C11orf21, a novel RUNX1 target gene, is down-regulated by RUNX1-ETO

Abstract

The fusion protein RUNX1-ETO is an oncogenic transcription factor generated by t(8;21) chromosome translocation, which is found in FAB-M2-type acute myeloid leukemia (AML). RUNX1-ETO is known to dysregulate the normal RUNX1 transcriptional network, which should involve essential factors for the onset of AML with t(8;21). In this study, we screened for possible transcriptional targets of RUNX1 by reanalysis of public data in silico, and identified C11orf21 as a novel RUNX1 target gene because its expression was down-regulated in the presence of RUNX1-ETO. The expression level of C11orf21 was low in AML patient samples with t(8;21) and in Kasumi-1 cells, which carry RUNX1-ETO. Knockdown of RUNX1-ETO in Kasumi-1 cells restored C11orf21 expression, whereas overexpression of RUNX1 up-regulated C11orf21 expression. In addition, knockdown of RUNX1 in other human leukemia cells without RUNX-ETO, such as K562, led to a decrease in C11orf21 expression. Of note, the C11orf21 promoter sequence contains a consensus sequence for RUNX1 binding and it was activated by exogenously expressed RUNX1 based on our luciferase reporter assay. This luciferase signal was trans-dominantly suppressed by RUNX1-ETO and site-directed mutagenesis of the consensus site abrogated the reporter activity. This study demonstrated that C11orf21 is a novel transcriptional target of RUNX1 and RUNX1-ETO suppressed C11orf21 transcription in t(8;21) AML. Thus, through this in silico approach, we identified a novel transcriptional target of RUNX1, and the depletion of C11orf21, the target gene, may be associated with the onset of t(8;21) AML.

Keywords: C11orf21; RNA-seq; RUNX1; RUNX1-ETO; t(8:21) AML.

Fig. 1

Differential gene expression analysis of published datasets revealed that C11orf21 expression is significantly suppressed in t(8;21) cells. (A) The expression of C11orf21 was measured by RNA-seq in Kasumi-1 cells after treatment with RUNX1-ETO siRNA. We used published RNA-seq data from GSE60131. (B, C) Reanalysis of C11orf21 expression in AML with t(8;21) and other karyotypes. Data sets were obtained from The Cancer Genome Atlas Research Network (TCGA) (B) and the Beat AML program (C). (D) Data were obtained from BloodSpot analysis using data sets GSE42519 for human normal hematopoiesis cells, and GSE13159, GSE15434, GSE61804, GSE14468, and TCGA for human AML cells.

Fig. 2

Knockdown of RUNX1-ETO in Kasumi-1 cells led to increased C11orf21 expression. In Kasumi-1 cells, RUNX1-ETO was knocked-down by siRNA, and RT-qPCR was performed for RUNX1-ETO, C11orf21 and Actin. Relative expression of RUNX1-ETO (A) and C11orf21 (B) is shown as bar graphs. RUNX1-ETO knocked-down results performed with other siRNAs, RUNX1-ETO siRNA No.2 (C and D) and RUNX1-ETO siRNA No.3 (E and F) are indicated. Data were normalized by expression levels of Actin. Data represent the means of triplicate experiments (bars, S.D).

Fig. 3

RUNX1-ETO directly bound the C11orf21 promoter region and down-regulated the transcription of C11orf21. (A) Chromatin immunoprecipitation (ChIP) data. Both RUNX1 and ETO binding peaks were detected on the C11orf21 promoter region in Kasumi-1 cells, suggesting that RUNX1-ETO bound this region. RUNX1 also bound the C11orf21 promoter region in CD34⁺ cells. Data were obtained from ChIP-Atlas. The structure of the C11orf21 gene is shown below the histograms of ChIP data. Black boxes show exons. The transcription start site is indicated by a black arrow and RUNX1-binding sites including TGTGGT sequences are shown by red arrows. (B) Chromatin immunoprecipitation (ChIP) data of ETO for the primary t(8;21) AML cells. Data were obtained from GSE34540 containing GSM850825 and GSM850826 via ChIP-Atlas database. (C) The schematic diagram of C11orf21 promoter that we used for the experiments. The length of promoter sequence C11orf21 was 3011 bp. The triangle indicates the RUNX1 binding site (TGTGGT). “+1″ means the transcription start site and ATG was the translation start site (TSS).

Fig. 4

C11orf21 expression was upregulated by RUNX1 overexpression. (A) Plasmid DNAs for RUNX1 and CBFβ were co-transfected into K562 cells, and C11orf21 (right panel) and RUNX1 (left panel) expression was measured by RT-qPCR. RUNX1 overexpression upregulated C11orf21 expression. (B) K562 cells were transfected with siRNA specific for RUNX1 by electroporation, and C11orf21 (right panel) and RUNX1 (left panel) expression was measured by RT-qPCR. The data are the means of triplicate determinations +/- S.D.

Fig. 5

RUNX1 transactivated the C11orf21 promoter through binding to the RUNX1-binding site. (A) pGL3-C11orf21 was transfected into K562 cells with control plasmid or RUNX1, RUNX1 R174Q-mutant, or RUNX1-ETO, with or without CBFβ expression plasmids. The result of the luciferase assay is shown as a bar graph. R174Q and RUNX1-ETO reduced C11orf21 promoter activity regardless of CBFβ status. (B) pGL3-C11orf21 and pGL3-C11orf21-mt were transfected into K562 cells. The result of the luciferase assay is shown as a bar graph. When the RUNX1-binding site was mutated (-mt), the promoter activity of pGL3-C11orf21 was attenuated. (C) pGL3-C11orf21 and pGL3-C11orf21-mt were transfected into K562 cells with control plasmid or RUNX1 in the presence of the CBFβ expression vector. The results of the luciferase assay are shown as a bar graph. Mutation of the RUNX1-binding site reduced the response for RUNX1. The data represent the mean of triplicate determinations +/- S.D.

Fig. 6

RUNX1 transactivation of C11orf21 was suppressed by RUNX1-ETO. (A) RUNX1-ETO suppressed RUNX1 transactivation of C11orf21 in a dose-dependent manner. Fixed doses of pGL3-C11orf21, pRc/CMV-RUNX1 and pRc/CMV-CBFβ were co-transfected into K562 cells with increasing doses of RUNX1-ETO. (B) pGL3-C11orf21 was transfected into Kasumi-1 cells with or without RUNX1 and CBFβ expression plasmids. RUNX1 expression restored the transcription activity of the C11orf21 promoter. (C) RUNX2, RUNX3, and RUNX1 were competent to activate the C11orf21 promoter in the luciferase assay experiments. The data are the mean +/- S.D (n = 3). (D) Chromatin immunoprecipitation (ChIP) data. RUNX1, RUNX2, RUNX3, and ETO binding peaks detected on the C11orf21 promoter region are shown. Data were reanalyzed by ChIP-Atlas. The structure of the C11orf21 gene is shown below the histograms of ChIP data. Black boxes indicate exons. Black arrow shows the transcription start site and red arrows show RUNX1-binding sites with TGTGGT sequences.

Fig. 7

The effects of C11orf21 expression on Kasumi-1 cells. (A) The CRISPR/Cas9-mediated gene knockout screens data were analyzed using The Cancer Dependency Map (DepMap). Knockout of C11orf21 did not affect the proliferation of t(8;21) AML cell lines such as Kasumi-1 (indicated by blue stars) or SKNO-1 (red stars). Stars were drawn by tracing the original data. (B, C and D) C11orf21 was overexpressed in Kasumi-1 cells. After 24 h of transfection, RT-qPCR for C11orf21 (B), cell counting (C) and cell cycle analysis by PI staining (D) were performed.