1
1 Functional Evidence Implicating Chromosome 7q22 Haploinsufficiency in
2 Myelodysplastic Syndrome Pathogenesis
3
4 Jasmine C. Wong1, Kelley M. Weinfurtner1, Pilar Alzamora1, Scott C. Kogan2, Michael R.
5 Burgess3,7, Yan Zhang4, Joy Nakitandwe5, Jing Ma5, Jinjun Cheng5, Shann-Ching Chen5,8,
6 Theodore T. Ho6, Johanna Flach6,9, Damien Reynaud6,10, Emmanuelle Passegué6, James
7 R. Downing5, Kevin Shannon1, *
8
9 Departments of 1Pediatrics, 2Laboratory Medicine, 3Division of Hematology/Oncology and
10 6Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research,
11 Department of Medicine, University of California, San Francisco, San Francisco, CA
12 94143, USA
13 4Unit of Hematopoietic Stem Cell and Transgenic Animal Models, Institut Pasteur of
14 Shanghai, Chinese Academy of Sciences, Shanghai 200025, China
15 5Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105,
16 USA
17 7Present address: Celgene Corporation, 1500 Owens Street, Suite 600 | San Francisco,
18 CA 94158, USA
19 8Present address: Thermo Fisher Scientific, South San Francisco, CA 94080, USA 2
20 9Present address: Institute of Experimental Cancer Research, Comprehensive Cancer
21 Center, 89081 Ulm, Germany
22 10Present address: Department of Pediatrics, Cincinnati Children’s Hospital Medical
23 Center, Cincinnati, OH 45229 USA
24
25 26 *Correspondence: Email address: [email protected], Tel (415) 476-7932; Fax:
27 (415) 502-5127
28 Running Title: Abnormal HSC Function in 5A3 (7q22) Mutant Mice
29 Word count: 1761
30 31 Competing Interests: No competing interests declared 3
32 Abstract 33 34 Chromosome 7 deletions are highly prevalent in myelodysplastic syndrome (MDS), and
35 likely contribute to aberrant growth through haploinsufficiency. We generated mice with a
36 heterozygous germline deletion of a 2 Mb interval of chromosome band 5A3 syntenic to a
37 commonly deleted segment of human 7q22, and show that mutant hematopoietic cells
38 exhibit cardinal features of MDS. Specifically, the long-term hematopoietic stem cell (HSC)
39 compartment is expanded in 5A3+/del mice, and the distribution of myeloid progenitors (MP)
40 is altered. 5A3+/del HSCs are defective for lymphoid repopulating potential and show a
41 myeloid lineage output bias. These cell autonomous abnormalities are exacerbated by
42 physiologic aging and upon serial transplantation. The 5A3 deletion partially rescues
43 defective repopulation in Gata2 mutant mice. 5A3+/del hematopoietic cells exhibit
44 decreased expression of oxidative phosphorylation genes, increased levels of reactive
45 oxygen species, and perturbed oxygen consumption. These studies provide the first
46 functional data linking 7q22 deletions to MDS pathogenesis.
47
48
49 4
50 Introduction
51 The myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized
52 by ineffective hematopoiesis, morphologic dysplasia, and a variable risk of progression to
53 acute myeloid leukemia (AML)(1). Monosomy 7 (-7) and deletions affecting the long arm of
54 chromosome 7 [del(7q)] are highly prevalent acquired cytogenetic abnormalities in de novo
55 and in therapy-related MDS and AML (t-MDS/t-AML)(2). The proportion of -7/del(7q) cells
56 is markedly increased in the hematopoietic stem cell (HSC) and progenitor compartments
57 of MDS patients relative to T and B lymphocytes (1, 3-6). Recent studies demonstrating
58 quantitative changes in the frequencies of phenotypic primitive long-term HSCs, common
59 myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) in MDS patients
60 with -7/del(7q) further support diverse effects on hematopoiesis (3, 7).
61 Multiple studies of MDS and AML specimens with interstitial deletions on 7q have
62 implicated three putative commonly deleted segments (CDS) at chromosome bands 7q22,
63 7q34 and 7q35-36 (Figure 1A)(8-15). Of these intervals, 7q22 is deleted most frequently in
64 cases of MDS or AML with a del(7q)(8). Targeted sequencing of candidate myeloid tumor
65 suppressor genes (TSGs) located within a 2.5 Mb 7q22 CDS delineated by Le Beau et alia
66 (8), and recent comprehensive genomic analyses of clinical specimens strongly implicates
67 a haploinsufficient role of 7q22 deletions in leukemogenesis (9, 10, 12-17). Consistent with
68 this proposed mechanism, biallelic inactivation of any 7q gene is rare in MDS patients with
69 -7/del(7q). For example, whole exome sequencing (WES) of 68 myeloid malignancies
70 characterized by -7/del(7q) uncovered recurrent mutations in only EZH2 (located at 7q36;
71 n =4) and LUC7L2 (at 7q34; n =3). Biallelic inactivation of these genes and of CUX1
72 (located at 7q22) was observed in a small cohort of patients with 7q isodisomy (9). 5
73 SAMD9L, RASA4, DOCK4 and MLL3 are other 7q genes that have been implicated as
74 contributing to leukemogenesis by haploinsufficiency or epigenetic transcriptional
75 repression (Figure 1A)(18-22). Here we demonstrate hematopoietic abnormalities in mice
76 with a germline deletion of a contiguous commonly deleted segment (CDS) of
77 chromosome band 5A3 (5A3+/del) syntenic to a 2.5 Mb 7q22 CDS delineated by Le Beau et
78 alia (8) that support a mechanistic role of 7q22 deletions in MDS pathogenesis.
79 Results and Discussion
80 Abnormal Differentiation and Repopulation of 5A3+/del Stem and Progenitor Cells
81 We generated mice carrying a 2 Mb germline 5A3 deletion that removes 13 genes
82 syntenic to a human 7q22 CDS (Figure 1A)(23). 5A3+/del mice are smaller than wild-type
83 (WT) littermates, and homozygous deletion of the 5A3 region causes embryonic lethality
84 before 10.5 dpc (data not shown). Total nucleated bone marrow (BM) cell counts as well
85 as spleen and thymus weights are reduced in mutant animals (Figure 1 B - D), which
86 maintain normal peripheral blood cell counts.
87 Differential expression of CD150 distinguishes HSC populations with different self-
88 renewal, differentiation, and repopulating potentials. Specifically, HSCs with a surface c-
89 kit+, lineage-, Sca-1+ (KLS) and CD150hi (CD150hi HSC) immunophenotype possess potent
90 self-renewal capacity, are predisposed to myeloid differentiation, and expand upon aging
91 (24-26). Strikingly, the proportion of CD150hi HSCs is increased in 5A3+/del BM with a
92 corresponding decrease in the percentage of CD150 negative multi-potent progenitors
93 (CD150neg MPP)(Figure 1E, Figure 1 – Figure Supplement 1A). This results in a normal
94 frequency of CD150hi HSCs in 5A3+/del mice, despite an overall reduction in the size of the
95 stem/progenitor compartment (Figure 1F). The proportion of common myeloid progenitors 6
96 (CMP) is elevated in 5A3+/del mice and the frequency of granulocyte-macrophage
97 progenitors (GMP) is decreased (Figure 1G – H, Figure 1 – Figure Supplement 1B), which
98 is consistent with changes in these populations in MDS patients (3, 7). Thus, the 5A3
99 deletion perturbs HSC and myeloid progenitor populations. By contrast, the proportions
100 and frequencies of common lymphoid progenitors (CLP) are similar in WT and 5A3+/del
101 mice (Figure 1 – Figure Supplement 1C - E).
102 We mixed donor 5A3+/del or WT BM cells with WT BM at ratios of 1:1 and 1:2, and
103 transplanted them into irradiated recipients. Whereas 5A3 mutant BM had markedly
104 reduced lymphoid repopulating capacity, these cells efficiently contributed to the c-kit+lin-
105 Sca-1+ (KLS) compartment (Figure 2 A, B). To investigate if the altered repopulating
106 potential of 5A3+/del BM is intrinsic to CD150hi HSC, we injected 15 of these cells into
107 lethally irradiated recipients with WT BM competitors. Similar to whole BM, CD150hi HSC
108 from 5A3+/del mice exhibited reduced overall repopulation potential due to defective
109 lymphoid reconstitution (Figure 2C - F). Importantly, however, 5A3+/del HSC efficiently
110 reconstituted KLS and myeloid compartments in both primary and secondary recipients
111 (Figure 2E - F). 5A3+/del CD150hi HSC exhibited markedly reduced lymphoid repopulating
112 potential in WT and 5A3+/del recipients, whereas WT cells restored lymphoid repopulation in
113 5A3+/del hosts almost as well as in WT recipients, demonstrating that the repopulation
114 defects in 5A3+/del CD150hi HSCs are cell intrinsic (Figure 2C - F).
115 WT and 5A3+/del HSCs generated similar numbers of myeloid colonies in
116 methylcellulose cultures supplemented with cytokines; however, colonies grown from
117 5A3+/del CD150hi HSC contained significantly fewer cells (Figure 2G, H). By contrast,
118 5A3+/del CD150neg MPP showed a 1.4-fold increase in colony forming activity but a similar 7
119 number of cells per colony as WT MPP (Figure 2G, I). In vivo labeling experiments
120 revealed similar rates of BrdU incorporation, cell division, and apoptosis in WT and 5A3+/del
121 K+L-S+ and MP cells (Figure 2 – Figure Supplement 1A – B and data not shown)(27).
122 Effects of Aging on 5A3+/del HSC and Interaction with Gata2 Haploinsufficiency
123 Physiologic aging is characterized by impaired HSC repopulating potential,
124 diminished lymphoid differentiation, the dominance of CD150hi HSCs that are skewed
125 toward myeloid differentiation, and a markedly increased risk of MDS (26, 28). Similarly,
126 the abnormal distribution of HSCs is exacerbated in 24 - 30 month old 5A3+/del mice
127 (Figure 3 A, B). Consistent with data from younger mice (Figure 2 A - F), aged 5A3 mutant
128 BM displayed markedly reduced lymphoid repopulating potential, but efficiently contributed
129 to myeloid reconstitution (Figure 3C, D). Old 5A3+/del BM cells also repopulated the KLS
130 compartment significantly better than WT BM in recipients analyzed 4 months after
131 transplantation, and 5A3+/del cells exhibited a two-fold increase in contribution to the KLS
132 and MP populations upon serial transplantation (Figure 3C, D). Despite these HSC
133 abnormalities, 5A3+/del mice have a normal lifespan, and the underlying causes of death
134 are similar to WT littermates (data not shown).
135 GATA2 mutations cause familial MDS, which is frequently characterized by -
136 7/del(7q)(29-31). We generated Gata2+/-; 5A3+/del mice, quantified HSC and progenitor
137 populations, and performed competitive repopulation experiments. Like 5A3+/del mutant
138 mice, Gata2+/-; 5A3+/del mice have reduced BM cellularity and spleen weights (Figure 3E,
139 F), and the CD150hi HSC bias is augmented by concomitant Gata2 deletion (Figure 3G).
140 Whereas lymphoid reconstitution is similarly impaired in 5A3+/del and compound mutant
141 mice, the 5A3 deletion partially rescues the repopulating deficit of Gata2+/- BM in the KLS, 8
142 MP, and myeloid compartments (Figure 3H)(32). These data suggest that the 7q22 CDS
143 contributes to transformation in familial MDS by impairing lymphoid differentiation while
144 also modestly enhancing the growth of GATA2 mutant HSC and their myeloid progeny.
145 Changes in Gene Expression and Metabolic Activities in 5A3+/del Hematopoietic Cells
146 Transcriptome (RNA-Seq) and Taqman quantitative real-time PCR analyses
147 revealed a ~50% reduction in the expression of multiple genes and of the long intergenic
148 non-coding RNA 503142E22Rik within the 5A3 interval in mutant HSC and MPP (Figure
149 4A, B)(33). Gene Set Enrichment Analysis (GSEA) of the RNA-Seq data from 5A3+/del
150 HSCs further demonstrated reduced expression of multiple gene sets related to oxidative
151 phosphorylation (OXPHOS) that are similarly down-regulated in the early stages of human
152 therapy-induced MDS and AML (Figure 4C)(34, 35). OXPHOS generates ATP, is
153 regulated by mitochondrial membrane potential, and defects in this metabolic pathway can
154 increase levels of reactive oxygen species (ROS). However, sorted WT and 5A3+/del HSC
155 and MPP showed similar ATP levels (Figure 4D). Membrane potential and intracellular
156 ROS levels were also similar in HSC and MPP from young mice, but ROS levels were
157 increased by ~50% in the HSC and MPP of aged 5A3+/del mice (Figure 4 E, F). Elevated
158 ROS levels in HSCs correlate with reduced self-renewal capacity, impaired multi-lineage
159 repopulating ability, and myeloid-biased differentiation (36). ROS levels are also increased
160 in t-MDS/AML BM (35, 37).
161 Down-regulation of OXPHOS genes is expected to reduce mitochondrial respiration
162 in HSC (38). We attempted to directly measure oxygen consumption rates (OCR) in
163 CD150hi and CD150lo HSC, but could not obtain reproducible results due to limiting cell
164 numbers. We therefore compared the OCRs of KLS, MP, B and T cells isolated from one 9
165 year-old WT and 5A3+/del mice. 5A3+/del KLS cells showed a similar basal OCR as their WT
166 counterparts, but a slightly lower maximal respiratory capacity that did not reach statistical
167 significance (p=0.2562)(Figure 4G). 5A3+/del MPs surprisingly showed a significantly higher
168 basal respiration and maximal respiratory capacity in comparison to WT cells, supporting
169 an overall increase in energy consumption (Figure 4H). Meanwhile, mature 5A3+/del B and
170 T cells have similar mitochondrial stress profiles as WT cells. We conclude that global
171 changes in OXPHOS gene expression exert a modest impact on cellular metabolism in
172 aged 5A3 mutant HSC and progenitors. Interestingly, treatment with N-acetyl-L-cysteine
173 (NAC) did not reverse the hematopoietic abnormalities in young 5A3+/del mice (data not
174 shown), suggesting that they are a direct consequence of the 5A3 deletion and are not
175 secondary to ROS production.
176 Segmental deletions are among the most frequent genetic alterations in human
177 cancer, and simultaneous loss of multiple haploinsufficient TSGs that individually have
178 minimal phenotypic consequences appears to underlie the growth advantage conferred by
179 most of these chromosomal losses (39). We show that a haploinsufficient deletion in mice
180 that models loss of a human 7q22 CDS causes hematopoietic abnormalities that include a
181 myeloid lineage output bias, impaired lymphoid repopulating potential, and a pronounced
182 age-associated expansion of HSC and myeloid progenitors. These functional abnormalities
183 support a role of 7q22 deletions in MDS pathogenesis. The seven genes within the deleted
184 5A3 segment that are expressed in HSC encode proteins that regulate diverse cellular
185 processes including transcription (Mll5 and Dnajc2), mitochondrial quality control (Pmpcb,
186 Armc10), protein degradation (Psmc2), biosynthesis of N-acylethanolamines (Napepld),
187 and DNA replication (Orc5)(40-48). Given this, it is likely that the haploinsufficiency for 10
188 multiple interacting genes leads to altered hematopoietic differentiation and function in
189 5A3+/del mice. Similar to the 5A3+/del mice described here, other mutations found in MDS
190 patients perturb hematopoiesis, but do not consistently induce hematologic disease in the
191 absence of cooperating mutations (49). This is not unexpected given the advanced age of
192 most MDS patients and the presence of multiple genetic lesions in diseased BM. The
193 5A3+/del mice reported here thus provide a novel resource for addressing how this common
194 deletion cooperates with other mutations to drive myeloid transformation, progression to
195 AML, and drug resistance.
196 Materials and Methods
197 Mice
198 We expressed Cre recombinase in embryonic stem (ES) cells containing LoxP sites and
199 HPRT sequences flanking the Fbxl13 and Srpk2 genes (23), and analyzed clones that
200 grew in HAT medium to identify the desired 2 Mb deletion. 5A3del/+ mice were generated by
201 standard blastocyst injection followed by mating coat color chimeras, and were
202 backcrossed for at least 10 generations onto a C57BL/6J background. Gata2+/- mice were
203 a generous gift from Dr. Stuart Orkin (Harvard Medical School)(50). Study mice were
204 housed in a specific pathogen–free facility at the University of California San Francisco,
205 and all animal experiments were conducted under protocols approved by the Institutional
206 Animal Care and Use Committee. Genotyping and disease monitoring were performed as
207 previously described (23).
208 Flow Cytometry
209 BM cells flushed from tibias and femurs were subjected to ammonium-chloride potassium
210 red cell lysis before staining with antibodies. For experiments requiring cell sorting, the 11
211 spinal cord, flat bone of the pelvis, humerus, and sternum of the mice were also crushed
212 and lysed. Low density mononuclear cells were separated using a HISTOPAQUE-1119
213 gradient (Sigma-Aldrich). For identification and sorting of CD150hi-HSC, CD150lo-HSC and
214 CD150neg-MPP, cells were pre-incubated with purified CD16/32 (2.4G2), followed by
215 staining with a lineage cocktail of PE-conjugated antibodies including B220 (RA3-6B2),
216 CD8 (53-6.7), Gr-1 (RB6-8C5), CD3 (17A2), Ter119 (TER-119), CD41 (MWReg30) and
217 CD48 (HM48-1), as well as PE-Cy7 c-kit (2B8), PacBlue Sca-1 (E13-161.7) and APC
218 CD150 (TC15-12F12.2) from Biolegend. For experiments requiring cell sorting, cells
219 expressing c-kit were enriched by magnetic cell sorting by staining with mouse CD117
220 microbeads, and positively selected on the MS columns (Miltenyi Biotec) according to
221 manufacturer’s protocol before antibody staining. Cells were classified as CD150hi-HSC,
222 CD150lo-HSC or CD150neg-MPP based in levels of CD150 expression.
223 CMP, GMP and MEP populations were identified by flow cytometry after staining
224 with a lineage cocktail of PE-conjugated antibodies including B220 (RA3-6B2), CD8 (53-
225 6.7), Gr-1 (RB6-8C5), CD3 (17A2), and Ter119 (TER-119), as well as APC-750 c-kit (2B8),
226 PacBlue Sca-1 (E13-161.7), Alexa Fluor 647 CD16/32 (93), biotin CD34 (MEC14.7) and a
227 streptavidin PE-Cy7 conjugate (Biolegend). Stained BM cells were analyzed in a FACS
228 LSRII instrument, and sorted in a FACS Aria3(Becton Dickinson). Flowjo software (Tree
229 Star, Inc.) was used to analyze and display the data.
230 Competitive Repopulation
231 BM cells from WT, 5A3+/del, Gata2+/-, Gata2+/-5A3+/del mice on a C57BL/6J background
232 (CD45.2) were used as donor cells. Competitor cells were isolated from 8 - 12 week old
233 BoyJ mice (CD45.1). Recipient F1 hybrid mice from a cross between C57BL/6J and BoyJ 12
234 mice (CD45.1+CD45.2) were at least 8 weeks old at the time of lethal irradiation (9.5 Gy
235 from a cesium source delivered in split dose 3 hours apart). After irradiation, the cells were
236 injected via the tail vein of recipient mice. For evaluation of the competitiveness of whole
237 BM, we injected 106 whole BM cells at a 1:1 or 1:2 donor to competitor ratio. To evaluate
238 the repopulating potential of purified CD150hi-HSC, we injected 15 CD150hi-HSC sorted
239 from 8 – 12 week old WT and 5A3+/del mice together with 2.5 x 105 BM competitor cells into
240 lethally irradiated recipients.
241 Blood was obtained from recipient mice every 30 days beginning 1 month after
242 transplant, and cells were stained with Alexa Fluor 700 CD45.2 (104), PE-Cy7 CD45.1
243 (A20), PacBlue B220 (RA3-6B2), FITC CD4 (GK1.5), FITC CD8 (53-6.7), PE Mac-1
244 (M1/70) and PE Gr-1 (RB6-8C5) to determine the precent donor cell contribution to
245 myeloid and B and T lymphoid lineages. Primary recipient mice were euthanized 6 months
246 after transplantation, BM were isolated from the tibiae and femur, and 2 x 106 BM cells
247 were injected into secondary recipients to test serial repopulation potential. To determine
248 the contribution of donor-derived cells in the K+L-S+ compartment, BM cells were stained
249 with V450 CD45.2 (104), APC-780 CD45.1 (A20), PE B220 (RA3-6B2), PE CD8 (53-6.7),
250 PE Gr-1 (RB6-8C5), PE CD3 (17A2), PE Ter119 (TER-119), PE-Cy7 c-kit (2B8) and APC
251 Sca-1 (E13-161.7). Secondary recipients were analyzed as in the primary recipients.
252 Methylcellulose Colony Assays
253 CD150hi-HSC, CD150lo-HSC and CD150neg-MPP were isolated as described above, and
254 100 cells were seeded into methylcellulose medium as described (51). Colonies were
255 counted on day 7 and the entire contents of a methylcellulose culture from an individual
256 plate were then flushed out using phosphate-buffered saline and counted in a 13
257 hemocytometer. Cells were also spun in a Cytospin 3 Cytocentrifuge (Shandon) at 400
258 rpm for 8 min, and differential cell counting and morphological analysis performed after
259 Wright Giemsa staining.
260 RNA Isolation and Expression
261 CD150hi-HSC, CD150lo-HSC and CD150neg-MPP were sorted as above into 500 µL of
262 TRIzol reagent (Life Technologies). RNA was isolated according to manufacturer’s
263 instructions and precipitated with the addition of glycogen (New England BioLabs). The
264 RNA was then treated with DNAse 1 (Ambion) and purified with the RNeasy MinElute
265 Cleanup Kit (Qiagen).
266 For TaqMan analysis, reverse transcription was carried out using the High Capacity
267 RNA-to-cDNA Master Mix (Life Technologies). Relative quantification of gene expression
268 was performed by performing quantitative real-time PCR using the following TaqMan Gene
269 Expression Assays (Applied Biosystems): Armc10 (Mm03011576_g1), Mll5
270 (Mm01129502_g1), Napepld (Mm00724596_m1), Orc5 (Mm00457242_m1), Psmc2
271 (Mm00803207_m1), Dnajc2 (Mm00494389_m1), Pmpcb (Mm01138654_m1), Srpk2
272 (Mm00486413_m1), Fbxl13 (Mm00622025_m1), Lrrc17 (Mm01167263_m1), Slc26a5
273 (Mm00446145_m1), Reln (Mm00465200_m1), Lhfpl3 (Mm03038441_m1) and Gapdh
274 (Mm99999915_g1) with the TaqMan Gene Expression Master Mix (ABI). PCR reactions
275 were performed on an ABI 7900HT Real-Time PCR System (Applied Biosystems) with the
276 Taqman Gene Expression Master Mix (Applied Biosystems) according to manufacturer’s
277 instructions. PCR cycling conditions were 2 minutes at 50°C and 10 minutes at 95°C,
278 followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. All reactions were
279 carried out in triplicate, and target quantities were determined using a relative standard 14
280 curve. The amounts of target were normalized to the endogenous control gene Gapdh and
281 compared with the corresponding WT BM (calibrator sample) to determine relative fold
282 differences.
283 For RNA-Seq analysis, total RNA (10 ng) were converted into double-stranded
284 cDNA using the Ovation® RNA Amplification System V2 (NuGen, CA) per manufacturer’s
285 recommendations. The amplified cDNA products were then used to generate RNA-seq
286 libraries using the TruSeq RNA Sample Preparation Kit v2 reagents (Illumina®, CA) per
287 manufacturer’s instructions, with 10 PCR amplification cycles. Library quality and quantity
288 were assessed by the Agilent DNA1000 Chip (Agilent, CA) and qPCR (Kappa Biosystems
289 Inc, MA). 10 pM of each library was sequenced using Illumina SBS chemistry at 2 x 100 bp
290 reads on the HiSeq2000 (Illumina®, CA).
291 The RNA-Seq paired-end reads were mapped to the mouse mm9 genome using an
292 in-house mapping and quality assessment pipeline (52). The expression of each gene was
293 estimated by the mean coverage of the highest-covered coding exon. Genes with low
294 expression level (<10) across all samples were filtered out, followed by quantile
295 normalization. Differential expression analysis was performed using limma (53) with
296 estimation of false-discovery rate (54). Gene Set Enrichment Analysis (55, 56) was used to
297 assess pathway enrichment.
298 ATP Quantification
299 HSC and MPP were sorted into PBS, and 600 CD150hi-HSC, 1000 CD150lo-HSC and
300 1000 CD150neg-MPP were aliquoted into a well of a 96-well plate in triplicate. ATP was
301 quantified using the Cell-Titer-Glo Luminescent Cell Viability Assay (Promega) following
302 manufacturer’s recommendations. Illumination was quantified with a Synergy 2 (Biotek). 15
303 Metabolic Studies
304 For flow cytometric analysis of ROS levels and membrane potential in CD150hi-HSC,
305 CD150lo-HSC and CD150neg-MPP, BM cells were isolated and enriched for c-kit positive
306 cells as described. c-kit positive cells were stained with an unconjugated cocktail of
307 purified antibodies including B220 (RA3-6B2), CD8 (53-6.7), Gr-1 (RB6-8C5), CD3 (17A2),
308 Ter119 (TER-119) and CD41 (MWReg30) from Biolegend, followed by PECy-5 IgG GOAT
309 anti-RAT pAb (HI47) antibody from Molecular Probes and IgG from rat serum (I4131
310 Sigma). After incubation with 50 nM CellROX Orange Reagent (Molecular Probes) or 20
311 nM Mitotracker Orange CMTMRos (Molecular Probes) for 30 min at 37°C in IMDM, cells
312 were washed and stained with 7-amino-actinomycin D, PE-Cy7 c-kit (2B8), PacBlue Sca-1
313 (E13-161.7), PE-Cy5 CD48 (HM48-1) and APC CD150 (TC15-12F12.2) from Biolegend.
314 The OCR was analyzed in an XF96 extracellular flux analyzer following
315 manufacturer’s protocol (Seahorse Biosciences). Freshly isolated K+L-S+ cells and K+L-S-
316 cells were cultured in StemSpan serum-free medium (STEMCELL Technologies)
317 supplemented with SCF (100 ng/mL) and Tpo (100ng/mL) while freshly isolated thymic
318 cells and B-220+ splenic cells were cultured in RPMI medium 1640 supplemented with
319 10% Fetal Calf Serum and incubated at 37° in a humidified atmosphere containing 8%
320 CO2 for 12 – 15 hours. Cells were then washed three times with Mito Stress Media (XF
321 base media supplemented with glucose (3 mg/mL), sodium pyruvate (1 mM) and Glutamax
322 (2 mM) adjusted to a pH=7.4), and seeded in XF96 microplates coated with poly-L-lysine
323 (Sigma). 60000 K+L-S+ cells, 100000 K+L-S- cells, 200000 thymic cells and 200000 B220+
324 splenic cells were plated per well respectively. K+L-S+ and K+L-S- cells were stimulated with
325 SCF (100 ng/mL) and Tpo (100 ng/mL), while thymic cells were stimulated with interleukin 16
326 (IL)-2 (20 ng/mL) and IL-7 (10ng/mL) and B-220+ splenic cells were stimulated with IL-7
327 (10 ng/mL) and maintained in a non-CO2 incubator for 1 hour before the assay. Five
328 baseline recordings were made, followed by sequential injection of Oligomycin (Sigma),
329 Carbonyl Cyanide 4-(trifluoromethoxy)phenylhydrazone (Sigma), and a combination of
330 Antimycin A (Sigma) and Rotenone (Sigma) to determine the mitochondrial respiration rate
331 under various conditions.
332 Statistical Analysis
333 Data are presented as means ± SEM unless stated otherwise. Statistical significance was
334 determined by performing two-tailed Student’s t-tests.
335 Acknowledgements
336 We are grateful to Michelle Le Beau, Qing Li, Scott Lowe, and Megan McNerney for
337 insightful suggestions, and to Stuart Orkin for providing the Gata2+/- mutant mice. This
338 work was supported by NIH grants P01 CA40046, R37 CA72614, T32 CA108462 and R01
339 HL092471, by the Kenneth and Mary Ellen Wilson St. Baldrick’s Research Grant, by a
340 Specialized Center of Research award from the Leukemia and Lymphoma Society (LLS
341 7019-04), and by the ALSAC of St. Jude Children’s Research Hospital. K.S. is an
342 American Cancer Society Research Professor, M.R.B. is supported by the American
343 Cancer Society Hillcrest Fellowship and E.P. is a Leukemia and Lymphoma Society
344 Scholar.
345 17
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538 Figure Titles and Legends
539 Figure 1. A heterozygous 5A3 deletion corresponding to human 7q22 perturbs
540 steady state hematopoiesis. (A) Top. Candidate 7q myeloid tumor suppressor genes
541 described previously (9, 10, 18, 20-22, 57, 58) appear above the diagram of chromosome
542 7q while commonly deleted segments (CDSs) within 7q22, 7q34, and 7q35-36 identified by
543 different research groups (8-15) are depicted by brackets immediately below it. Middle.
544 Dotted lines demarcate the segments of mouse chromosome 5A3 corresponding to the
545 human 7q22 CDS targeted in this study. Bottom. Expressing Cre recombinase in ES cells
546 doubly targeted with LoxP sequences within the Fbxl13 and Srpk2 genes excised a 2Mb
547 interval. Gene order is based on the Ensembl database and is not drawn to scale. (B) 24
548 Total numbers of BM cells from 2 femurs and 2 tibiae in 5A3+/del mice and WT littermates
549 at 8 - 12 weeks of age. (C and D) Spleen (C) and thymus (D) weights in 5A3+/del mice and
550 WT littermates at 8 - 12 weeks of age. (E - F) Percent contributions (E) and frequencies (F)
551 of cells with high (CD150hi HSC), low (CD150lo HSC) and absent CD150 expression
552 (CD150neg MPP) within the K+L-S+CD41-CD48- compartment of WT and 5A3+/del mice at 8-
553 12 weeks of age (n = 6 of each genotype). (G) Percent contribution of CMP, GMP and
554 MEP within the Lin-Sca1+c-kit+ compartment of 5A3+/del mice and WT littermates. (H)
555 Frequencies of CMP, GMP and MEP in WT and 5A3+/del BM. The error bars indicate
556 S.E.M. with significant differences between WT and 5A3+/del mice designated by asterisks
557 as follows: *p<0.05, **p<0.01, ***p<0.001.
558
559 Figure 2. Defective repopulating potential of 5A3+/del BM and CD150hi HSC. (A - B) BM
560 cells from WT or 5A3+/del mice (n = 9 per genotype) were mixed at ratios of 1:1 and 1:2 with
561 WT competitor cells and transplanted into 2 -3 irradiated WT recipients. Percent
562 contribution to the K+L-S+ (KLS), K+L-S- (MP), myeloid, B and T cell lineages in the BM of
563 recipient mice 6 months after primary (A) and secondary (B) transplants are shown. (C)
564 Leukocyte chimerism after competitive transplantation of 15 5A3+/del or WT CD150hi HSC
565 into WT or mutant recipients (n = 12 for WT HSC in WT recipients; n = 12 for 5A3+/del HSC
566 in WT recipients; n = 8 for WT HSC in 5A3+/del recipients; n = 9 for 5A3+/del HSC in 5A3+/del
567 recipients). (D - F) Relative proportions of donor-derived B, T, and myeloid cells in the
568 blood of recipient mice 6 months after transplantation (D). Percent contribution to the K+L-
569 S+ (KLS), K+L-S- (MP), myeloid, B and T cell lineages in the BM of recipient mice 6 months
570 after primary (E) and secondary (F) transplants. Data shown are means ± SEM of results 25
571 from four independent experiments. (G - I) One hundred CD150hi HSC, CD150lo HSC, and
572 CD150neg MPP from 8 – 12 week old 5A3+/del mice and their WT littermates were plated
573 into methylcellulose medium supplemented with cytokines (n = 6 for each genotype). The
574 total number of colonies (G) and the average number of cells per colony (H - I) were
575 assessed after 7 days. Dots represent individual mice, and the horizontal lines indicate
576 median values. Data shown are means ± SEM of results from three independent
577 experiments with significant differences between WT and 5A3+/del mice designated by
578 asterisks as follows: *p<0.05, **p<0.01, ***p<0.001.
579
580 Figure 3. Effects of aging and Gata2 inactivation on 5A3+/del hematopoietic cells. (A -
581 B) Percent contributions (A) and frequencies (B) of CD150hi HSC, CD150lo HSC, and
582 CD150neg MPP within the K+L-S+CD41-CD48- compartment in 5A3+/del mice (n = 12) and
583 their WT littermates (n = 11) at 24 - 30 months of age. (C - D) Competitive transplantation
584 of BM cells from 24 - 30 month old WT or 5A3+/del mice. Percent donor contribution of
585 5A3+/del cells to K+L-S+ (KLS), K+L-S- (MP), myeloid, B cell, and T cell populations in the BM
586 of recipient mice 4 months after primary (C) and secondary (D) competitive transplantation
587 (n = 5 donors and 10 recipients of each genotype). Data shown are means ± SEM of
588 results from two independent experiments. (E) Total numbers of BM cells from 2 femurs
589 and 2 tibiae in 5A3+/del mice, Gata2+/- mice, compound Gata2+/- ; 5A3+/del mice and WT
590 littermates at 8 - 12 weeks of age. (F) Spleen weights in 5A3+/del mice, Gata2+/- mice,
591 compound Gata2+/- ; 5A3+/del mice, and WT littermates at 8 - 12 weeks of age. (G) Percent
592 contributions of cells with high (CD150hi HSC), low (CD150lo HSC) and absent CD150
593 expression (CD150neg MPP) within the K+L-S+CD41-CD48- compartment of WT, 5A3+/del, 26
594 Gata2+/-, and compound Gata2+/-; 5A3+/del mice at 8 - 12 weeks of age (n = 5 of each
595 genotype). (H) BM cells from WT, 5A3+/del, Gata2+/- or compound Gata2+/-; 5A3+/del mice (n
596 = 5 of each genotype) were each mixed at ratios of 1:1 with WT competitor cells and
597 transplanted into 2 irradiated WT recipients. Percent contribution to the K+L-S+ (KLS), K+L-
598 S- (MP), myeloid, B and T cell lineages in the BM of recipient mice 6 months after primary
599 transplants. Data shown are means ± SEM from five independent experiments with
600 significant differences designed by asterisks as follows: *p<0.05, **p<0.01, ***p<0.001.
601 The enhanced repopulating ability of compound Gata2+/-; 5A3+/del versus Gata2 singly
602 mutant HSC achieved borderline statistical significance in three myeloid populations (KLS
603 (p=0.09), MP (p=0.09), and myeloid cells (p=0.12).
604
605 Figure 4. Changes in gene expression and oxidative phosphorylation in 5A3+/del HSC
606 and MP. (A) Relative mRNA abundances for genes within the deleted 5A3 interval
607 expressed at detectable levels in sorted HSC populations were determined by Taqman
608 reverse transcriptase PCR (n = 3 per genotype). (B) Expression levels of genes located
609 within and flanking the deleted interval measured by RNA-Seq in sorted CD150hi HSC and
610 CD150lo HSC from 5 mice of each genotype. Each column presents data from an
611 individual mouse, and genes within the 5A3 deleted region are delimited with a black box.
612 Three non-coding RNAs (6030443J06Rik, AC112688.1 and 5031425E22Rik) are located
613 within the 5A3 deletion. Two of these (6030443J06Rik and AC112688.1 are expressed at
614 extremely low levels in HSC, and the other (5031425E22Rik) showed ~50% lower
615 expression in 5A3+/del HSC. 5031425E22Rik is homologous to the human KMT2E (a.k.a.
616 MLL5) antisense RNA1. Expression levels of the flanking Fbxl13 and Srpk2 genes are 27
617 modestly up-regulated in 5A3+/del HSC, which is consistent with the targeting strategy used
618 to generate the segmental deletion. (C) GSEA analysis of 5A3+/del CD150hi HSCs revealed
619 negative enrichment for genes associated with oxidative phosphorylation (OXPHOS).
620 False discovery rate (FDR) q-val, nominal p-value (NOM p-value) and normalized
621 enrichment scores (NES) are indicated. (D) ATP levels in HSC and MPP from 8-12 week
622 old WT (n = 6) and 5A3+/del (n = 5) mice. Data shown are means ± SEM of results from two
623 independent experiments. (E) Fold change in the mean Mitotracker Orange fluorescence
624 levels in 5A3+/del cells normalized to values in WT cells analyzed in the same experiment.
625 (F) Fold change in the mean fluorescence level (MFI) of 5A3+/del cells that are CellROX
626 Orange positive normalized to values in WT cells analyzed in the same experiment. For
627 the Mitotracker and CellROX experiments, n = 13 for WT and n = 12 for 5A3+/del young
628 mice, three independent experiments; n = 5 for WT and n = 6 for 5A3+/del aged mice, two
629 independent experiments. Data shown are means ± SEM of results from independent
630 experiments. (G - H) Oxygen consumption rate (OCR) was assessed basally and in
631 response to the mitochondrial inhibitors oligomycin (oligo), carbonyl cyanide 4-
632 (trifluoromethoxy) phenylhydrazone (FCCP), and antimycin A and rotenone (A/R) for (G)
633 KLS and (H) MP cells. Data are shown as mean ± SEM of n = 5 mice of each genotype
634 from 2 independent experiments.
635 Inventory of Supplemental Information:
636 Figure 1 – Figure Supplement 1. Gating strategy for hematopoietic stem and progenitor
637 populations.
638 Figure 2 – Figure Supplement 1. Proliferation and divisional kinetics of 5A3+/del HSCs.
639 28
640 Figure Supplements Legend
641 Figure 1 – Figure Supplement 1. Gating strategy for hematopoietic stem and
642 progenitor populations. (A) Gating strategy for CD150hi-HSC, CD150lo-HSC, and
643 CD150neg-MPP. K+L-S+CD41-CD48- cells were separated based on CD150 expression into
644 CD150hi, CD150lo, and CD150neg populations. Representative plots from WT (top) and
645 5A3+/del (bottom) mice are shown. (B) Gating strategy for CMP, GMP and MEP. K+L-S- cells
646 were subdivided by CD34 and FcγR expression into CMP (CD34+FcγRlo), GMP
647 (CD34+FcγRhi), and MEP (CD34-FcγRlo). Representative plots from WT (top) and 5A3+/del
648 (bottom) mice are shown. (C) Gating strategy for CLP. Lin- cells were gated for CLP
649 (Flk2+IL7Rα+). (D) Percent contribution of CLP within the Lin- compartment of WT (n = 6)
650 and 5A3+/del (n = 6) mice. (E) Frequencies of CLP in WT (n = 6) and 5A3+/del (n = 6) BM.
651 For CLP staining experiments, data shown are means ± SEM of results from three
652 independent experiments. For gating strategy figures, the numbers are expressed as
653 percentages of the parental gates.
654 Figure 2 – Figure Supplement 1. Proliferation and divisional kinetics of 5A3+/del
655 HSCs. (A) BrdU labeling frequencies of K+L-S+ and K+L-S- cells from WT and 5A3+/del
656 mice taken 24 hours after intraperitoneal injection of BrdU. (B) HSCs were labeled with EZ-
657 Link Sulfo-NHS-LC-LC-biotin (biotin) in vivo, and the in vivo proliferative behavior of HSCs
658 was tracked by dilution of the biotin label caused by cell division. Biotin intensities on
659 CD150hi HSC, CD150lo HSC, and CD150neg MPP taken 15 minutes and 6 days after
660 intravenous injection of biotin (representative plots from one of 3 mice in each group).
661 Following 6 days of chase, the reduction in biotin label intensity is similar in WT and
662 5A3+/del CD150hi HSC, CD150lo HSC, and CD150neg MPP. 29
663 Supplemental Experimental Procedures
664 Antibodies for flow cytometry of CLP cells
665 CLP were identified and evaluated by flow cytometry by staining with a lineage cocktail of
666 PE-conjugated antibodies including B220 (RA3-6B2), CD8 (53-6.7), Gr-1 (RB6-8C5), CD3
667 (17A2), and Ter119 (TER-119), as well as APC-750 c-kit (2B8), PacBlue Sca-1 (E13-
668 161.7), APC IL-7Rα (A7R34) and biotin Flk2 (A2F10) and a streptavidin PE-Cy7 conjugate
669 (Biolegend).
670 BrdU Staining
671 Mice received an initial intraperitoneal injection of BrdU (Sigma-Aldrich, 1 mg/6 g mouse
672 weight) and were then maintained on 1.0mg/mL BrdU in the drinking water 24 hours prior
673 to sacrifice. To measure BrdU incorporation, BM were enriched for c-kit positive cells using
674 c-kit antibody-conjugated microbeads (Miltenyi). Enriched BM cells were pre-incubated
675 with purified CD16/32 (2.4G2), followed by staining with a lineage cocktail of PE-
676 conjugated antibodies including B220 (RA3-6B2), CD8 (53-6.7), Gr-1 (RB6-8C5), CD3
677 (17A2), Ter119 (TER-119), as well as PECy7 c-kit (2B8) and PacBlue Sca-1 (E13-161.7)
678 (Biolegend). BrdU incorporation was then assayed according to manufacturer’s
679 instructions of the APC BrdU Flow Kit (BD Pharmingen).
680 NHS-biotin Staining
681 NHS-biotin (EZ-Link Sulfo-NHS-LC-LC-biotin; Life Technologies) was dissolved at
682 10mg/mL in normal saline, and injected into mice intravenously via the tail vein at 1mg/6 g
683 mouse weight. To assess biotin dilution in the HSC subpopulations, BM were enriched for 30
684 c-kit positive cells using c-kit antibody-conjugated microbeads (Miltenyi). Enriched cells
685 were pre-incubated with purified CD16/32 (2.4G2), followed by staining with a lineage
686 cocktail of PE-conjugated antibodies including B220 (RA3-6B2), CD8 (53-6.7), Gr-1 (RB6-
687 8C5), CD3 (17A2), Ter119 (TER-119), CD41 (MWReg30) and CD48 (HM48-1), as well as
688 PerCP/Cy5.5 c-kit (2B8), PacBlue Sca-1 (E13-161.7), APC CD150 (TC15-12F12.2) and
689 PECy-7-conjugated Streptavidin (Biolegend).
690
691
692
693 SAMD9L A CUX1 DOCK4 EZH2 Figure 1 RASA4 LUC7L2 MLL3 Genes
Human 7q q11 q21 q22 q31 q32 q33 q34 q35 q36 CDS
Mouse 5
Lrrc17/FbxL13Armc10 Napepld Pmpcb Dnajc2 Psmc2 Slc26a5Reln Orc5 Lhfpl3 Mll5 Srpk2
5’Hprt-loxP-neo Cre puro-loxP-3’Hprt-GFP
Lrrc17/FbxL13Srpk2 5’Hprt-loxP-3’Hprt-GFP
B 10 C 0.10 ** D 0.06 * *
) 8 0.08 7 0.04 6 0.06
4 0.04 0.02 BM cells (X10 Spleen Weight (g) Weight Spleen
2 0.02 Thymus Weight (g)
0 0.00 0.00 WT +/del WT +/del WT +/del 5A3 5A3 5A3
85 450 E ** F WT - 80 WT 400 75 5A3+/del 350 5A3+/del 70 300 CD48 - 65 250 BM cells BM
20 6 200
CD41 ** 80 + 15 S - 60 L + 10 40 No. per 10 per No.
% K 5 20 0 0
CD150hi CD150lo CD150neg CD150hi CD150lo CD150neg
G 60 WT H 8000 WT +/del +/del 5A3 5A3 * 6000
- 40 S
- * BM cells L 6 + 4000
% K 20 2000 No. per 10 per No.
0 0 CMP GMP MEP CMP GMP MEP Figure 2
WT 1:1 WT 1:2 A 80 WT 1:1 WT 1:2 B 100 5A3+/del 1:1 5A3+/del 1:2 5A3+/del 1:1 5A3+/del 1:2 *** * 80 60 *** *** 60 ** *** 40 ** * ** 40 20 20 % donor contribution % donor % donor contribution % donor
0 0 KLS MP B cell T cell KLS MP Myeloid Myeloid B cell T cell
C 50 WT(D)WT(R) D T cell B cell Myeloid 5A3+/del(D)WT(R) WT(D)5A3+/del(R) 100 40 5A3+/del(D)5A3+/del(R)
30 50 20
% donor leukocytes % donor 10
Lineage Chimerism Ratio (%) Ratio Lineage Chimerism 0 l l +/de (R) +/de (R) 0 +/del (D)WT(R) WT(D)WT(R) +/del (D)5A3 1 2 3 4 5 6 5A3 WT(D)5A3 5A3 WT(D)WT(R) +/del WT(D)WT(R) +/del F WT(D)5A3 (R) E 80 WT(D)5A3 (R) 100 5A3+/del(D)WT(R) +/del +/del +/del +/del +/del 5A3 (D)5A3 (R) 5A3 (D)WT(R) 5A3 (D)5A3 (R) * 80 * 60 * * * 60 40 * ** 40 20 20 % donor contribution % donor % donor contribution % donor
0 0 KLS MP B cell T cell KLS MP Myeloid Myeloid B cell T cell GHI 40 10000 60000 * * 30 8000 40000 6000 20 4000 20000
No. of colonies of No. 10 2000 Average no. of cells / colony / cells of no. Average 0 colony / cells of no. Average 0 0 +/del +/del +/del WT WT WT WT +/del WT +/del WT +/del 5A3 5A3 5A3 5A3 5A3 5A3 CD150hi CD150lo CD150neg CD150hi CD150lo CD150neg Figure 3 A 80 B 400 - WT *** WT 5A3+/del 5A3+/del 60 300 CD48 - * BM cells 40 6 200 CD41 ** * + S - L + 20 100 No. per 10 per No. % K 0 0
CD150hi CD150lo CD150neg CD150hi CD150lo CD150neg C 100 D 100 WT * WT ** +/del 80 ** 5A3+/del 80 5A3
60 60 *** 40 *** 40 20 20 % donor contribution % donor % donor contribution % donor
0 0 KLS MP KLS MP B cell T cell Myeloid B cell T cell Myeloid E * F * 10 * 0.10 * * * ** 0.08 ) 8 * 7
6 0.06
4 0.04 BM cells (X10 cells BM 2 (g) Weight Spleen 0.02
0 0.00 l +/del +/de +/del +/del +/- ; WT +/- ; WT WT; WT +/- ; 5A3 WT; WT +/- ; 5A3 WT; 5A3Gata2 WT; 5A3Gata2 Gata2 Gata2 G H WT; WT Gata2+/-; WT WT; WT * WT; 5A3+/del Gata2+/-; 5A3+/del WT; 5A3+/del * ** 80 50 * ** ** - 75 Gata2+/-; WT * ** * ** 70 Gata2+/-; 5A3+/del 40 * 65 *** * CD48
- *** 60 * *** * 30 55 * ** * CD41
+ * *
S 20 - 20 * L
+ * 10 10 % donor contribution % donor % K
0 0 hi lo neg l KLS MP CD150 CD150 CD150 Myeloid B cell T cel Figure 4
A 8
7 *
6
5
4 * ** ** 3 Fold difference relative to Whole BM 2 ** ** ** ** * ** ** * 1
0 Mll5 Armc10 Psmc2 Dnajc2 Orc5 Pmpcb Napepld
WT CD150hi (n=3) WT CD150lo (n=3) WT CD150neg (n=3) 5A3+/del CD150hi (n=3) 5A3+/del CD150lo (n=3) 5A3+/del CD150neg (n=3)
WT +/del 5A3 WT B CD150hiCD150lo CD150hi CD150lo Gene C FDR q-val = 0 D ) 8 5A3+/del Speer4c NOM p-val = 0 -14 Hgf NES = 2.58 Speer4f Sema3c 6 Cd36 Gnat3 Gnai1 4 Magi2 Phtf2 Tmem60 Rsbn1l WT 2 Ptpn12 Pion 5A3+/del
Ccdc146 100Mol / ATP cells (X10 Fgl2 0
upstream Fam185a FFbxl13bxl13 CD150hi CD150lo CD150neg LLrrc17rrc17 AArmc10rmc10 1.5 WT 2.0 NNapepldapepld E F WT PPmpcbmpcb 5A3+/del 5A3+/del ** DDnajc2najc2 1.5 5A3 PPsmc2smc2 ReRelnln 1.0 OOrc5rc5 LLhfpl3hfpl3 1.0 MMll5ll5 SSrpk2rpk2 Pus7 0.5 Fold change in change Fold Fold change in change Fold
Rint1 Mitotracker MFI 0.5 Tomm7 CellROX Orange MFI Orange CellROX Fam126a Klhl7 0.0 0.0 Nupl2 Kcnh2 CD150hiCD150loCD150negCD150hiCD150loCD150neg Nos3 CD150hiCD150loCD150negCD150hiCD150loCD150neg Atg9b young Abcb8 young old old Accn3
downstream Slc4a2 Fastk +/del +/del Tmub1 WT 5A3 WT 5A3 Agap3 G H Gbx1 60 150 A/R A/R
Asb10 Oligo Oligo FCCP 4931409K22Rik FCCP Abcf2 40 100 Max ** Max -3 -2 -1 0 1 2 3 OCR * 20 50 OCR OCR (pmol/min) Genes not expressed Basal (pmol/min) OCR Basal X OCR OCR 0 0 0 20406080100 0 20406080100 time (min) time (min)