bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
1 A coordinated function of lncRNA HOTTIP and miRNA-196b underpinning
2 leukemogenesis by targeting Fas signaling
3
4 Ajeet P. Singh1, 2 # *, Huacheng Luo1 *, Meghana Matur1, Melanie Eshelman1, Arati
5 Sharma3, 4, 5, Suming Huang1, 5 #
6
7 1Division of Pediatric Hematology/Oncology, Department of Pediatrics, Pennsylvania
8 State University, College of Medicine, Hershey, PA 17033, USA
9 2Department of Biochemistry & Molecular Biology, Pennsylvania State University, College
10 of Medicine, Hershey, PA 17033, USA
11 3Department of Medicine, Division of Hematology and Oncology, Pennsylvania State
12 University, College of Medicine, Hershey, PA, USA
13 4Department of Pharmacology, Pennsylvania State University, College of Medicine,
14 Hershey, PA, USA
15 5Penn State Cancer Institute, Pennsylvania State University, College of Medicine,
16 Hershey, PA 17033, USA
17
18 Key words: Long non-coding RNA, micro-RNA, Epigenetics, Cancer, Leukemia
19
20 *Equally contributed
21
22 #Correspondence: [email protected],
1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
24 Abstract
25 MicroRNAs (miRNAs) may modulate more than 60% of human coding genes and act as
26 negative regulators, while long non-coding RNAs (lncRNAs) regulate gene expression on
27 multiple levels by interacting with chromatin, functional proteins, and RNAs such as
28 mRNAs and microRNAs. However, the crosstalk between lncRNA HOTTIP and miRNAs
29 in leukemogenesis remains elusive. Using combined integrated analyses of global miRNA
30 expression profiling and state-of-the-art genomic analyses of chromatin such as ChIRP-
31 seq., (genome wide HOTTIP binding analysis), ChIP-seq., and ATAC-seq., we found that
32 miRNA genes are directly controlled by HOTTIP. Specifically, the HOX cluster miRNAs
33 (miR-196a, miR-196b, miR-10a and miR-10b), located cis & trans, were most dramatically
34 regulated and significantly decreased in HOTTIP–/– AML cells. HOTTIP bound to the miR-
35 196b promoter, and HOTTIP deletion reduced chromatin accessibility and enrichment of
36 active histone modifications at HOX cluster associated miRNAs in AML cells, while
37 reactivation of HOTTIP restored miR gene expression and chromatin accessibility in the
38 CTCF-boundary-attenuated AML cells. Inactivation of HOTTIP or miR-196b promotes
39 apoptosis by altering the chromatin signature at the FAS promoter and increasing FAS
40 expression. Transplantation of miR-196b knockdown MOLM13 cells in NSG mice
41 increased overall survival compared to wild-type cells. Thus, HOTTIP remodels the
42 chromatin architecture around miRNAs to promote their transcription, consequently
43 repressing tumor suppressors and promoting leukemogenesis.
44
45 Introduction
2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
46 Non-coding RNAs are emerging as important regulators of gene expression in multiple
47 cellular processes, especially in cancer1. In particular, long non-coding RNAs (lncRNAs)
48 are involved in regulating chromatin dynamics, gene expression, cell growth,
49 differentiation, and development2. Overexpression of HOXA9 is a dominant factor driving
50 certain subtypes of human acute myeloid leukemia (AML)3. Moreover, the aberrant
51 activation of posterior HOXA gene, HOXA9, and its cofactor, MEIS1, following a variety
52 of genetic alterations, including MLL-translocations, NUP98-fusion, CDX dysregulation,
53 and monocytic leukemia zinc-finger fusion, was associated with poor prognosis and
54 treatment response4. LncRNAs regulate transcription through recruiting histone modifiers
55 and chromatin remodeling factors that play active roles in various aspects of development
56 and disease state5-10. The lncRNA HOTTIP, located at the posterior end of HOXA gene
57 cluster, acts as a scaffold to recruit the WDR5-MLL-complex to the promoters of posterior
58 HOXA genes and positively regulates their expression in normal hematopoiesis and AML
59 leukemogenesis5,11. In contrast, loss of HOTTIP strongly inhibits posterior (HOXA9-
60 HOXA13) compared to anterior HOXA gene expression (e.g., HOXA1-HOXA7)11.
61
62 HOTTIP provides a basis for transcriptional activation and three-dimensional (3D)
63 chromatin organization in the 5’ HOXA gene loci by acting downstream of the CBS7/9
64 boundary (CTCF binding site located between HOXA7 and HOXA9)11. Our previous data
65 indicated that depleting HOTTIP reduces active histone marks (H3K4me3 and
66 H3K79me2) and enhances repressive histone marks (H3K27me3) resulting in a switch
67 from an active to a repressive chromatin state in the promoter region of the 5’ HOXA
68 genes in MOLM13 cells. Genome-wide binding analysis of HOTTIP lncRNA using
3 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
69 chromatin isolation by RNA purification combining deep sequencing (ChIRP-seq)
70 revealed that HOTTIP directly regulates its target genes through cis and trans binding at
71 HOXA9-13, RUNX1 and MEIS1 genes. Consequently, HOTTIP also binds in cis and trans
72 regulatory patterns to non-coding regions and certain annotated genes involved in
73 chromatin organization, hematopoiesis, myeloid cell differentiation, cell-cycle
74 progression, and JAK-STAT and WNT signaling pathways. These findings suggested that
75 HOTTIP lncRNA might control gene expression by interacting with and regulating non-
76 coding regulatory elements such as microRNAs (miRNAs) in cis and trans11.
77
78 Growing evidence indicates that non-coding RNAs, in particular lncRNAs and
79 microRNAs, regulate one another and cooperate to influence the levels of target mRNAs
80 in a cell-type specific manner12. LncRNAs process, interact with, and regulate miRNAs at
81 both transcriptional and post-transcriptional levels13. LncRNAs can function as miRNA
82 sponges, acting as decoys to impair the functional interaction of a miRNA and its target
83 mRNA, thereby preventing suppression of gene expression14. Additionally, lncRNAs can
84 be precursors of miRNAs and regulate miRNA biogenesis at different points. Primary
85 miRNAs (pri-miRNAs) are transcribed in the genome (i) either within the body of another
86 gene and often their expression is linked to the expression of the parent transcript, or (ii)
87 from independent miRNA genes, similar to mRNA, where transcription is primarily
88 controlled by RNA polymerase II-driven promoters15. However, the molecular
89 mechanisms, particularly those mediated by lncRNAs, regulating miRNAs transcription
90 remains elusive.
91
4 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
92 MicroRNAs (miRNAs) are 20-22 nucleotide non-coding RNAs that inhibit target gene
93 expression post-transcriptionally either by translational repression or mRNA degradation
94 via binding at complementary seed sequences mainly located at the 3’ UTR. Widespread
95 dysregulation of certain miRNAs associated with hematological malignancies, including
96 acute myeloid leukemia16. Besides the role of lncRNA HOTTIP in regulating
97 hematopoietic genes, miRNAs also control leukemic and tumor suppressor gene
98 expression17. In particular, miR-196b, which is located adjacent to HOXA9, targets
99 HOXA9 and its cofactor MEIS118. It also targets proapoptotic factor FAS19, suggesting a
100 double-edged sword (miR-196b) that could simultaneously repress both the oncogenic
101 and tumor suppressor target genes. Recent studies have shown the importance of miR-
102 196b in AML; however, the mechanism of its transcriptional regulation remains unknown
103 in AML. Thus, in this study we characterize the mechanisms by which HOTTIP controls
104 the expression and function of HOXA9 and FAS through miR-196b in human AML.
105
106 Results
107
108 HOTTIP lncRNA differentially regulates microRNAs in AML
109 To define the mechanism by which HOTTIP regulates gene expression in AML, we
110 compared genome wide microRNA expression patterns between wild-type (WT) and
111 HOTTIP–/– MOLM13 AML cells by performing small-RNA-sequencing (smRNA-seq)
112 analysis (Figure 1A). Differential expression analysis shows that a total 569 miRNAs
113 changed more than 2-fold in HOTTIP–/– cells compared to WT cells, including 333 down-
114 regulated miRNAs and 236 upregulated miRNAs (Figure 1B, C). These altered miRNAs
5 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
115 in HOTTIP–/– cells play vital roles in molecular and cellular processes, including
116 hematopoiesis and leukemogenesis, suggesting that HOTTIP controls AML pathogenesis
117 by regulating miRNAs. Gene ontology (GO) analysis revealed that HOTTIP-regulated
118 miRNAs were involved in many biological processes including the metabolic process,
119 regulation of gene expression, transcription, and RNA processing (Figure 1D).
120 Next, we sought to determine whether HOTTIP-regulated miRNAs control the
121 expression of HOTTIP-regulated mRNAs. In silico analysis revealed that HOTTIP-
122 regulated miRNAs are predicted to directly target 4347 mRNAs. We compared these with
123 863 mRNAs that we previously demonstrated are transcriptionally regulated by
124 HOTTPI11. Strikingly, 357 genes are co-regulated by both HOTTIP-associated miRNAs
125 and HOTTIP itself in AML, including hematopoietic and leukemic signature genes like
126 HOXA factors (e.g., HOXA9, HOXA13), MYC, ETS1, VEGFA, ZEB2, TEAD4, WNT5a,
127 WNT2 and FAS (Figure 1E, F).
128
129 Depletion of HOTTIP inhibits the HOX clusters’ miRNAs in cis – trans
130 Within the HOX cluster several non-coding transcripts, including miRNAs, are transcribed.
131 Six miRNAs are transcribed within the intergenic regions of the HOX clusters (Figure 2A).
132 Loss of HOTTIP impaired posterior HOXA gene expression (e. g. HOX13-HOXA9) but did
133 not affect anterior HOXA1-7 genes, HOXB, HOXC, or HOXD genes11. We asked whether
134 depletion of HOTTIP affects miRNAs that are transcribed in either cis or trans in the
135 intergenic region of the HOX cluster genes. Disruption of HOTTIP inhibits several HOX
136 cluster-related miRNAs, including miR-196b-5p, miR-196a-5p, miR-10b-5p, and miR-
137 10a-5p/3p (Figure 2B), suggesting these miRNAs are positively regulated by HOTTIP in
6 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
138 AML. Interestingly, while HOX cluster-associated miRNAs were altered, several HOX
139 genes targeted by these miRNAs remained unaltered in HOTTIP–/– cells (Figure 1E). We
140 performed correlation analysis of HOXA9, its cofactor MEIS1, miR-196b and HOTTIP
141 according to the RNA-seq dataset of acute myeloid leukemia samples18. Our data
142 analysis indicated that miR-196b correlates with expression of HOTTIP, posterior HOXA
143 genes, MEIS1, and PBX3, which are aberrantly overexpressed in MLL-associated AML
144 (Figure 2C, D). In contrast, pro-apoptotic genes, including FAS, CASP3, and CASP9,
145 were downregulated in MOLM13 cells relative to miR-196b (Figure 2D). Additionally, miR-
146 196a/b was enriched in MLL-AF9 MOLM13 cells compared to non-MLL-rearranged
147 leukemia cells such as KASUMI-1/t (8;21) (Figure 2E). However, the question remains
148 whether miR-196b targets pro leukemic gene HOXA9 in non-MLL-rearranged leukemia
149 cells. The co-expression of HOTTIP and miR-196b and their inverse co-relation with pro-
150 apoptotic genes suggests that HOTTIP and miR-196b may co-target these genes in MLL-
151 AF9 rearranged MOLM13 AML cells. Likewise, perhaps HOTTIP counteracts miR196b
152 mediated repression of HOXA9 in AML.
153
154 HOTTIP established chromatin signature on HOX clusters related miRNAs loci
155 To further define the mechanism by which HOTTIP regulates miRNA expression, we
156 employed ChIRP-sequencing (chromatin isolation by RNA purification) to identify
157 HOTTIP binding genome-wide11. Our previous HOTTIP CHIRP-seq data indicated that
158 HOTTIP mainly binds to non-coding regions, including promoter and intergenic regions.
159 The question then arises whether HOTTIP binds to regulatory loci of miRNA to induce the
160 differential expression we observed in HOTTIP–/– compared to WT MOLM13 cells. Our
7 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
161 data suggests that loss of HOTTIP greatly reduces its binding at the upstream promoter
162 region of miR-196b compared to WT MOLM13 cells (Figure 3A), which is correlated with
163 attenuated expression of miR-196b upon HOTTIP-depletion (Figure 2C). Furthermore, de
164 novo binding motif analysis revealed that the HOTTIP bound genomic locus at miR-196b
165 is occupied by transcription factors, CTCF, GATA-2, MYC and CEBPB, as well as many
166 other chromatin remodeling factors (Supplementary Figure 1).
167 Next, we asked whether HOTTIP controls the chromatin signature on its targeted
168 miRNA loci, using ChIP-seq (chromatin immunoprecipitation sequencing) for histone
169 modifications (H3K4me3, H3K79me2; active; and H3K27me3; repressive) and ATAC-seq
170 (assay for transposase-accessible chromatin using sequencing) to compare chromatin
171 signatures between WT and HOTTIP–/– MOLM13 cells11. Depletion of HOTTIP resulted
172 in marked decreases in H3K4me3 and H3K79me2 enrichment, while H3K27me3 levels
173 were elevated on the genomic loci of all four HOX cluster miRNAs (Figure 3B-E). The
174 changes in histone marks coincide with transcriptional changes (Figure 3B-E) and
175 HOTTIP binding pattern alteration. Concomitantly with decreased HOTTIP binding and
176 active histone marks in HOTTIP–/– cells, chromatin accessibility at regulatory loci of all
177 four HOX cluster miRNAs was also reduced (Figure 3G-K). Therefore, HOTTIP governs
178 its target genes by regulating the chromatin signature of specific miRNAs in the MLLr+
179 AML.
180
181 HOTTIP reactivation in CBS7/9+/– AML cells rescued the miRNA expression coupled
182 with restored chromatin signature
8 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
183 We previously demonstrated that HOTTIP coordinates with its upstream regulatory
184 element the CTCF binding site located between HOXA7 and HOXA9 (CBS7/9) to activate
185 posterior HOXA genes and other hematopoietic oncogenes11,20. Reactivation of
186 endogenous HOTTIP expression using sgRNA targeted dCAS9-VP160 mediated
187 promoter activation in the CBS7/9+/– MOLM13 cells efficiently restored posterior HOX
188 gene (HOXA9-HOXA13) expression. These findings led us to test whether HOTTIP
189 reactivation in CBS7/9+/– also affects miRNA expression and alters chromatin dynamics
190 at the miRNA loci. Indeed, levels of all HOX cluster miRNAs were significantly repressed
191 in CBS7/9+/– MOLM13 cells and completely restored in CBS7/9+/–-HT-VP160 cells with
192 reactivated HOTTIP (Figure 4A).
193 Next, we carried out ATAC-seq data analysis using WT, CBS7/9+/-, and HOTTIP-
194 reactivated CBS7/9+/– MOLM13 cells. Repression of HOTTIP in CBS7/9+/– cell reduced
195 chromatin accessibility at the HOX cluster-associate miRNA loci, while accessibility was
196 largely restored in HOTTIP-activated CBS+/–-HT-VP160 MOLM13 (Figure 5B). Hence,
197 HOTTIP function is critical for regulating the chromatin accessibility and expression levels
198 of HOX locus miRNAs.
199
200 miR-196b simultaneously targets oncogenes and tumor suppressors to maintain
201 MLL-AF9 AML
202 miR-196b represses a subset of targets with tumor suppressor activity in vivo and is
203 selectively enriched by cooperation with MLL-AF9 to promote leukemogenesis18.
204 Surprisingly, miR-196b also targets HOXA9 and MEIS1 that play essential oncogenic
205 roles18. Because HOTTIP positively regulates both miR-196b and its target HOXA9 and
9 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
206 MEIS111, we investigated the miR-196b function in leukemogenesis. Expression of miR-
207 196b was strongly suppressed in MOLM13 cells treated with LNA196b compare to NTC
208 (non-targeting control). Although, MOLM13 cells have high levels of HOXA9 expression,
209 we assessed the regulatory effects of miR-196b on HOXA9 and MEIS1 in MOLM13 cells.
210 Both HOXA9 and MEIS1 were significantly elevated upon miR-196b inhibition (Figure 5a).
211 Reduction of miR-196b also resulted in increased expression of HOXA13 in MOLM13
212 cells (Figure 5A). This suggests that repressive function of miR-196b might fine tune
213 expression of HOTTIP activated HOX genes in acute myeloid leukemia.
214 To further understand the pro-oncogenic role of miR-196b, we investigated the
215 expression of tumor suppressor FAS, which is a verified target of miR-196b in AML
216 subtypes and colon cancer cells18,19. Expression of FAS and its downstream genes
217 Caspase3 (CASP3) and Caspase9 (CASP9) was significantly elevated in cells treated
218 with LNA196b compare to NTC (Figure 5A). However, growth competition shows no
219 difference in the proliferation rate of cells treated with LNA196b compared to NTC.
220 Next, we assessed whether overexpression of miR-196b simultaneously
221 represses the expression of both oncogenes and tumor suppressors. Forced expression
222 of miR-196b significantly represses HOXA9, MEIS1, FAS, CASP3 and CASP9 in
223 MOLM13 cells (Figure 5B). To reveal the mechanism underlying the oncogenic role of
224 miR-196b, we analyzed the apoptosis rate in MOLM13 cells treated with either LNA196b
225 or NTC. Repression of miR-196b manifested a higher rate of apoptosis compared to NTC-
226 treated MOLM13 cells (Figure 5C). These results indicate that miR-196b promotes
227 leukemogenesis by down-regulating FAS, and its downstream genes, thus suppressing
228 apoptosis in AML.
10 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
229
230 HOTTIP inhibits FAS expression to maintain MLL-AF9 leukemia
231 We previously reported that enforced expression of HOTTIP aberrantly elevated HOXA9-
232 HOXA13 genes, resulting in impaired hematopoietic stem cell (HSC) function and an
233 increased leukemia stem cell (LSC) population in vivo11. Thus, we intend to define the
234 mechanisms by which HOTTIP might control the LSC pool. Since HOTTIP regulates the
235 chromatin signature and expression of miR-196b, which targets both oncogenes and pro-
236 apoptotic genes (e.g., FAS) simultaneously, we investigated whether HOTTIP also
237 targets FAS in AML. The mRNA level of FAS and its downstream pathway genes (e.g.,
238 CASP3, CASP8, and CASP9) significantly increased in HOTTIP–/– relative to WT
239 MOLM13 cells (Figure 6A). Further, we found that expression of the pro-apoptotic was
240 enhanced in CBS7/9+/– cells in which HOTTIP expression is significantly repressed
241 (Figure 6B). Intriguingly, when comparing CBS7/9+/– cells to WT cells (Figure 6B), mRNA
242 levels of CASP3 and CASP9 were significantly elevated in while CASP8 was unaffected.
243 We next tested whether CRISPR-mediated endogenous gene activation of HOTTIP in
244 these cells represses the pro-apoptotic genes. Reactivation of HOTTIP in the CBS7/9+/–
245 cells strongly repressed CASP3 and CASP9 while the CASP8 level remained unchanged
246 (Figure 6B). Next, we carried out western blot analysis using WT, HOTTIP–/–, CBS+/–, and
247 the HOTTIP-activated CBS7/9+/– MOLM13 cells. Cleaved CASP3 protein level was
248 markedly increased in HOTTIP–/– and CBS7/9+/– compare to WT cells, whereas the
249 Cleaved CASP3 protein level was undetectable upon HOTTIP reactivation, which closely
250 resembles WT MOLM13 cells (Figure 6C). Then we examined the elevated level of
251 cleaved CASP3 upon HOTTIP–/– induced apoptotic cell death in MOLM13 cells. FACS
11 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
252 analysis clearly showed an increase in apoptotic cell death in HOTTIP–/– cells compared
253 to WT cells (Figure 6D). Thus, HOTTIP directly controls AML cell survival and apoptosis
254 by regulating the FAS-Caspase axis.
255 To evaluate how HOTTIP directly regulates FAS expression, we investigated the
256 HOTTIP binding and chromatin signature at the promoter of FAS in WT, HOTTIP–/–,
257 CBS7/9+/–, and CBS7/9+/–HT-VP160 MOLM13 cell lines. Although, enrichment of the
258 HOTTIP binding on the FAS promoter occurs at relatively low level, HOTTIP depletion
259 affects histone modifications associated with active and repressive chromatin.
260 Consistently, relative enrichment of active histone modifications (H3K4me3 and
261 H3K79me2) was increased, whereas repressive histone modification (H3K27me3) was
262 decreased in HOTTIP–/– compare to WT cells. Additionally, chromatin accessibility, as
263 determined by ATAC-seq, was increased at the HOTTIP binding site on FAS in CBS7/9+/–
264 , whereas reactivation of HOTTIP reduced accessibility, resembling WT MOLM13 cells.
265 This suggests that HOTTIP maintains a repressive chromatin signature at the promoters
266 of tumor suppressor genes to promote leukemogenesis.
267
268 MiR-196b repression delays leukemogenesis in MOLM13 cell transplantation
269 miR-196b functions downstream of HOTTIP, and together they coordinate to repress FAS
270 tumor suppressor in MOLM13 cells. We previously showed that transplantation of
271 HOTTIP–/– MOLM13 cells into irradiated NSG mice delayed leukemogenesis compared
272 to WT MOLM13 cells. To further assess the function of miR-196b downstream of HOTTIP
273 in leukemogenesis in vivo, we knocked down miR-196b in MOLM13-YFP-luciferase cells
274 and then transplanted them into immunodeficient NOD-scid IL2rγnull (NSG) mice for
12 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
275 luciferase-based imaging of leukemogenic burden over time and, ultimately, survival
276 analysis. Inhibition of miR-196b inhibited leukemia cell engraftment, whereas
277 transplantation of WT MOLM13-YFP-luc cells showed remarkable expansion in vivo
278 (Figure 7A, B, C). We found that depletion of miR196b resulted in elongated survival (19
279 days, OS), whereas mice receiving WT MOLM13 cells died on day 15 (overall survival,
280 OS) (Figure 7D). The spleen size was dramatically smaller in mice receiving miR-196b
281 KD MOLM13-YFP-luc cells than in mice receiving WT MOLM13-YFP-luc cells (Figure
282 7E). Fluorescence-activated cell sorting (FACS) analysis showed that CD45+ bone
283 marrow (BM) and spleen cells were drastically reduced in mice receiving miR-196b KD
284 cells compare to WT cells (Figure 7F). Thus, miR-196b functions downstream of HOTTIP
285 and coordinates with the lncRNA to repress FAS signaling, and miR-196b inhibition
286 reduces the AML leukemia burden in vivo (Figure 7G).
287
288 Discussion
289
290 Long-non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have emerged as
291 biomarkers, drivers, and potential therapeutic targets for a wide array of complex
292 disorders, including leukemia16. A growing number of publications demonstrate that
293 miRNAs interact with lncRNAs thereby influencing their target gene expression21. The
294 role of lncRNAs in gene silencing is well established; however, less is known about their
295 function in active genomic loci where miRNAs are transcribed5. HOTTIP (HOXA transcript
296 at the distal TIP), a lncRNA, has been known to interact with WDR or WDR/MLL protein
297 complex to epigenetically regulate the 5’ HOXA gene by methylating histone H3K4, which
13 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
298 is associated with active transcription. Recent studies from our laboratory combining a
299 variety of molecular biology and bioinformatics techniques have revealed the regulatory
300 networks activated by HOTTIP in malignant hematopoiesis and acute myeloid leukemia.
301 Depletion of HOTTIP effectively inhibited the posterior HOXA genes (HOXA9-HOXA13),
302 and its inhibitory effects gradually diminished on genes towards the anterior end (HOXA1-
303 HOXA7)11,22. However, whether miRNA genes are regulated by HOTTIP in a similar
304 manner to protein coding genes remained a question relevant to hematopoiesis and
305 leukemogenesis. LncRNA can influence miRNA function in many ways: (1) lncRNAs can
306 serve as a source to produce mature miRNAs (2) lncRNAs act as sponge, binding miRNA
307 to prevent them from repressing their target mRNA genes and (3) lncRNA alter miRNA
308 gene transcription by binding on their promoter16. To profile HOTTIP regulated miRNA in
309 MOLM13 cells, we carried out small RNA sequencing that revealed that loss of HOTTIP
310 significantly affects expression of several miRNAs when comparing WT and HOTTIP–/–
311 MOLM13 AML cells. Intriguingly, several ncRNAs, including lncRNAs and miRNAs, are
312 transcribed from the intergenic regions of the HOX clusters. However, it remains unknown
313 how HOTTIP may mediate ncRNA transcription, particularly of miRNAs, and how the
314 HOTTIP-miRNA axis governs gene expression in malignant hematopoiesis.
315 HOXA9 expression is positively regulated by mixed lineage leukemia (MLL)
316 methyltransferase, which trimethylates histone 3 lysine 4 (H3K4me3) at the HOXA9
317 promoter23. This mechanism is directly antagonized by the sequential activity of polycomb
318 repressive complexes PRC1 and PRC2 that trimethylated histone 3 lysine 27
319 (H3K27me3). Moreover, topologically associating domains (TAD) of the HOX loci within
320 the nucleus also have an important role in coordinating expression. Long non-coding RNA
14 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
321 HOTTIP interacts with the WDR5-MLL complex and localizes the complex to the 5’HOXA
322 locus. To determine whether HOTTIP provides a basis for transcriptional activation and
323 three-dimensional (3D) chromatin organization in posterior HOX gene loci, we screened
324 all CTCF sites and lncRNAs important for HOXA9 expression within all four HOX gene
325 loci in MLL-AF9 rearranged MOLM13 AML cells using a CRISPR/CAS9 lentivirus
326 screening library11,24. The HOTTIP lncRNA acts downstream of the CBS7/9 boundary to
327 regulate expression of genes located in cis and trans, including HOXA13-HOXA9, MEIS1,
328 and RUNX1, which are important for hematopoiesis, and leukemia11. Furthermore,
329 ChIRP-seq analysis revealed that HOTTIP binds to its target genes11 and a cohort of
330 miRNAs. This data raises the question of whether HOTTIP also controls the expression
331 of miRNAs that are involved in the management of malignant hematopoiesis.
332 Our analysis demonstrated that inhibition of HOTTIP in MOLM13 cells significantly
333 inhibited several miRNAs, including HOX cluster miRNA, miR-196b, which targets both
334 oncogenic HOXA9 and the FAS tumor suppressor. Expression profiling of primary AML
335 patient samples showed strong correlation among miRNA, HOTTIP, and their target HOX
336 genes. Furthermore, ATAC-seq (assay for transposase-accessible chromatin using
337 sequencing) data analysis defined chromatin signatures at differentially expressed
338 miRNAs bound by HOTTIP in cis and trans, including HOX clusters miR-196b. HOTTIP
339 binding on the promoter region of miRNAs that are differential expressed in HOTTIP–/–
340 MOLM13 cells suggests that HOTTIP directly controls transcriptional regulation of
341 miRNAs.
342 Dynamics of chromosomal structure play important roles in gene control. A number
343 of proteins modulate chromatin dynamics by contributing to structural interactions
15 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
344 between gene promoters and their enhancer elements. Enhancer/promoter
345 communications for specific transcription programs are enabled by topological associated
346 domains (TADs), which are basically structural and functional chromosomal units. Often
347 inappropriate promoter/enhancer interactions result from altered TADs, which lead to
348 aberrant transcription of oncogenes or tumor suppressor genes. Binding of transcription
349 factor CTCF in chromatin boundaries plays an important role in defining TADs and
350 chromatin signature within TADs. We previously reported that CTCF binding located in
351 between of HOXA7 and HOXA9 defines posterior HOXA locus TADs and chromatin
352 signature within the TADs. Deletions of CBS7/9 impaired chromatin structure and altered
353 posterior HOXA gene expression due to lacking function of HOTTIP lncRNA11. By virtue
354 of CBS7/9’s role in regulating posterior HOXA genes and lncRNA HOTTIP expression,
355 we show that HOX cluster miRNAs are altered in CBS7/9–/– and that HOTTIP over-
356 expression restored CBS7/9-mediated HOX cluster miRNA expression and chromatin
357 signature. Apart from the cis coding miR-196b, HOTTIP lncRNA also bound and regulated
358 trans HOX and non-HOX cluster miRNAs.
359 To decipher the mechanisms by which HOTTIP exerts miRNAs to control target
360 genes, we performed bioinformatics analysis that revealed a large number of differentially
361 expressed genes in HOTTIP–/– MOLM13 cells, which are co-regulated by HOTTIP and
362 miR-196b. Although HOTTIP positively regulates expression of their co-expressed
363 oncogenes, several of them are negatively regulated by miR-196b. Thus, it seems that
364 negative regulation of the co-expressed 5’ HOXA genes in MOLM13 cells might be fine-
365 tuned by miR-196b in normal hematopoiesis18. Notably, single miRNA (or groups of
366 miRNAs) target multiple genes including oncogenes and tumor suppressors
16 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
367 simultaneously or sequentially. Partial repression of HOXA9 and MEIS1 by miR-196b in
368 the human MLL-rearranged leukemia may not potent enough to affect their function to
369 induce and maintain leukemia. The tumor suppressor targets (e.g., FAS) of miR-196b
370 could significantly inhibit cell transformation and leukemogenesis. Indeed, miR-196b
371 inhibition induced FAS expression and Cleaved Caspase-3. As a result, the apoptotic cell
372 death increased upon miR-196b knockdown in MOLM13 cells. Similarly, induced
373 expression of FAS and Cas3 in HOTTIP–/– and CBS7/9–/– is associated with increased
374 cell death. HOTTIP modulates epigenetic marks on the FAS promoter and thereby
375 controls chromatin accessibility and gene expression. However, HOTTIP represses FAS
376 expression perhaps through activation of miR-196b that directly targets FAS. miR-196b-
377 deficient MOLM13 cell transplantation in NSG mice delayed leukemogenesis. All mice
378 transplanted with WT MOLM13 cells died within 15 days, while mice receiving miR-196b-
379 depleted cells survived longer and exhibited fewer CD45+ cells. Transgenic
380 overexpression of HOTTIP lncRNA in mice affected HSC function and increased
381 leukemia stem cell (LSC) pool, inducing leukemia-like disease11. Thus, HOTTIP and miR-
382 196b deletion reduces the AML leukemic burden in vivo, and both coordinate to regulate
383 FAS expression at the transcriptional and post-transcriptional level to promote
384 leukemogenesis.
385 Taken together, we report a mechanism mediated by HOTTIP to regulate miR-
386 196b expression in AML. Our study revealed a regulatory model in which HOTTIP-miR-
387 196b axis repress expression of tumor suppressor FAS that circumvent the negative
388 effects of HOXA9 repression by miR-196b in AML. The aberrant upregulation of both
389 HOTTIP and miR-196b by MLL fusion results in the persistent repression of its tumor-
17 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
390 suppressor targets (e.g., FAS) along with dual control (transactivation and inhibition) of
391 their oncogenic co-targets (HOXA9/MEIS1). This inhibits differentiation, disrupts cell
392 homeostasis, and promotes cell proliferation via inhibiting apoptosis, consequently
393 maintaining leukemia stem cell pools. Apart from miR-196b, HOTTIP also bound and
394 regulated a subset of non-HOX cluster miRNAs while a subset of miRNAs was
395 upregulated in HOTTIP-inhibited MOLM13 cells. Future studies should aim to investigate
396 the mechanism by which HOTTIP modulates expression of candidate miRNA in
397 hematopoiesis vs leukemia.
398
399 Figure Legends
400
401 Figure 1. LncRNA-HOTTIP regulates miRNAs target genes control leukemogenic
402 program in AML cells. (A) Principal component analysis (PCA) of the miRNAs
403 differentially expressed in HOTTIP–/– vs WT AML cells. (B) Heatmap of miRNAs changed
404 >2-fold up- and down upon HOTTIP–/– by small-RNA sequencing (smRNA-seq). (C)
405 Select number of mRNAs up- and down regulated in HOTTIP–/– MOLM13 cells. (D) GO
406 (Gene Ontology) analysis of miRNAs differentially expressed in HOTTIP–/– cells. (E)
407 Number of genes regulated by either miRNAs or HOTTIP. (F) Select number of DEGs in
408 HOTTIP–/– are the direct target of miRNAs that are regulated by HOTTIP.
409
410 Figure 2. HOTTIP–/– perturbs HOX-cluster miRNAs, mediate oncogenic program. (A)
411 Schematics represent HOX cluster coding and non-coding genes. HOX genes are
412 indicated as numbered boxes, miRNAs are shown as triangles and lncRNAs are
18 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
413 presented as rectangles. (B,) Expression level of HOX cluster miRNAs in WT and
414 HOTTIP–/– MOLM13 cells. (C) Expression correlation between miR-196b, HOTTIP, and
415 HOXA genes in de novo AML and normal control dataset. (D) Relative expression level
416 of the indicated mRNAs, miRNAs and lncRNA in the MOLM13 cells. (F) Relative
417 expression level of the indicated HOX cluster miRNAs in MOLM13 and KASUMI cells.
418
419 Figure 3. HOTTIP controls the epigenetic modifications of HOX cluster miRNAs. (A)
420 ChIRP-seq analysis shows HOTTIP binding on promoter region of miR196b in WT and
421 HOTTIP–/– MOLM13 cells. (B, C, D, E, F) Enrichment of the active histone marks
422 (H3K4me3 and H3K79me2) and repressive histone mark (H3K27me3) on genomic loci
423 of the HOX cluster miRNAs. (G, H, I, J, K) ATAC-seq analysis shows chromatin
424 accessibility on the HOX-cluster miRNAs upon HOTTIP–/– in MOLM13 cells, and WT cells.
425
426 Figure 4. CBS7/9 boundary regulates HOX cluster miRNAs chromatin
427 neighborhood. (A) qRT-PCR analysis of HOX cluster miRNAs in WT, CBS7/9+/–, and
428 CBS7/9+/–+HOTTIP-VP MOLM13 cells. (B) ATAC-seq analysis shows chromatin
429 accessibility on the loci of HOX cluster miRNAs in WT, CBS7/9+/–, and dCas9-VP160-
430 mediated HOTTIP-activated MOLM13 cells.
431
432 Figure 5. Inhibition of miR-196b induced apoptosis in MOLM13 cells. (A) qRT-PCR
433 analysis of miR-196b targets in MOLM13 cells treated with either negative control or
434 locked nuclei acids (LNA) against miR-196b. (B) qRT-PCR analysis of miR-196b targets
435 in MOLM13 cells treated with either mimics of specific miRNA or control. (C) Bar graph
19 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
436 shows FACS (florescence activated cell sorting) evaluated percentage of annexin stained
437 apoptotic population of MOLM13 cells in control and miR-196b knockdown groups.
438
439 Figure 6. HOTTIP targets FAS, and CBS7/9 boundary play role in maintaining the
440 HOTTIP targets-chromatin neighborhood. (A) qRT-PCR of the genes associated with
441 apoptosis in WT and HOTTIP–/– MOLM13 cells. (B) Expression level of Caspase genes
442 in WT, CBS7/9+/–, and dCas9-VP160-mediated HOTTIP-activated MOLM13 cells. (C)
443 Western blot shows protein level of ß-actin and Cleaved Caspase-3 in WT, CBS7/9+/–,
444 and dCas9-VP160-mediated HOTTIP-activated MOLM13 cells. (D) Bar graph shows
445 percentage of FACS evaluated annexin stained apoptotic population in WT and HOTTIP–
446 /– MOLM13 cells. (E) ChIP-seq analysis shows histone marks enrichment on the FAS
447 promoter in WT and HOTTIP–/– MOLM13 cells. (F) ATAC-seq analysis of chromatin
448 accessibility on the FAS promoter in WT and HOTTIP–/– MOLM13 cells. (G) ATAC-seq
449 analysis of chromatin accessibility on the FAS promoter in WT, CBS7/9+/–, and dCas9-
450 VP-160-mediated HOTTIP-activated MOLM13 cells.
451
452 Figure 7. Inhibition of miR-196b inhibits in vivo leukemogenesis. (A) Schematic
453 representation of the MOLM13 transplantation in NSG mice and imaging. Luciferase
454 expressing MOLM13 cells either WT or miR196b KD were injected into NSG mice,
455 followed by in vivo imaging to assess the knockdown effects of miR-196b in
456 leukemogenesis (B, C) Representative images of leukemogenesis in NSG mice
457 transplanted with MOLM13 cells treated with either LNA196b or negative control. (D)
458 Overall survival (OS) of NSG mice injected with MOLM13 cells. (E) Images of spleens of
20 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
459 NSG mice 12 days after transplantion with MOLM13 cells treated with either LNA196B or
460 neg control. (F) Human CD45+ cells from bone marrow (BM) and spleen isolated 12 days
461 after transplantation were analyzed by FACS. (G) Model of HOTTIP/miR26b regulation
462 of AML. HOTTIP activation in MLLr+ AML activates expression of HOXA9, MEIS1, and
463 miR296b and suppresses FAS. miR296b, in turn, represses HOTTIP target gene to
464 maintain a proliferative state and drive leukemogenesis. In the absence of HOTTIP, these
465 pathways are inactive, therefore, HOXA9 and MEIS1 levels decrease, and FAS levels
466 increase, resulting in leukemic cells undergoing apoptosis.
467
468 Supplementary Figures
469
470 Supplementary Figure 1. UCSC gene track shows transcription factor binding motifs on
471 the miR-196b genomic locus and posterior HOXA genes.
472
473 Supplementary Figure 2. FACS analysis of annexin stained cells upon knockdown of
474 miR-196b, and control.
475
476 Supplementary Figure 2. FACS analysis of annexin stained HOTTIP–/– and wild type
477 MOLM13 cells.
478
479 Supplementary Figure 2. FACS analysis of CD45 stained bone marrow and spleen cells,
480 harvested from NSG mice transplanted with either WT or miR-196b knocked down miR-
481 196b MOLM13 cells.
21 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
482
483
484 Material and Methods
485
486 Tissue and Cell culture
487 MOLM13 cells were cultured and maintained in RPMI medium supplemented with 10%
488 FBS and 1% penicillin and streptomycin solution. All medium and supplements were
489 purchased from Thermo Fisher Scientific.
490
491 SsecCRISPR-Cas9 Mediated HOTTIP lncRNA Knock-Out and Lentivirus Production
492 The detailed protocol of generating HOTTIP knockout (KO) MOLM13 leukemia cells were
493 described previously in Luo et. al. 2019. In brief, CRISPR-RNA (crRNA) and tracrRNA
494 were mixed and annealed at 950C for 5 min and the cooled down to room temperature.
495 Subsequently, crRNA: tracrRNA duplex and S.p. Cas9 Nuclease components were
496 combined together and then mixed with 500,000 AML cells for electroporation with Neon®
497 System. The DNA was extracted after 24 hrs. or 96hrs. from 100ul of transfected cells
498 using Qiagen Quick Extract kit and processed for Sanger sequencing for verification of
499 mutation. The targeted deletion of HOTTIP: HOTTIP–/– -#1 targeted region is Chr7:
500 27241953-27241985; HOTTIP-/- -#2 targeted region is Chr7: 27240098-27240123.
501
502 dCas9-Mediated Overexpression of HOTTIP in AML Cells
503 HOTTIP promoter targeting guide RNA was designed using the Zhang laboratory web
504 tool (http://crispr.mit.edu, and cloned into lentiviral vector the pLKO5.sgRNA.EFS.tRFP
22 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
505 vector (Addgene #57824). The gRNA plasmid encoding puromycin was co-transfected
506 with a plasmid encoding dCas9-VP160 (pAC94-pmax-dCas9VP160-2A-puro, addgene
507 plasmid number #48226) in MOLM13 and OCI-AML3 cells. Transfected MOLM13 cells
508 were selected with 2ug/ml puromycin for 48 hrs. post-transfection, and then FACS sorted
509 for RFP+ cells. RNA was extracted from RFP+ cells, and gene expression was analyzed
510 by qRT-PCR using specific primer sets (Luo et al. 2019).
511
512 MiRNA Knockdown and Over Expression
513 Locked Nucleic Acids (LNA) and miRNA mimics and scrambled LNA/mimic (negative
514 control) was purchased from Qiagen and MOLM13 cells were transfected with either
515 LONZA nucleofector devise (program X-001) or Lipofectamine 3000 (Invitrogen). Cells
516 were harvested post 72 hrs. of transfection for RNA extraction and gene expression
517 analysis.
518
519 Western Blot Analysis
520 Whole cell lysate extract (total protein) was prepared using RIPA buffer and quantified
521 using Bradford method. Total protein lysates were fractionated on 4%-20% on tris-glycine
522 polyacrylamide gradient gel and transferred onto PVDF membrane. The bot was exposed
523 to specific antibodies to detect the endogenous protein level using chemiluminescence
524 method and BioRad imaging system: anti-ß-actin (A2066-100U); Sigma at 1: 5000, anti-
525 Cleaved Caspase-3 (9661); Cell Signaling at 1:1000 dilution.
526
527 Apoptosis Analysis
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528 MOLM13 cells transfected with either LNA against specific miRNA or scrambled non-
529 targeting control were seeded and cultured in 6-well dishes. Cells were harvested 72hrs
530 post transfection and washed with PBS. Apoptotic cells were detected by Accuri C6
531 fluorescence-activated cell sorting (FACS) using Annexin V-APC Apoptosis Detection Kit
532 (BD #), according to the manufacturers protocol.
533
534 RNA Extraction and Real-Time qPCR
535 Norgen Biotek RNA purification kit was used to isolate total RNA as per manufacturer’s
536 instructions. cDNA (reverse transcription - RNA to cDNA) was made using High
537 Capacity RNA to cDNA kit (Life Technologies, Grand Island, NY). miRNA and mRNA
538 qPCR were performed using TaqMan (Life Technologies) and Sybr Green assay
539 respectively with either Taqman Universal PCR Master Mix (miRNA qPCR) or BioRad
540 SsoAdvanced Universal Sybr Green Supermix according to the manufacturer’s protocol
541 on a Bio-Rad CFX96 Touch Real Time PCR Detection System (Bio-Rad Laboratories,
542 Richmond, CA). PCR reactions were performed in triplicate using either U6 (miRNA
543 qPCR), or GAPDH (human messenger RNA qPCR) as the normalizer.
544
545 Small RNA library preparation and sequencing analysis
546 The small RNA-seq of the MOLM13 WT and HOTTIP–/– cells was conducted at Genome
547 science Facility, Pennsylvania State University College of Medicine. Small RNA-seq.
548 libraries were prepared using BioO from Perkin Elmer and Qiagen kits. Small RNAs were
549 sequenced using a TruSeq Small RNA Sequencing Kit (Illumina, San Diego, CA, USA)
550 according to manufacturer instructions. The quality of libraries was assesses based on
24 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
551 size distribution and concentration using 2100 Bioanalyzer with DNA 1000 chip (Agilent
552 Technologies. All samples were sequenced 25M reads on an Illumina NovaSeq 6000
553 Sequencer using the 2x50bp paired-end platform.
554 All of raw binary base call files from the sequencer were transformed into FASTQ format
555 and de-multiplexing using Illumina bcl2fastq2 Conversion Software v2.20
556 (https://support.illumina.com/downloads/bcl2fastq-conversion-software-v2-20.html).
557 Quality of the sequenced reads was evaluated using FastQC developed by Babraham
558 Bioinformatics (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Then these
559 fastq files were performed by adapter trimming using the FASTQ Toolkit App version 1.0
560 of Illumna BaseSpace (http://basespace.illumina.com/apps/) and sequence alignment
561 with GRCh37 human genome assembly database using OASIS2.0
562 (https://oasis.dzne.de/index.php).
563 Differential expression analysis was performed using the DESeq2 algorithm 25 and the
564 expression was normalized using de Variance Stabilizing Transformation from the
565 DESeq2 algorithm in R. The differential expression of miRs with adjusted p values < 0.05,
566 and fold change (FC)≥2 representing positive log2FC (>1.0) and negative log2FC (<−1.0)
567 were considered to be significantly different.
568 The potential targets of miRs were derived from the miRTarBase 26 in human genome
569 (http://mirtarbase.cuhk.edu.cn/php/index.php). Raw sequencing data and miRNA
570 quantification tables can be accessed through GEO record #.
571
572 Chromatin Immunoprecipitation (ChIP) Assay
25 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
573 ChIP were performed as described in Luo et. al., 2019. Briefly, chromatin prepared from
574 MOLM13 cells were immunoprecipitated with antibodies against MLL1, H3K4me3,
575 H3K9me2, H3K27me3, and H3K79me2, separately. The MLL1, H3K4me3, H3K79me2
576 and H3K27me3, H3K79me2 ChIP-DNA libraries were prepared using illumina’s TruSeq
577 ChIP Sample Preparation Kit according to the manufacturers’ instructions (Cat # IP-202-
578 1012). Agilent TapeStation was used to check the quality of the libraries as per
579 manufacturer’s instruction. Final libraries were submitted to paired-end sequencing of 100
580 bp length on an Illumina HiSeq 3000.
581
582 Chromatin Isolation by RNA Immunoprecipitation (CHIRP) Assay
583 The detailed methods of CHIRP assay were described in Luo et. al. 2019, briefly
584 sonicated chromatin materials diluted using hybridization buffer and hybridized with
585 100pmol of biotinylated DNA probes targeting HOTTIP or LacZ containing 100 mL of
586 Streptavidin-magnetic C1 beads (Invitrogen). RNA and DNA hybrids and RNA binding
587 proteins were subjected to analysis by qRT-PCR and western blot with respective
588 antibodies respectively. Illumina’s TruSeq ChIP Sample Preparation Kit was used
589 according to manufacturer’s instructions for preparation of CHIRP libraries and submitted
590 to paired-end sequencing of 100 bp length on an Illumina HiSeq 2500. All sequencing
591 genomics datasets were deposited in the NCBI GEO under accession number
592 (GSE114981).
593
594 ChIP-seq and ChIRP-seq Data Analysis
26 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
595 ChIP-seq and ChIRP-seq data processing and analysis was described in Luo et. al. 2019.
596 Briefly, all sequencing tracks were viewed using the Integrated Genomic Viewer
597 (Robinson et al., 2011). Peaks annotation was carried out with the command
598 ‘‘annotatePeaks.pl’’ from HOMER package (Heinz et al., 2010). For ChIRP-seq motif
599 analysis, the de novo motif analysis was performed by the ‘‘findmotifsgenome.pl’’ from
600 the HOMER motif discovery algorithm (Heinz et al., 2010). The genes and pathways
601 regulated by the HOTTIP bound promoters or intergenic regions were analyzed and
602 annotated by the Gene Ontology analysis with the Database for Annotation, Visualization
603 and Integrated Discovery (DAVID) tool (https://david.ncifcrf.gov/, Version 6.8) (Huang da
604 et al., 2009a; Huang da et al., 2009b). Each GO term with a p value more than 1 x 10^-3
605 is used for cutoff (threshold: 10^-3). All genomics datasets were deposited in the NCBI
606 GEO under accession number (GSE114981).
607
608 Assay for Transposase-Accessible Chromatin Using Sequencing (ATAC-seq)
609 ATAC-seq was previously described in Luo et. al. 2019 using the Nextera DNA library
610 preparation kit. Libraries were quantified using qPCR (Kapa Library Quantification Kit for
611 Illumina, Roche), and purified with AMPure beads (Beckman Coulter). Quality of the DNA
612 library was examined by Agilent bio-analyzer 2100 prior to sequencing in duplicates with
613 2x100 bp paired-end reads on an Illumina NextSeq 500.
614
615 ATAC-seq Analysis
616 Detailed method of ATAC-seq analysis was previously described in Luo et. al. 2019.
617 Briefly, all sequencing tracks were viewed using the Integrated Genomic Viewer
27 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
618 (IGV/2.4.19) (Robinson et al., 2011). Peaks annotation was carried out with the command
619 ‘‘annotatePeaks.pl’’ from HOMER package (version 4.8) (Heinz et al., 2010) and GREAT
620 (McLean et al., 2010). DEseq2 (Benjamini-Hochberg adjusted p< 0.05; FoldChangeR2)
621 were also performed to find the differential binding sites between two peak files, including
622 control and treatment groups with C+G normalized and ‘‘reads in peaks’’ normalized data
623 (Ross-Innes et al., 2012). The de novo motif analysis was performed by the
624 ‘‘findmotifsgenome.pl’’ from the HOMER package (Heinz et al., 2010). For each genomic
625 feature (peaks or chromVAR annotation), we calculated the chromatin accessibility
626 median deviation z-score (for chromVAR features) or fragment counts (for peaks) in
627 control and treatment groups with chromVAR package in R language (Rubin et al., 2019;
628 Schep et al., 2017). Pearson’s correlation coefficient and Pearson’s c2-test were carried
629 out to calculate overall similarity between the replicates of ATAC-seq global open
630 chromatin signatures. All genomics datasets were deposited in the NCBI GEO under
631 accession number (GSE114981).
632
633 Xenotransplantation of Human Leukemic Cells and Patient-Derived Xenografts
634 (PDX)
635 MOLM13 cells - WT or miR-196b KD were injected into the tail vein of the NSG mice (2-
636 3 months old). Cells were resuspended into DPBS and injected at 5 x 105 cells (in 150 to
637 200ul DPBS) per mouse. Mice were monitored daily for symptoms of disease (ruffled
638 coat, hunched back, weakness and reduced motility) to determine the time of killing for
639 injected mice with sign of distress. Experimentally used NSG mice were humanely killed
640 at the time of moribund. Blood was collected into microtubes for automated process with
28 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
641 K2EDTA. bone (tibias, femurs and pelvis) and spleen were dissected. BM cells were
642 isolated by flushing the bones. Spleens were mashed through a 70-mm mesh filter and
643 made into single cell suspensions. Human CD45 (BD #) chimerism in these hematopoietic
644 tissues was analyzed by Accuri C6 flow cytometry.
645
646 Acknowledgements
647
648 This work was supported by the grants from National Institutes of Health (S.H.,
649 R01DK110108, R01CA204044, HL141950)
650
651 Author Contributions
652
653 A.P.S., H.L., and S.H. conceptualized the study and formal data analysis. A.P.S. designed
654 and performed the experiments. H.L. and M.E. contributed bioinformatics analysis. M.M.
655 helped with qRT-PCR. A.P.S. and A.S. performed mouse transplantation studies. Writing-
656 original draft, A.P.S., Review and editing, A.P.S., H.L., M.E., S.H., Visualization, A.P.S.,
657 H.L., M.E., S.H., Project supervision, A.P.S. and S.H...
658
659 References
660
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31 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
A C Figure 1
Up regulated Down 41% (236) regulated 59% (333)
B D WT KO Gene ontology enrichment for differentially expressed miRNAs - Biological Processes GO:BP_nucleobase-containing compound catabolic… GO:BP_RNA processing GO:BP_positive regulation of macromolecule… GO:BP_regulation of RNA metabolic process GO:BP_transcription, DNA-templated GO:BP_regulation of cellular metabolic process GO:BP_regulation of biosynthetic process GO:BP_negative regulation of biological process GO:BP_RNA catabolic process GO:BP_regulation of metabolic process GO:BP_cellular component biogenesis GO:BP_cellular nitrogen compound biosynthetic process GO:BP_cellular biosynthetic process GO:BP_organelle organization GO:BP_nucleic acid metabolic process GO:BP_organic cyclic compound metabolic process GO:BP_cellular metabolic process 0 2 4 6 8 10 12 14 16 18
CO TARGETS OF HOTTIP AND MIRs E F MYC HOXA1 S100A8 WNT5A WDR3 HSP90AA1 ACSS1 EHD1 R3HDM2 EIF1 UNG FADS1 FZD8 IGSF1 CD320 HIVEP2 KIF1B HSPA1B PSAT1 RRM2 863 357 4347 PTK2 ETS1 ZNF395 LRRC59 (29.2%) SQLE HK1 BCL3 ZNF493 SLC2A11 KIAA1147 CCNE2 TUSC1 FDPS NT5DC3 IGFBP2 HIPK2 RNA_seq_DEG TUBB4B CDRT4 VEGFA ROPN1L smRNA_seq_DEG_targets GPM6B PAM EGFL7 ATF7IP2 NUDT8 FMNL3 HOXA9 ZNF594 TUBA1B BMF MYO6 TMEM45A miR-10b- HOXB8, COX8A, HOXA10, NPC1, PHKA1 CCNE1 SOCS7 PLXDC2 miR-196a FLT3, AKT1, NPM1, DROSHA, MED13L TIMM10 ALDH3A1 ZNF426 AGO2, NFYC, PAX7, FAS JAZF1 RHOV F11R H1F0 OCLN STC2 BNIP3 WNT2 HOXA1, HOXA7, HOXA9, HOXA10, miR-196a NT5DC2 NRARP HLTF TEAD4 HOXB4, HOXB7, HOXB8, HOXC8, miR-196b HOXD4, HOXD10, FAS IRS2 SORT1 MAP2K6 CCNA1 PSMD2 HOXA13 ZEB2 CXCR2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.
miR-196b A CTCF Figure 2 HOXA 1 2 3 4 5 6 7 9 10 11 7p15.2 13 HOTAIRM1 HOTTIP miR-10a miR-196a-1 HOXB 1 2 3 4 5 6 7 8 9 17q21.32 13 PRAC miR-615 miR-196a-2 HOXC 4 5 6 8 9 10 12q12-q13 11 12 13 miR-10b HOTAIR HOXD 10 2q31.1 1 3 4 8 9 11 12 13
Transcription B C
MOLM13 (WT) MOLM13 (HOTTIP–/–) 1.5
1
0.5 ** ** 0 ** ** miR-196a miR-196b miR-10a miR-10b Rel. mRNA ex. level ex. mRNA Rel.
D MOLM13 0.08
0.06
0.04
0.02 Rel. expression
normalized to GAPDH 0
FAS Hottip HOXA9 MEIS1 CASP3 CASP9 miR-196bmiR-196a E 1.4 1.2 1 ** ** 0.8 0.6 MOLM13 0.4 KASUMI Rel. fold change 0.2 0
miR-196a miR-196b bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 3 A CHIRP-HT+/+ Binding - CHIRP-HT–/–-R1 CHIRP-HT–/–-R2
B HOTTIP H3K4me3-HT+/+ H3K4me3-HT–/– +/+
SEQ H3K27me3-HT - H3K27me3-HT–/–
ChIP H3K79me2-HT+/+ H3K79me2-HT–/–
C H3K4me3-HT+/+ H3K4me3-HT–/– H3K27me3-HT+/+ SEQ - H3K27me3-HT–/– H3K79me2-HT+/+ ChIP H3K79me2-HT–/– D H3K4me3-HT+/+ H3K4me3-HT–/– H3K27me3-HT+/+ SEQ - H3K27me3-HT–/– H3K79me2-HT+/+ ChIP H3K79me2-HT–/–
E H3K4me3-HT+/+ H3K4me3-HT–/– H3K27me3-HT+/+ SEQ - H3K27me3-HT–/– H3K79me2-HT+/+ ChIP H3K79me2-HT–/– F H3K4me3-HT+/+ H3K4me3-HT–/– H3K27me3-HT+/+ SEQ - H3K27me3-HT–/– +/+
ChIP H3K79me2-HT H3K79me2-HT–/– bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 3 G HOTTIP+/+ SEQ - HOTTIP–/–-R1
ATAC HOTTIP–/–-R2
H HOTTIP+/+
SEQ –/– - HOTTIP -R1
ATAC HOTTIP–/–-R2
I HOTTIP+/+
SEQ –/–
- HOTTIP -R1
ATAC HOTTIP–/–-R2
J HOTTIP+/+
–/– SEQ HOTTIP -R1 -
ATAC HOTTIP–/–-R2
K HOTTIP+/+
HOTTIP–/–-R1 SEQ -
–/– ATAC HOTTIP -R2 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeFigure 4 A B available under aCC-BY-NC-ND 4.0 International license.
miR-196b HOTTIP+/+ 2.5 * SEQ 2 - CBS7/9–/– 1.5 * RQV 1 ATAC HOTTIP-VP 0.5 0
HOTTIP+/+ SEQ - CBS7/9–/–
ATAC HOTTIP-VP
miR-196a HOTTIP+/+ 1.5 * * SEQ 1 - CBS7/9–/–
RQV 0.5 ATAC HOTTIP-VP 0
HOTTIP+/+ miR-10a
2.5 * SEQ - 2 CBS7/9–/– 1.5
* ATAC
RQV 1 0.5 HOTTIP-VP 0
HOTTIP+/+ miR-10b SEQ 2.5 - CBS7/9–/– 2 *
1.5 * ATAC
RQV 1 HOTTIP-VP 0.5 0 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made A available under aCC-BY-NC-NDB 4.0 International license. HOXA9 – MOLM13 HOXA13 – MOLM13 Figure 5 HOXA9 – MOLM13 MEIS1 – MOLM13 10 4 * 0.06 * 1.6 1.2 8 3 1.4 1 1.2 6 1 0.8 2 * * 0.8 0.6 RQV 4 RQV 0.6 * 1 * 0.4 2 0.4 * 0.2 * 0.2 * * 0 0 0 0
Cont Cont Neg contLNA196ALNA196B Neg contLNA196ALNA196B LNA196A+B LNA196A+B MIMIC196AMIMIC196B MIMIC196AMIMIC196B MIMIC196A+B MIMIC196A+B MEIS1 – MOLM13 FAS – MOLM13 FAS – MOLM13 Casp3 – MOLM13 10 50 * 1.4 1.4 8 40 1.2 1.2 1 1 6 30 0.8 0.8
RQV 0.6 0.6 RQV 4 * 20 * * 0.4 * 0.4 * * 2 10 * 0.2 * * 0.2 * 0 0 0 0
Cont Cont
Neg contLNA196ALNA196B Neg contLNA196ALNA196B MIMIC196AMIMIC196B MIMIC196AMIMIC196B LNA196A+B LNA196A+B MIMIC196A+B MIMIC196A+B Casp3 – MOLM13 Casp9 – MOLM13 Casp9 – MOLM13 120 16 1.2 100 * 1 12 * 80 0.8 60 8 0.6 RQV RQV 40 0.4 * * * 4 0.2 20 * * 0 0 0
Cont
Neg contLNA196ALNA196B Neg contLNA196ALNA196B MIMIC196AMIMIC196B LNA196A+B LNA196A+B MIMIC196A+B
% APC + cells C
* P-value 0.02678203
0 10 20 30 40 50 60 miR-196b KD WT bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 6 A 4 B MOLM13 C – 2.5 * / 3 * * – /+ – * 2 * WT ns Ladder WT HOTTIP CBS CBS.HT.VP 2 HOTTIP–/– 1.5 ns * ns WT 50kda β-actin : IB 1 ns CBS7/9+/– Rel. fold change 1 CBS HT VP Cleaved Rel. fold change 0.5 19kda Caspase-3 : IB 17kda 0 0
FAS Casp3 Casp8 Casp9 Casp3 Casp8 Casp9 D E H3K4me3-WT –/–
30 H3K4me3-HT H3K27me3-WT SEQ P-value - H3K27me3-HT–/– 0.24455934 H3K79me2-WT 25 ChIP H3K79me2-HT–/–
F 20 WT HOTTIP+/+ SEQ – / - –/–
– HOTTIP 15 HOTTIP–/– ATAC APC+ cells APC+ HOTTIP 10 G
HOTTIP+/+ 5 SEQ - CBS7/9-KO ATAC 0 HOTTIP-VP bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Figure 7 A B
MOLM13-YFP-luc MOLM13-YFP-luc miR196b KD WT
72 hrs
Tail vein Day5
D-Luciferin WT MOLM13-YFP-luc miR-196b KD MOLM13-YFP-luc
Imaging D Day 5, 12 Female C
N=4 C P-value 0.018126457
Male Day12
N=4 WT MOLM13-YFP-luc miR-196b KD MOLM13-YFP-luc P-value 0.0134
E WT miR-196b KD G
% CD45+ cells F Bone marrow
P-value Spleengiolitis0.25575772 Spleen P-value 0.31558326
0 10 20 30 40 50 miR-196b KD WT bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary Figure 1
Potential TF motifs in miR196b loci bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary Figure 2
Control unstained
LNA Control LNA 196B bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary Figure 3
D Control unstained
Wild Type Hottip–/– bioRxiv preprint doi: https://doi.org/10.1101/2021.07.15.452559; this version posted July 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Supplementary Figure 4 IgG WT
Spleengiolitis 196b KD - miR
Spleen CD45 WT 196b KD - miR
Bone marrow CD45