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| Status |
Public on Oct 06, 2020 |
| Title |
RUNX1 and CBFβ-SMMHC transactivate target genes together in abnormal myeloid progenitors for leukemogenesis |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Inversion of chromosome 16 is a consistent finding in patients with acute myeloid leukemia subtype M4 with eosinophilia (AML M4Eo), which generates a CBFB-MYH11 fusion gene. It is generally considered that CBFβ-SMMHC, the fusion protein encoded by CBFB-MYH11, is a dominant negative repressor of RUNX1. However, recent findings challenge the RUNX1-repression model for CBFβ-SMMHC mediated leukemogenesis. To definitively address the role of Runx1 in CBFB-MYH11 induced leukemia, we crossed conditional Runx1 knockout mice (Runx1f/f) with conditional Cbfb-MYH11 knockin mice (Cbfb+/56M). Upon Mx1-Cre activation in hematopoietic cells induced by poly (I:C) injection, all Mx1-CreCbfb+/56M mice developed leukemia in 5 months while no leukemia developed in Runx1f/fMx1-CreCbfb+/56M mice, and this effect was cell autonomous. Importantly, the abnormal myeloid progenitors (AMPs), a leukemia initiating cell population induced by Cbfb-MYH11 in the bone marrow, decreased and disappeared in Runx1f/fMx1-CreCbfb+/56M mice. RNA-seq analysis of AMP cells showed that genes associated with proliferation, differentiation blockage and leukemia initiation, were differentially expressed between Mx1-CreCbfb+/56M and Runx1f/fMx1-CreCbfb+/56M mice. In addition, with chromatin immunocleavage sequencing (ChIC-seq) assay, we observed a significant enrichment of RUNX1/CBFβ-SMMHC target genes in Runx1f/fMx1-CreCbfb+/56M cells, especially among down-regulated genes, suggesting that RUNX1 and CBFβ-SMMHC mainly function together as activators of gene expression through direct target gene binding. These data indicate that Runx1 is indispensable for Cbfb-MYH11 induced leukemogenesis by working together with CBFβ-SMMHC to regulate critical genes associated with the generation of a functional AMP population.
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| Overall design |
For bulk RNA-sequening: Total RNA from AMP cells was extracted with AllPrep DNA/RNA/Protein Mini Kit (QIAGEN). Poly-A selected stranded mRNA libraries were constructed using the Illumina TruSeq Stranded mRNA Sample Prep Kits according to manufacturer’s instructions. Unique barcode adapters were applied to each library. All libraries were combined in equimolar proportion into one pool for sequencing with a NovaSeq6000 sequencer; For ChIC-seq: AMP cells were fixed with 1% PFA and subjected to ChIC-seq as described.7 Specifically, antibody+PA-MNase complex with 50,000 cells was used for each sample. RUNX1 (Abcam, AB_2184205), CBFβ (Diagenode, C15310002), MYH11 (Diagenode, C15310254), H3K27Ac (Abcam, AB_2118291) and H3K27me3 (Millipore Sigma, AB_310624) antibodies were used to identify the binding sites of the indicated proteins. All libraries were combined in equimolar proportion into one pool for sequencing with a HiSeq 3000 sequencer; For scRNA-seq: 10X genomics chromium platform was used to capture isolated AMP cells and all steps were performed according to manufacturer’s instructions. The Chromium Single Cell 3’ Library & Gel Bead Kit v2 was used. Libraries were sequenced on an Illumina NextSeq 550 sequencer
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| Contributor(s) |
Zhen T, Cao Y, Ren G, Zhao L, Hyde RK, Lopez G, Alemu L, Zhao K, Liu PP |
| Citation(s) |
32929473 |
| Submission date |
Jun 16, 2020 |
| Last update date |
Oct 06, 2020 |
| Contact name |
Tao Zhen |
| E-mail(s) |
zhentao0526@gmail.com
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| Organization name |
NIH
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platforms (3) |
| GPL21493 |
Illumina HiSeq 3000 (Mus musculus) |
| GPL21626 |
NextSeq 550 (Mus musculus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (33)
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| GSM4618347 |
RNA-seq CBFB-SMMHC knock-in Runx1 knock-out rep1 |
| GSM4618348 |
RNA-seq CBFB-SMMHC knock-in Runx1 knock-out rep2 |
| GSM4618349 |
RNA-seq CBFB-SMMHC knock-in Runx1 knock-out rep3 |
| GSM4618350 |
ChIC-seq CBFB-SMMHC knock-in Cbfb rep1 |
| GSM4618351 |
ChIC-seq CBFB-SMMHC knock-in H3K27ac rep1 |
| GSM4618352 |
ChIC-seq CBFB-SMMHC knock-in H3K27me3 rep1 |
| GSM4618353 |
ChIC-seq CBFB-SMMHC knock-in IgG rep1 |
| GSM4618354 |
ChIC-seq CBFB-SMMHC knock-in MYH11 rep1 |
| GSM4618355 |
ChIC-seq CBFB-SMMHC knock-in Runx1 rep1 |
| GSM4618356 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out Cbfb rep1 |
| GSM4618357 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out H3K27ac rep1 |
| GSM4618358 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out H3K27me3 rep1 |
| GSM4618359 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out IgG rep1 |
| GSM4618360 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out MYH11 rep1 |
| GSM4618361 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out Runx1 rep1 |
| GSM4618362 |
ChIC-seq CBFB-SMMHC knock-in Cbfb rep2 |
| GSM4618363 |
ChIC-seq CBFB-SMMHC knock-in H3K27ac rep2 |
| GSM4618364 |
ChIC-seq CBFB-SMMHC knock-in H3K27me3 rep2 |
| GSM4618365 |
ChIC-seq CBFB-SMMHC knock-in MYH11 rep2 |
| GSM4618366 |
ChIC-seq CBFB-SMMHC knock-in Runx1 rep2 |
| GSM4618367 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out Cbfb rep2 |
| GSM4618368 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out H3K27ac rep2 |
| GSM4618369 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out H3K27me3 rep2 |
| GSM4618370 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out IgG rep2 |
| GSM4618371 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out MYH11 rep2 |
| GSM4618372 |
ChIC-seq CBFB-SMMHC knock-in Runx1 knock-out Runx1 rep2 |
| GSM4618373 |
10x scRNA-seq CBFB-SMMHC knock-in rep1 |
| GSM4618374 |
10x scRNA-seq CBFB-SMMHC knock-in rep2 |
| GSM4618375 |
10x scRNA-seq CBFB-SMMHC knock-in Runx1 knock-out rep1 |
| GSM4618376 |
10x scRNA-seq CBFB-SMMHC knock-in Runx1 knock-out rep2 |
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| Relations |
| BioProject |
PRJNA639776 |
| SRA |
SRP267560 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE152573_IgG.bedpe.gz |
89.8 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_KI_Cbfb.bedpe.gz |
103.2 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_KI_H3K27ac.bedpe.gz |
517.8 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_KI_H3K27me3.bedpe.gz |
384.6 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_KI_MYH11.bedpe.gz |
124.4 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_KI_Runx1.bedpe.gz |
142.9 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_RAW.tar |
73.6 Mb |
(http)(custom) |
TAR (of H5) |
| GSE152573_RNA-seq_gene_exp.diff.gz |
1.0 Mb |
(ftp)(http) |
DIFF |
| GSE152573_RNA-seq_gene_exp.txt.gz |
854.0 Kb |
(ftp)(http) |
TXT |
| GSE152573_Rx1KI_Cbfb.bedpe.gz |
191.0 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_Rx1KI_H3K27ac.bedpe.gz |
467.2 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_Rx1KI_H3K27me3.bedpe.gz |
633.9 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_Rx1KI_MYH11.bedpe.gz |
68.3 Mb |
(ftp)(http) |
BEDPE |
| GSE152573_Rx1KI_Runx1.bedpe.gz |
234.3 Mb |
(ftp)(http) |
BEDPE |
SRA Run Selector |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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