB clusters contain multiple binding sites for the chromatin
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68 organizing factor CTCF, and chromatin conformation experiments suggest these events
69 mediate local chromatin loops in AML cells with MLL rearrangements (8). In addition,
70 heterozygous deletion of a single CTCF binding site in the HOXA cluster in MLL-rearranged
71 AML cells resulted in altered chromatin structure and reduced HOXA gene expression (9).
72 These studies suggest that MLL fusion proteins directly activate the HOXA locus in ways that
73 require specific CTCF binding events or their associated chromatin structures.
74
75 While these mechanistic insights have provided valuable information about HOXA regulation in
76 MLL-rearranged AML, this molecular subtype accounts for <5% of all adult AML patients and
77 only 25% of AMLs that express HOXA genes (1). Although AMLs with the NPM1 mutations
78 nearly always express HOXA genes (along with HOXB genes), it is unclear whether HOXA
79 expression in these cells shares similar regulatory factors and chromatin structures that appear
80 to be critical for HOXA expression in MLL-rearranged AML cells. In this study, we investigated
81 histone modifications and chromatin domains at the HOXA locus in NPM1-mutated AML
82 samples vs. other AML subtypes, and used a NPM1-mutant AML cell line model to determine
83 whether CTCF binding at multiple sites at the HOXA locus is required to maintain HOXA gene
84 expression and chromatin structure in NPM1-mutated AML cells.
85
86 Materials and Methods
87 Primary AML samples, normal hematopoietic cells, and AML cell lines. Primary AML samples
88 and normal hematopoietic cells were obtained from bone marrow aspirates from AML patients
89 at diagnosis or healthy volunteers following informed consent using protocols approved by the
90 Human Research Protection Office at Washington University and have been described
91 previously ((10,11); see Table S1). OCI-AML3 cells were cultured at 0.5-1 x 106 cell/mL in MEM-
92 alpha media with 20% FBS and 1% penicillin-streptomycin. The presence of NPM1c mutation in
93 the parental OCI-AML3 line was verified by targeted sequencing at the beginning of the study
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94 and in RNA-seq data from wild type and mutant clones. Kasumi-1, IMS-MS2, and MOLM13 cell
95 lines were cultured in RPMI-1640 with 1% penicillin-streptomycin and fetal calf serum (20% for
96 Kasumi-1 and MOLM13, 10% for IMS-MS2).
97
98 ChIP-seq preparation and analysis. ChIP-seq was performed using the ChIPmentation method
99 (12) with the following ChIP-grade antibodies: CTCF (2899S), H3K27me3 (9733S), and
100 H3K27ac (8173S) from Cell Signaling Technology and H3K4me3 (ab1012) from Abcam. All
101 libraries were sequenced on a NovaSeq 6000 (Illumina, San Diego, CA) to obtain ~50 million
102 150 bp paired end reads, and data were analyzed via adapter trimming with trim galore and
103 alignment to the GRCh38 human reference sequence using bwa mem (13). Preparation of
104 normalized coverage for visualization and analysis used the deeptools ‘bamCoverage’ tool (14),
105 and peaks were called with macs2 using default parameters (15) for CTCF and epic2 (16) for all
106 histone marks. Statistical comparisons were performed with DESeq2 (17) using raw fragment
107 counts at identified peak summits. Visualizations were prepared using the Gviz package in R
108 (18).
109
110 Targeted deletion of CTCF binding sites via CRISPR-Cas9. Targeted deletions in OCI-AML3
111 cells were generated using CRISPR/Cas9 with guide RNAs from the UCSC genome browser
112 (19,20) that overlapped CTCF ChIP-seq peaks from this study (see Table S2). Mutagenesis
113 was performed via either an inducible Cas9-expressing OCI-AML3 cell line (Lenti-iCas9-neo
114 vector; Addgene 85400) with lentiviral sgRNA expression (Addgene 70683), or transient
115 transfection of OCI-AML3 cells with Cas9 protein complexed with synthetic tracrRNA/crRNA
116 hybrids (Alt-R system, IDT, Coralville, IA). For the latter, RNAs were complexed with Cas9
117 protein using the manufacturer’s protocol with 1 million cells and 28 μM of Cas9/RNA for either
118 transfection (CRISPRMAX; Thermofisher Scientific, Waltham, MA) or nucleofection (SG Amaxa
119 Cell Line 4D-Nucleofector Kit, Lonza, Basel, Switzerland). Mutation efficiency was assessed in
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120 bulk cultures via DNA extraction, PCR with tailed primers (see Table S2), and sequencing to
121 obtain 2x250 bp reads on an Illumina MiSeq instrument. Deletion abundance was assessed
122 using custom scripts to analyze insertions/deletion in minimap2-based alignments (21). Single
123 cell sorting into 96 well plates via FACS was used for expansion of individual mutant clones.
124 Cells from single wells were screened via direct lysis by proteinase K (P8107S; NEB) in 20 μl of
125 single cell lysis buffer (10 mM Tris-HCl pH 7.6, 50 mM NaCl, 6.25 mM MgCl2, 0.045% NP40,
126 0.45% Tween-20), PCR amplification, and gel electrophoresis; clones with evidence for
127 deletions from the amplicon band size were sequenced, and clones with deletions were
128 expanded for analysis.
129
130 RNA analysis. RNA extractions were performed using ~1 million cells using Quick-RNA
131 MicroPrep Kit (Zymo Research, Irvine, CA). RT-qPCR for HOXA9 used 100 ng of RNA for
132 cDNA synthesis (Applied Biosystems, Foster City, CA) and qPCR in duplicate in a StepOnePlus
133 PCR System (Applied Biosystems) for HOXA9 exons 1-2 with a GUSB internal control (IDT).
134 RNA-seq libraries were generated using 300 ng of RNA with the Kapa Hyper total stranded
135 RNA library kit for Illumina (Roche) following the manufacturer’s instructions. Libraries were
136 sequenced on a NovaSeq 6000 to obtain ~50 million 2x150 bp reads. Reads were trimmed with
137 trim galore, aligned with STAR (22), and transcript-level expression values in TPM were
138 obtained with stringtie (23).
139
140 In situ Hi-C. Hi-C libraries were prepared as described previously (24) using 4-5 million cells as
141 input. Final libraries were first assessed by sequencing ~1 million reads on a MiSeq instrument
142 (using metrics recommended by Rao et al., (24)); passing libraries were sequenced to obtain
143 >400 million 2x150 bp reads on a NovaSeq 6000. Hi-C data were analyzed on GRCh38 with
144 juicer ((25); see Table S4). All analyses used contact matrices (mapping quality >30),
145 deduplicated chimeric reads from the ‘merged_pairs.txt’ file, and chromatin loops and contact
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146 domains identified using HICCUPS and arrowhead, respectively, with default parameters (25).
147 Chromatin loops from wild type and mutant OCI-AML3 cells were merged using bedtools
148 ‘pairToPair’ function (26) with 5,000 bp overlap. Pairwise and joint comparisons of chromatin
149 loop intensities were performed with hicCompare (27) and multiHicCompare (28), respectively,
150 using raw contact counts from chromosome 7 obtained from the juicer hic file at 25 kbp
151 resolution. Statistical comparisons used chromosome-wide FDR corrections for multiple
152 hypothesis testing. Visualizations used the HiTC (29), GenomicInteractions and Gviz R
153 packages.
154
155 Data availability. All processed sequence data from this study (e.g., bigwig files for ChIP-seq,
156 TPM values for RNA-seq, and Hi-C contact matrices) are available for public download at the
157 following site: https://wustl.box.com/v/ghasemiCTCFHOXA. Raw sequence files for cell lines will
158 be made available upon request.
159
160 Results
161 CTCF defines dynamic chromatin domains at the HOXA locus in primary AML cells
162 We previously showed that specific regions in the HOXA gene cluster have accessible
163 chromatin in primary AML samples that coincide with CTCF binding sites in other human cell
164 types (1). To confirm the presence of CTCF at these loci in primary AML cells, we performed
165 CTCF ChIP-seq on bone marrow samples from AML patients with the NPM1c insertion mutation
166 in NPM1, t(9;11) and t(11;19) MLL rearrangements, and t(8;21) creating the RUNX1-RUNX1T1
167 gene fusion, which displayed the expected HOXA and HOXB, HOXA only, and no HOX
168 expression patterns, respectively (Table S1, Figure S1A). This confirmed CTCF binding in
169 chromatin-accessible regions between HOXA6 and HOXA7 (“CTCF Binding Site CBSA6/7”),
170 and HOXA7 and HOXA9 (“CBSA7/9”), near the transcriptional start site of HOXA10 (“CBSA10”),
171 and in the 5’ UTR of HOXA13 (“CBSA13”) (Figure 1A). Additional CTCF peaks with lower
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172 signals were present near the promoters of HOXA4, and HOXA5. All four major CTCF peaks
173 were present in all AML samples regardless of HOXA expression status, and quantification of
174 the CTCF signal demonstrated similar occupancy among the different AML types (Figures 1B-
175 D).
176
177 We next determined whether these CTCF binding events defined chromatin domains in primary
178 AML samples by performing ChIP-seq for H3K4me3 and H3K27me3 to measure active and
179 repressed chromatin, respectively. This identified a distinct region of active chromatin in the
180 center of the HOXA cluster that overlapped CBSA6/7 and CBSA7/9, and was conserved in the
181 MLL-rearranged and NPM1-mutant AML samples, and normal CD34+ cells (which also express
182 HOXA and HOXB genes) (Figures 2A, S2A). The H3K4me3 signal was continuous across this
183 interval, including non-promoter sequences, and was also marked with H3K27ac in primary
184 AML samples (Figure S2B). Regions adjacent to this active chromatin domain possessed
185 repressive H3K27me3 marks in all AML samples and in CD34+ cells, which correlated with the
186 expression levels of the overlapping genes (see Figures S1A, S2A). Histone ChIP-seq using
187 AML samples with the RUNX1-RUNX1T1 gene fusion and low HOXA expression showed that
188 the active chromatin domain is dynamic, with little H3K4me3 signal and increased H3K27me3 in
189 this AML type (Figure 2B; blue tracks). This was also observed in ChIP-seq data from FACS-
190 purified normal promyelocytes and mature neutrophils (10) that also do not express HOXA
191 genes (Figures 2B, pink and green tracks, Figure S2A). Interestingly, H3K4me3 was not
192 completely absent in these cells, and similar H3K4me3 retention was observed in the RUNX1-
193 RUNX1T1 AML samples, although to a lesser extent. This suggests that regulation of HOXA
194 genes in both AML cells and normal hematopoietic cells is associated with a poised chromatin
195 state in CTCF-defined chromatin regions.
196
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197 Targeted deletions at the HOXA locus eliminate CTCF binding but do not affect viability
198 in NPM1-mutant OCI-AML3 cells
199 We sought to define the sequences that are required for CTCF binding at the HOXA locus, and
200 determine whether loss of these DNA elements has functional consequences in NPM1-mutant
201 AML cells. To this end, we used the OCI-AML3 cell line with a canonical NPM1 insertion
202 mutation and that expresses MEIS1 and genes in both the HOXA and HOXB clusters (ref. 30;
203 see Figure 3A). This pattern was unique to OCI-AML3 cells and was not observed in cell lines
204 with other mutation-associated HOX expression phenotypes, including MLL-rearranged
205 MOLM13 cells that expressed only HOXA genes, and the RUNX1-RUNX1T1-containing
206 Kasumi-1 cell line, which had low HOX expression. ChIP-seq for CTCF using OCI-AML3 cells
207 identified the four conserved CTCF sites observed in primary AML samples (Figures 3B, S3A)
208 and peaks in the anterior HOXA cluster that were variably present in the primary AML samples
209 (see Figure 1A). ChIP-seq for H3K4me3 and H3K27me3 also demonstrated an active chromatin
210 domain between HOXA9 and HOXA13 (Figure 3B), which was consistent with the patterns of
211 gene expression in this cell line, but different from chromatin domain boundaries in primary AML
212 samples. However, the histone modifications between CBSA7/A9 and CBSA10 were shared
213 between OCI-AML3 cells and NPM1-mutated primary AML samples, indicating that regulatory
214 mechanisms in this specific region are conserved between primary AML samples and OCI-
215 AML3 cells.
216
217 We next used CRISPR/Cas9-mediated editing to delete the three conserved CTCF binding sites
218 in OCI-AML3 cells (CBSA6/7, CBSA7/9 and CBSA10). The resulting mutations did not
219 appreciably alter markers of cell maturation in the edited cells (Figure S3B), and the deletion
220 frequency at CBSA7/9 and CBSA10 remained stable after 7 and 14 days; CBSA6/7 deletion
221 was less efficient, and showed a modest decrease in deletion frequency over time (Figure S3C).
222 Cells sorted from bulk cultures were expanded and screened for deletions, which identified at
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223 least 5 individual clonal lines with homozygous deletions at each of the three sites (see Table
224 S3), with deletions as small as 25, 10, and 9 bp sufficient to eliminate nearly all CTCF ChIP-seq
225 signal from CBSA6/7, CBSA7/9, and CBSA10, respectively (Figure 3C-H). Additional
226 experiments using single deletion mutants resulted in 10 doubly homozygous mutants and 7
227 triple mutants with homozygous deletions at all three sites (Figures 3I). None of the mutant OCI-
228 AML3 clones showed overt defects in growth or maturation status, despite complete loss of
229 CTCF binding in the posterior HOXA cluster.
230
231 CTCF binding is not required for maintenance of HOXA gene expression or chromatin
232 boundaries in NPM1-mutant AML cells
233 We selected 45 OCI-AML3 deletion mutants for HOXA9 expression analysis via RT-qPCR to
234 assess whether loss of CTCF binding affected HOXA9 expression. Surprisingly, HOXA9
235 expression was largely unchanged in all lines with deletions, with little evidence for consistent
236 reduction in expression across the homozygous mutant clones and no trend toward decreased
237 expression between wild type, heterozygous, and homozygous single mutants (Figure 4A) or
238 multiply mutated clones (Figure 4B). Expression analysis via RNA-seq showed modest
239 increases in the expression of the anterior HOXA genes HOXA1-HOXA7 in lines with CBSA7/9
240 deletions, and subtle increases in the posterior HOXA9-HOXA13 genes when CBSA10 was
241 deleted (Figures 4E, 4D, S4A). However, most mutant lines showed few expression changes.
242 Furthermore, a heterozygous single nucleotide polymorphism (SNP) in the HOXA9 gene
243 showed balanced expression of both alleles in wild type OCI-AML3 cells, and in all mutant lines,
244 except for two with large mutations that involved the posterior HOXA cluster (Figure S4B).
245
246 ChIP-seq for H3K4me3 and H3K27me3 was also performed on multiple mutants to determine
247 whether loss of CTCF binding altered chromatin boundaries. H3K4me3 signal was reduced
248 specifically at the CBSA7/9 site, but was otherwise intact. H3K27me3 was also still present, but
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249 was modestly decreased across the anterior HOXA genes in CBSA7/9 mutants; few changes
250 were evident in other mutant lines, including triple mutants (Figure 4G, 4H, S4C-S4E). Mutants
251 were also analyzed for other histone modifications, including H3K79me2 and H3K27ac, which
252 were intact compared to wild type OCI-AML3 cells (Figure S4F).
253
254 CTCF deletions result in compensatory HOXA chromatin loops
255 Given the role of CTCF in regulating chromatin architecture, we used in situ Hi-C (24) to define
256 the chromatin interactions at the HOXA locus in wild type OCI-AML3 cells, CBSA7/9 single
257 mutants, double mutants with deletions of CBSA6/7 and CBSA7/9 or CBSA7/9 and CBSA10,
258 and two triply mutant lines (see Table S4). Analysis of the chromatin contacts from these cells
259 identified a mean of 9,797 chromatin loops and 5,160 contact domains at 10 kbp resolution (ref.
260 25; Table S4). The HOXA cluster on chromosome 7p was located at the edge of a contact
261 domain boundary (Figure 5A), with centromeric loops connecting HOXA13 to the promoter of
262 TAX1BP1 and intronic sequences of HIBADH and JAZF1. The telomeric loops involved the
263 remaining HOXA genes (HOXA1-HOXA11), which contacted regions in the SKAP2, and SNX10
264 genes, and an intergenic region 1.4 Mbp upstream with no associated gene annotations. Similar
265 chromatin loops were observed in Hi-C data generated from the MLL-rearranged MOLM13 cell
266 line, and in previously reported data from normal human HSPCs (31) (Figures S5A, S5B), which
267 both express HOXA genes.
268
269 We next analyzed Hi-C data from the OCI-AML3 deletion mutants to determine whether loss of
270 CTCF binding altered chromatin architecture at the HOXA gene cluster. Although the general
271 contact domain and chromatin loop structure remained intact, loops involving the HOXA locus
272 were altered in the deletion mutants (Figure 5B). Pairwise comparisons of normalized
273 chromosome 7 interaction frequencies between each mutant line vs. wild type cells identified
274 decreased long-range interactions and increased interactions with the more proximal SKAP2
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275 gene (Figure 5B and S5C; red loops indicate chromosome-wide FDR<0.05). These findings
276 were confirmed via statistical analysis of joint normalized contact frequencies from all samples,
277 which demonstrated that mutant cells had significantly reduced interaction frequencies between
278 the HOXA cluster and SNX10 and the intergenic region, and increased interactions with two
279 SKAP2 loci (chromosome-wide FDR<0.05; Figure 5C). To define the specific HOXA genes
280 involved in these changes, we mapped the positions of HOXA-SKAP2 interacting reads within
281 the HOXA locus. This showed that SKAP2-HOXA loops involved the anterior HOXA genes and
282 intron 1 of SKAP2 in wild type cells (Figure 5D, top panel, shown in black); however, in mutant
283 cells there were increased interactions between the posterior HOXA genes HOXA9-HOXA13
284 and intron 11 of SKAP2 (Figure 5D, top panel, shown in red). Interacting reads with SNX10 and
285 the intergenic locus showed the opposite pattern, and were decreased in the posterior HOXA
286 cluster (Figure S5D), indicating that loss of CTCF binding resulted in ‘spreading’ of the proximal
287 SKAP2 interactions to include the posterior HOXA genes (Figure 5E). There were no clear
288 differences between cells with only CBSA7/9 deletions only, vs. double or triple deletions,
289 suggesting that CBSA7/9 is critical for defining long-distance chromatin interactions with the
290 anterior vs. posterior HOXA locus.
291
292 Posterior HOXA chromatin loops correlate with expression and involve enhancer loci in
293 primary AML samples
294 To determine the functional relevance of chromatin loops involving the posterior HOXA genes,
295 we performed in situ Hi-C on the Kasumi-1 cell line with low expression of HOXA genes (see
296 Figure 3A) to assess the correlation between chromatin architecture and posterior HOXA gene
297 expression. Although the interactions in these cells displayed similar overall patterns in the
298 HOXA region, the posterior HOXA genes did not participate in any statistically supported
299 chromatin loops (Figures 6A, 6B), and reads involved in HOXA contacts mapped primarily to the
300 anterior HOXA genes (Figure 6C). Loops to the posterior HOXA cluster were also present in the
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301 NPM1-mutant IMS-M2 cell line with high HOXA expression (Figures S6A, S6B), further
302 supporting the observation that expression of these HOXA genes is associated with long-
303 distance chromatin interactions in NPM1-mutant AML cells.
304
305 We next analyzed ChIP-seq data for H3K27ac from primary AML samples to determine whether
306 loci that interact with HOXA genes may have enhancer properties. Indeed, both intron 1 and 11
307 of SKAP2 displayed H3K27ac signals in NPM1-mutant AML samples, as well as MLL-
308 rearranged samples that also express HOXA genes (Figure 6D). Multiple loci in the SNX10
309 gene also possessed this mark, including a non-coding RNA downstream of the 3’ UTR of
310 SNX10 (Figure 6E), but H3K27ac was not present at other regions that formed contacts with the
311 HOXA cluster, including the distal intergenic locus (Figure S6C). Although the enhancer
312 modifications observed at interacting regions had relatively low signal, they were clearly absent
313 in the RUNX1-RUNX1T1 samples, which lack HOXA expression. This provides evidence that
314 these specific regions interact with the HOXA cluster in primary AML samples that express
315 HOXA genes, and may therefore contribute to HOXA gene regulation.
316
317 Discussion
318 HOX transcription factors are drivers of self-renewal in AML cells and are highly expressed in
319 AMLs with NPM1c mutations. In this study, we demonstrated that the chromatin organizing
320 factor CTCF is bound equally to specific sites at the HOXA locus in NPM1-mutant primary AML
321 samples compared to other AML subtypes, and define a dynamic chromatin domain in primary
322 AML samples and normal hematopoietic cells. Targeted deletions in the NPM1-mutant OCI-
323 AML3 cell line eliminated CTCF binding, but surprisingly did not disrupt HOXA gene expression.
324 This was true when multiple binding sites were deleted individually, or in combination, and was
325 supported by independent mutant clones that showed little evidence for consistent decreases in
326 HOXA gene expression or changes to the histone modifications we measured. However, loss of
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327 CTCF binding did result in clear alterations to the chromatin loops involving the posterior HOXA
328 genes, including HOXA9 and HOXA10. Specifically, long-range loops were diminished, and
329 were replaced by compensatory interactions with regions of the SKAP2 gene. Some of these
330 candidate enhancers have been reported in other studies (32), but have not previously been
331 shown to be active in hematopoietic cells. Further investigation of these sequences, and their
332 associated regulatory proteins, may shed light into factors that promote HOXA gene activation
333 in normal and malignant myeloid cells.
334
335 The central observation in this study is that CTCF binding at the HOXA cluster is not absolutely
336 required for maintenance of HOXA expression in NPM1-mutated AML cells. Deletion of CTCF
337 site CBSA7/9 was previously shown to affect HOXA expression in the MLL-rearranged
338 MOLM13 cell line (9). These discrepant findings may be due to fundamental differences in how
339 HOX genes are regulated in MLL-rearranged vs. NPM1-mutant AML cells. Indeed, the HOX
340 expression phenotype of these AML subtypes is strikingly different: MLL rearrangements are
341 associated with only HOXA gene expression, whereas both HOXA and HOXB genes are
342 expressed in NPM1-mutant AML cells. HOXA and HOXB genes are simultaneously
343 downregulated during normal myeloid maturation (1), which implies that the clusters may be
344 controlled by common factors that may function in different ways in NPM1-mutant vs. MLL-
345 rearranged AML cells. We also observed differences in HOXA cluster expression patterns in
346 MOLM13 vs. OCI-AML3 cells, including higher expression of HOXA11, HOXA13, and the
347 HOTTIP long noncoding RNA in OCI-AML3 cells. HOTTIP has an established role in HOXA
348 gene regulation (33,34), and its higher expression may influence the requirement for certain
349 CTCF-mediated chromatin architectures, thereby making these CTCF binding sites dispensable
350 for steady-state expression of posterior HOXA genes in NPM1-mutant AML cells.
351
14 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
352 Three-dimensional architecture is an important component of the regulatory control of HOXA
353 genes during development. Our analysis of Hi-C data from OCI-AML3 cells identified long-range
354 loops between the active posterior HOXA genes HOXA9-HOXA13 and sequences with
355 enhancer-associated epigenetic marks in the SNX10 and SKAP2 genes. Additional loops were
356 identified at other loci, but these did not possess active chromatin modifications in primary AML
357 samples. AML cells without HOXA expression did not display these loops, and lacked active
358 histone marks at the interacting loci, which provides evidence that these contacts may be
359 functionally relevant for HOXA gene expression in AML. Interestingly, loss of CTCF within the
360 HOXA cluster revealed the plasticity of these interactions, implying that multiple loci may serve
361 as exchangeable HOXA enhancers, which is reminiscent of other well-studied, complex
362 regulatory systems, and has been implicated in developmental HOXA gene regulation (35,36).
363
364 Our data showed that transcriptional activity and active histone modifications remained highly
365 localized to the posterior HOXA cluster in OCI-AML3 cells, even after elimination of key CTCF
366 boundaries. In fact, there was a tendency for deletion mutants to show increased HOXA gene
367 expression levels compared to wild type cells. This suggests the posterior HOXA cluster
368 possesses intrinsic properties that maintain its active state. It is unclear whether this activity is
369 caused by the compensatory interactions we observed, or if these chromatin loops are
370 consequences of persistent gene expression that is mediated by autonomous, sequence-
371 specific factors that recruit transcriptional machinery. The HOXA cluster contains many highly
372 conserved noncoding elements beyond CTCF binding sites. Additional studies will be required
373 to better understand whether these elements provide key signals that promote and maintain
374 HOXA gene expression in NPM1-mutant AML cells.
375
15 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
376 Acknowledgements
377 This work was supported by the American Society of Hematology (ASH Scholar Award), the
378 American Cancer Society (Institutional Research Grant), and the National Cancer Institute
379 (K08CA190815) to D.H.S. Primary AML samples were provided by the Genomics of AML
380 Program Project (P01CA101937, T. Ley, PI). Technical assistance was provided by the Siteman
381 Cancer Center Tissue Procurement and Cell Sorting cores (NCI Cancer Center Support Grant
382 P30CA91842).
383
384
16 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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21 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
512 Figure 1. CTCF is bound to chromatin accessible sites at the HOXA locus in primary AML
513 samples. A. Chromatin accessibility by ATAC (top tracks highlighted in white; ref. 1) and ChIP-
514 seq for CTCF (bottom tracks highlighted in yellow) from primary AML samples with either t(8;21)
515 creating the RUNX1-RUNX1T1 gene fusion (blue), MLL rearrangements (green), or a normal
516 karyotype and NPM1 mutation (purple). CTCF sites CBSA6/7, CBSA7/9, CBSA10, and CBSA13
517 are indicated by the dashed boxes. B-D. Scatter plots comparing the CTCF peak summit counts
518 between each AML type in log2 normalized read counts. Red points indicate ChIP-seq signal for
519 the four CTCF binding sites highlighted in panel A, which are similar across AML samples.
520 Dashed orange lines indicate a two-fold change between the samples.
521
22 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
522 Figure 2. CTCF defines chromatin boundaries in AML samples and normal CD34+ cells
523 with HOXA gene expression. A. Top track shows CTCF ChIP-seq from a NPM1-mutant
524 primary AML sample. Tracks highlighted in yellow show ChIP-seq for H3K4me3 from primary
525 samples with high HOXA expression, including AML samples with MLL rearrangements (green)
526 and NPM1 mutations (purple), and primary CD34+ hematopoietic stem/progenitor cells (HSPCs)
527 purified from normal donor bone marrow samples (gray; GSE104579). Tracks highlighted in
528 blue show H3K27me3 ChIP-seq from the same set of samples. B. H3K4me3 and H3K27me3
529 ChIP-seq from primary samples with no HOXA expression, including AML samples with t(8;21)
530 creating the RUNX1-RUNX1T1 fusion, and normal promyelocytes (CD14-, CD15+, CD16 low)
531 and neutrophils (CD14-, CD15+, CD16 high) from healthy donor individuals. Dashed box
532 indicates the region of dynamic chromatin that correlates with HOXA gene cluster expression.
533
534
23 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
535 Figure 3. Targeted deletions eliminate CTCF binding in the NPM1-mutant OCI-AML3 cell
536 line. A. RNA-seq expression of HOXA and HOXB genes in OCI-AML3 cells, which display the
537 canonical mutant NPM1-associated HOXA/HOXB expression phenotype. Also shown are the
538 MLL-rearranged MOLM13 cell line that expresses only HOXA genes, and the RUNX1-
539 RUNX1T1-containing Kasumi-1 cell line that has low HOXA and HOXB gene expression. B.
540 ChIP-seq data from OCI-AML3 cells for CTCF (black), H3K4me3 (highlighted in yellow) and
541 H3K27me3 (highlighted in blue), which show conserved CTCF binding sites and distinct regions
542 of active (H3K4me3) and repressed (H3K27me3) chromatin. C-E. Targeted deletions that
543 disrupt CTCF binding in OCI-AML3 cells at sites CBSA6/7 (in C), CBSA7/9 (in D), and CBSA10
544 (in E). Bottom panels show allele pairs from homozygous or compound heterozygous deletion
545 mutants at each site; top panels show CTCF ChIP-seq signal from these mutant cell lines (multi-
546 colored lines) compared to wild type OCI-AML3 cells (in gray). F-H. Normalized CTCF ChIP-seq
547 for all CTCF peaks from deletion mutants (Y axis) vs. wild type OCI-AML3 cells, showing
548 dramatically reduced CTCF ChIP-seq signal in deletion mutants at all three sites, with the
549 exception of clones A101 and A091.31, which only partially eliminates CTCF signal at site
550 CBSA6/7. I. CTCF ChIP-seq tracks from double (in purple) and triple mutants (in blue),
551 generated via sequential targeted deletion experiments. CTCF ChIP-seq from wild type OCI-
552 AML3 cells is shown in black at the top for reference.
553
554
24 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
555 Figure 4. CTCF binding is not required to maintain gene expression or chromatin
556 boundaries in the HOXA gene cluster. A. qPCR for HOXA9 in single mutants with
557 heterozygous or homozygous deletions at CBSA6/7, CBSA7/9, or CBSA10. HOXA9 expression
558 from the Kasumi-1 cell line is shown in blue as a no-HOXA9 expressing control. B. qPCR for
559 HOXA9 in double and triple mutants at the CTCF binding sites indicated. Kasumi-1 cells are
560 included in blue as in panel A. C-F. RNA-seq expression of all HOXA genes in single mutants
561 (C-E) and triple mutants (D). Expression level is shown in log2 transcripts per million (TPM). G.
562 ChIP-seq for H3K4me3 and H3K27me3 in deletion mutants lacking CTCF at site CBSA7/9 (in
563 red; N=2). Mean ChIP-seq signal from wild type OCI-AML3 cells (N=2) is shown in gray. H.
564 Mean ChIP-seq for H3K4me3 and H3K27me3 in triple mutants (N=2), with ChIP-seq from wild
565 type cells shown in gray, as in panel G.
566
25 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
567 Figure 5. CTCF-mediated chromatin architecture at the HOXA locus in NPM1-mutant OCI-
568 AML3 cells. A. Contact matrix, contact domains, and chromatin loops for a 2.5 Mbp region of
569 chromosome 7p that contains the HOXA gene cluster from in situ Hi-C using wild type OCI-
570 AML3 cells. Top panel shows the KR-normalized contact matrix at 5kb resolution. Tracks below
571 the matrix show the contact domains and statistically supported chromatin loops identified using
572 established methods (25). Loops shown in black are anchored within the HOXA gene cluster. B.
573 Contacts and HOXA region loops from in situ Hi-C from wild type OCI-AML3 cells and
574 homozygous (biallelic) single mutants at CBSA7/9, double mutants at CBSA6/7 and CBSA7/9,
575 and triple mutants at CBSA6/7, CBSA7/9, and CBSA10. Loops in red indicate statistically
576 significant differences in pairwise comparisons between each mutant and wild type cells with a
577 chromosome-wide FDR<0.05. C. Normalized interaction frequencies for all datasets for 6
578 telomeric loops. Top panel shows normalized interaction frequencies for individual Hi-C libraries
579 from wild type OCI-AML3 cells (N=2), single mutants at CBSA7/9, CBSA6/7-CBSA7/9 and
580 CBSA7/9-CBSA10 double mutants, and two CBSA6/7-CBSA7/9-CBSA10 triple mutant lines
581 (N=5 total mutant lines). Dashed boxes highlight loops that were statistically different in
582 comparisons of all mutant libraries vs. two wild type OCI-AML3 libraries (chromosome-wide
583 FDR<0.5). Mean interaction frequencies for chromatin loops from wild type and mutant lines are
584 shown graphically in the lower panel in solid and dashed lines, respectively. D. Relative read
585 depth (reads per million) across the HOXA locus for reads that interact between HOXA and
586 SKAP2. Depth for wild type OCI-AML3 cells is shown in black and deletion mutants are shown
587 in red. CTCF ChIP-seq signal from wild type OCI-AML3 cells and HOXA genes are shown in the
588 bottom tracks. CTCF site deletions result in new interactions between the posterior HOXA
589 cluster (genes HOXA9-HOXA13) with the SKAP2 gene. E. Graphical representation of HOXA
590 chromatin loops in wild type OCI-AML3 cells (left) and deletion mutants lacking HOXA CTCF
591 binding sites (right).
592
26 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
593 Figure 6. Long-distance interactions with posterior HOXA genes correlate with
594 expression and involve loci with enhancer-associated histone acetylation. A. Chromatin
595 interactions at chromosome 7p from the Kasumi-1 AML cell line that contains t(8;21)/RUNX1-
596 RUNX1T1 and has low expression of HOXA genes. The HOXA cluster resides at the boundary
597 of a topologically associated domain, but genes in the posterior HOXA cluster do not participate
598 in chromatin loops in this cell line. B. Focused view of HOXA chromatin loops in the Kasumi-1
599 and OCI-AML3 cell lines with low and high posterior HOXA expression, respectively, which
600 display the differences in chromatin loop structure and loop anchors. C. Relative read depth in
601 reads per million of Hi-C reads between the HOXA locus and telomeric chromatin loops in
602 Kasumi-1 (in blue) and OCI-AML3 cells (in black). Interacting reads in Kasumi-1 cells are
603 localized to the central HOXA cluster, compared to interacting reads in OCI-AML3 cells that
604 map to the central and posterior HOXA locus. D. Enhancer-associated histone H3 lysine 27
605 acetylation (H3K27ac) at HOXA interacting loci from primary AML samples with and without
606 HOXA gene expression. Primary AML samples include patients with NPM1c (purple; N=2
607 distinct patients) and MLL rearrangements (t(9;11) and t(11;19)) (green; N=2 distinct patients)
608 with high HOXA expression, and samples with t(8;21)/RUNX1-RUNX1T1 (N=2 distinct patients).
609 Regions highlighted in the dashed boxes were shown to interact with the HOXA cluster in the
610 OCI-AML3 cell line model (loop track, top), and display H3K27ac signal suggesting they may
611 represent functional genomic elements. E. High-resolution view of two loop anchor regions in
612 SKAP2 intron 1 and downstream of the SNX10 gene, which possess the enhancer-associated
613 H3K27ac mark in HOXA-expressing AML samples but not in samples with no HOXA
614 expression.
615
27 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 1
A RUNX1-RUNX1T1 MLLX NPM1c CBSA6/7 CBSA7/9 CBSA10 CBSA13
100 80 60 40 20 0 100 80 60 40 20 0 −120 15
Chromatin 10 accessibility 5 0 80 60 40 20 0 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 100 80 60 40 20 0 150 CTCF ChIP-seq 100 50 0 100 80 60 40 20 0 100 80 60 40 20 0 HOXA1 HOXA3 HOXA7 HOXA10 HOXA13 HOXA2 HOXA4 HOXA5 HOXA9 HOXA11 HOXA6 27.09 mb 27.11 mb 27.13 mb 27.15 mb 27.17 mb 27.19 mb
27.1 mb 27.12 mb 27.14 mb 27.16 mb 27.18 mb 27.2 mb B C D Normalized CTCF peak signal Normalized CTCF peak signal Normalized CTCF peak signal
● HOXA CTCF sites ● HOXA CTCF sites ● HOXA CTCF sites NPM1c RUNX1−RUNX1T1 RUNX1−RUNX1T1 6 8 10 12 6 8 10 12 14 6 8 10 12 14
6 8 10 12 6 8 10 12 6 8 10 12
MLLX MLLX NPM1c bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 2
A MLLX NPM1c CD34 100 80 60 CTCF ChIP-seq 40 20 0 200 150 100 50 0 120 100 80 60 40 20 0 100 80 60 40 20 0 150 100 50 0 60 50 40 30 20 10 0
H3K4me3 ChIP-seq 150 100 50 0 50 40 30 20 10
50 40 30 20 10
25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 5 0 25 20 15 10 H3K27me3 ChIP-seq 5 0 25 20 15 10 5 0 25 20 15 10 5 0 HOXA1 HOXA3 HOXA7 HOXA10 HOXA13 HOXA2 HOXA4 HOXA5 HOXA9 HOXA11 HOXA6 27.09 mb 27.11 mb 27.13 mb 27.15 mb 27.17 mb 27.19 mb
27.1 mb 27.12 mb 27.14 mb 27.16 mb 27.18 mb 27.2 mb B RUNX1-RUNX1T1 Pro PMN 100 80 CTCF ChIP-seq 60 40 20 0 40 30 20 10 0
30 20 10 0 50 40 30 20 10 0 50 40 30 20 10 0 80 60 40 20 H3K4me3 ChIP-seq 0 60 40 20 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 30 25 20 15 10 5 0 30 25 20 15 10 5
H3K27me3 ChIP-seq 0 30 25 20 15 10 5 0 HOXA1 HOXA3 HOXA7 HOXA10 HOXA13 HOXA2 HOXA4 HOXA5 HOXA9 HOXA11 HOXA6 27.09 mb 27.11 mb 27.13 mb 27.15 mb 27.17 mb 27.19 mb
27.1 mb 27.12 mb 27.14 mb 27.16 mb 27.18 mb 27.2 mb bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 3
CBSA7/9 A B CBSA6/7 CBSA10 100 80 OCI−AML3 80 MOLM13 60 40 Kasumi−1 CTCF 60 20 0
30 40 20 10 H3K4me3 0 30 20 25 20 RNA−seq expression (TPM) RNA−seq expression 15 10 0 5 H3K27me3 0 HOXA1 HOXA4 HOXA6 HOXA9 HOXA11 HOXA2 HOXA5 HOXA10
MEIS1 HOXA3 HOXA13 HOXA1 HOXA2 HOXA3 HOXA4 HOXA5 HOXA6 HOXA7 HOXA9 HOXB1 HOXB2 HOXB3 HOXB4 HOXB5 HOXB6 HOXB7 HOXB8 HOXB9 HOTTIP HOXA10 HOXA11 HOXA13 HOXB13 HOXA7
C D E CBSA6/7 CBSA7/9 CBSA10
100 WT OCI-AML3 100 WT OCI-AML3 100 WT OCI-AML3 80 80 80 60 60 60 40 40 40 20 20 20 CTCF signal CTCF signal CTCF signal 0 0 0 A100 A056 A073 A101 A058 A074 A076 A091.31 A059 A077 A091.33 A060 A079 A095.12.1 A091 A082 A095.12.2 A095 A083 700 800 A086 A089
600 F G H
I CBSA6/7 CBSA7/9 CBSA10 100 80 WT OCI-AML3 60 40 20 0 100 80 ∆CBSA6/7, ∆CBSA7/9 60 40 20 0 100 80 ∆CBSA7/9, ∆CBSA10 60 40 20 0 100 80 ∆CBSA6/7, ∆CBSA7/9, ∆CBSA10 60 40 20 0 100 CTCF ChIP-seq signal 80 ∆CBSA6/7, ∆CBSA7/9, ∆CBSA10 60 40 20 0 100 80 ∆CBSA6/7, ∆CBSA7/9, ∆CBSA10 60 40 20 0 HOXA1 HOXA3 HOXA7 HOXA10 HOXA13 HOXA2 HOXA4 HOXA6 HOXA9 HOXA11 HOXA5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 4
A B 0.6 HOXA9 RT-qPCR: single site deletions 0.6 HOXA9 RT-qPCR: multiple site deletions
0.4 ∆CBSA6/7 ∆CBSA7/9 ∆CBSA10 0.4 HOXA9 expression HOXA9 expression 0.2 0.2 Relative Relative
0.0 0.0
Kasumi-1 Kasumi-1 ∆CBSA6/7 ∆CBSA7/9 ∆CBSA6/7 Heterozygous Homozygous Heterozygous Homozygous Homozygous ∆CBSA7/9 ∆CBSA10 ∆CBSA7/9 WT OCI-AML3 WT OCI-AML3 ∆CBSA10
C CTCF A6/7 mutants D CTCF A7/9 mutants G 200 WT OCI-AML3 WT OCI-AML3 WT OCI-AML3 ∆CBSA7/9 (N=2) Deletion mutants Deletion mutants 150 ∆CBSA7/9 100 H3K4me3 DataTrack 50 ∆CBSA6/7 0 12 WT OCI-AML3 10 ∆CBSA7/9 (N=2) 8 6 4 RNA−seq expression (log2 TPM) RNA−seq expression RNA−seq expression (log2 TPM) RNA−seq expression DataTrack H3K27me3 2 0 2 4 6 8 0 2 4 6 8 0 HOXA1 HOXA4 HOXA7 HOXA11 HOXA2 HOXA5 HOXA9 HOXA13 HOXA1HOXA2HOXA3HOXA4HOXA5HOXA6HOXA7HOXA9 HOXA1HOXA2HOXA3HOXA4HOXA5HOXA6HOXA7HOXA9 HOXA3 HOXA10 HOXA10HOXA11HOXA13 HOXA10HOXA11HOXA13 HOXA6 E CTCF A10 mutants F CTCF A6/7, A7/9 & A10 mutants H 200 WT OCI-AML3 WT OCI-AML3 ∆CBSA10 WT OCI-AML3 ∆CBSA10 ∆CBSA6/7, ∆CBSA7/9, ∆CBSA10 (N=2) Deletion mutants Deletion mutants 150 ∆CBSA7/9 100 H3K4me3 DataTrack 50 ∆CBSA6/7 0 12 WT OCI-AML3 10 ∆CBSA6/7, ∆CBSA7/9, ∆CBSA10 (N=2) 8 6 4 RNA−seq expression (log2 TPM) RNA−seq expression RNA−seq expression (log2 TPM) RNA−seq expression DataTrack H3K27me3 2 0 2 4 6 8 0 2 4 6 8 0 HOXA1 HOXA4 HOXA7 HOXA11 HOXA2 HOXA5 HOXA9 HOXA13 HOXA1HOXA2HOXA3HOXA4HOXA5HOXA6HOXA7HOXA9 HOXA1HOXA2HOXA3HOXA4HOXA5HOXA6HOXA7HOXA9 HOXA10HOXA11HOXA13 HOXA10HOXA11HOXA13 HOXA3 HOXA10 HOXA6 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 5 A B Normalized interactions WT
0 5 I.F.
∆CBSA7/9
* *
MUT vs. WT P<0.05 I.F.
∆CBSA6/7 ∆CBSA7/9 *
MUT vs. WT P<0.05 I.F.
∆CBSA6/7 Contact domains ∆CBSA7/9 All loops ∆CBSA10 * HOXA loops
MUT vs. WT P<0.05 HOXA cluster I.F. HNRNPA2B1 SKAP2 HIBADH NFE2L3 EVX1 TAX1BP1 HOXA cluster HNRNPA2B1 SKAP2 EVX1 HIBADH CBX3 JAZF1 NFE2L3 TAX1BP1 SNX10 CBX3 JAZF1 SNX10 26 mb 27 mb 28 mb 26 mb 27 mb 28 mb
26.5 mb 27.5 mb 26.5 mb 27.5 mb C D HOXA-SKAP2 interacting reads ● WT OCI−AML3 ● Deletion mutants 5 ● WT OCI-AML3 Deletion mutants ● 3e-5 5e-3 0.02 ● 4 ● ● ● ● ● ● ●● ● ● ● ● ●●● 3 ● ● ● ● ● ●● ● ●● ● Norm. Freq. Interaction ● ● ●● ●● ●● ●●●●● 0 100 200
DataTrack 2
1 WT OCI-AML3 Relative read depth (rpm) Deletion mutants (mean) 0 CBSA7/9 CBSA10 CBSA6/7 100 80 CTCF ChIP-seq 60 40 Signal 20
HOXA cluster Normalized Norm. interaction freq. 0 HNRNPA2B1 SNX10 SKAP2 NFE2L3 HOXA1 HOXA4 HOXA6 HOXA9 HOXA11 CBX3 HOXA2 HOXA5 HOXA10 HOXA13 HOXA3 HOXA7 E TEL SNX10 SKAP2 SKAP2 CBSA7/9 deletion intergenic intergenic enhancer enhancer TEL SNX10 CEN CEN Anterior HOXA CTCF Posterior HOXA Anterior HOXA Posterior HOXA
CTCF bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952390; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 6 A B Kasumi-1 Kasumi-1 Normalized interactions SKAP2
0 5 SNX10
SKAP2 OCI-AML3
SNX10, distal enhancer
HOXA1 HOXA4 HOXA6 HOXA9 HOXA11 HOXA2 HOXA5 HOXA10 HOXA13 HOXA3 HOXA7 C HOXA interacting reads 5 WT OCI-AML3
See Panel B 4 Kasumi-1 HOXA cluster HNRNPA2B1 SKAP2 EVX1 HIBADH 3 NFE2L3 TAX1BP1 CBX3 JAZF1 SNX10 2 26 mb 27 mb 28 mb 1
26.5 mb 27.5 mb Relative read depth (rpm) 0 CBSA6/7 CBSA7/9 CBSA10 100 80 60 D 40 signal 20 HOXA proximal loops 0
NPM1c MLLX Normalized HOXA1 HOXA4 HOXA6 HOXA9 HOXA11 HOXA2 HOXA5 HOXA10 RUNX1-RUNX1T1 HOXA3 HOXA13 HOXA7
80 60 E 40 NPM1c MLLX RUNX1-RUNX1T1 20 0 HOXA loop anchor HOXA loop anchor 80 60 40 80 40 20 60 30 0 40 20 20 10 80 0 0 60 80 40 40 60 30 40 20 20 20 10 0 0 0 40 80 40 30 60 30 40 20 20 20 10 10 0 0 0 40 40 30 30 40 20 H3K27ac signal 20 30 10 10 20 0 0 40 40 H3K27ac signal 10 H3K27ac signal 30 30 0 20 20 40 10 10 30 0 0 40 40 20 30 30 10 20 20 0 10 10 0 0
SNX10 SNX10 SKAP2 HOXA cluster SKAP2 AC004540.2