RUNX1 induces DNA replication independent active DNA demethylation at SPI1 regulatory regions
© The Author(s) 2017
Received: 2 November 2016
Accepted: 28 March 2017
Published: 4 April 2017
SPI1 is an essential transcription factor (TF) for the hematopoietic lineage, in which its expression is tightly controlled through a −17-kb upstream regulatory region and a promoter region. Both regulatory regions are demethylated during hematopoietic development, although how the change of DNA methylation status is performed is still unknown.
We found that the ectopic overexpression of RUNX1 (another key TF in hematopoiesis) in HEK-293T cells induces almost complete DNA demethylation at the −17-kb upstream regulatory region and partial but significant DNA demethylation at the proximal promoter region. This DNA demethylation occurred in mitomycin-C-treated nonproliferating cells at both regulatory regions, suggesting active DNA demethylation. Furthermore, ectopic RUNX1 expression induced significant endogenous SPI1 expression, although its expression level was much lower than that of natively SPI1-expressing monocyte cells.
These results suggest the novel role of RUNX1 as an inducer of DNA demethylation at the SPI1 regulatory regions, although the mechanism of RUNX1-induced DNA demethylation remains to be explored.
KeywordsSPI1 RUNX1 DNA demethylation Endogenous expression
SPI1 is a hematopoietic lineage-specific TF belonging to the ETS family . It plays an important role in the development of myeloid and lymphoid cells and is highly expressed in monocytes [2, 3]. The expression of SPI1 is regulated by the combined activity of a proximal promoter and an upstream regulatory element (URE) located −17-kb upstream of the transcription start site (also known as the distal promoter region) in human [3, 4]. Deletion of the URE region contributes to the exacerbation of acute myeloid leukemia (AML) or erythroleukemia [5, 6]. The SPI1 regulatory regions are differentially methylated in SPI1-expressing and nonexpressing cell lines [7, 8]. This differential pattern is also maintained during hematopoietic differentiation, in which ES cells are hypermethylated while hematopoietic stem cells become hypomethylated [7–9]. Meanwhile, abnormal hypermethylation of the SPI1 regulatory regions is frequently observed in myeloma cell lines with downregulated SPI1 . Thus, this methylation change seems to be important for SPI1 expression. However, no molecular mechanism behind the change in DNA methylation status at SPI1 regulatory regions has been reported yet.
RUNX1 is another TF that is essential for the regulation and maintenance of mammalian hematopoiesis . A previous study reported that RUNX1 regulates SPI1 at both transcriptional and epigenetic levels . RUNX1 binding at the conserved sites in the URE of SPI1 is critical for the onset of SPI1 expression during hematopoietic stem cell formation, making SPI1 the direct downstream target of RUNX1 . At the pre-hematopoietic or hemangioblast stage, the inception of RUNX1 expression induces chromatin remodeling at the regulatory regions of SPI1, in which the binding of RUNX1 to the SPI1 regulatory region is essential . Moreover, several studies have revealed that chromatin remodeling is coupled with DNA demethylation during embryonic development . Therefore, we hypothesize that RUNX1 may also be involved in recruiting the DNA methylation status change at the SPI1 regulatory regions.
Here, we describe that the ectopic expression of RUNX1 in HEK-293T cells induces DNA demethylation at the two functionally active regulatory regions of SPI1 in a replication-independent manner.
RUNX1 overexpression induced DNA demethylation at SPI1 −17-kb URE
RUNX1 overexpression induced partial DNA demethylation at SPI1 proximal promoter
Molecular mechanism of RUNX1-induced DNA demethylation at SPI1 regulatory regions
RUNX1 induces significant SPI1 expression, but much less than in monocytes
In this study, we found that the ectopic expression of RUNX1 in wild-type HEK-293T cells converts the methylation status of both SPI1 regulatory regions from hypermethylated to hypomethylated; the results also showed that the induced demethylation was replication-independent active DNA demethylation. Further, RUNX1 overexpression did not change gene expression of enzymes involved in both DNA methylation and demethylation. It has been reported that chromatin remodeling of the SPI1 URE region in hemangioblasts is induced by the binding of RUNX1, which also accompanies the DNA demethylation of this region . This suggests that RUNX1 binding directly recruits the DNA demethylating machinery. Actually, several TFs have recently shown to be involved in DNA demethylation by recruiting DNA demethylation machinery . On the other hand, the proximal promoter region revealed partial but significant DNA demethylation, although it does not contain any binding sites for RUNX1 and neither any mechanism of its binding is known [12, 14]. Thus, it was interesting to ponder how this replication-independent active DNA demethylation occurs at the proximal promoter region of SPI1 by the overexpression of RUNX1. Previous chromatin immunoprecipitation (ChIP) data have shed light on the binding of various transcription factors in the URE and proximal promoter regions . Therefore, we speculate that other RUNX1-regulated transcription factor(s) may also be able to induce DNA demethylation at both regulatory regions. Thus, we can conclude that RUNX1 could be directly or indirectly responsible for inducing DNA demethylation at the SPI1 regulatory regions, although the actual process remains to be confirmed.
Our study also showed that RUNX1 overexpression is not sufficient for inducing the higher level of endogenous SPI1 expression in HEK-293T cells. The partial demethylation at the proximal promoter region may be responsible for the low level of SPI1 expression. In fact, the proximal promoter region of SPI1 is completely hypomethylated in monocytes, in which SPI1 is expressed at a higher level [18, 19]. Previous reports also suggest the positive correlation between DNA demethylation at gene regulatory regions and gene expression, revealing that active genes are generally hypomethylated while inactive genes are generally hypermethylated at their promoter regions [7, 20]. Furthermore, the cells with high SPI1 expression regulate this expression by forming an autoregulatory loop between its URE and the proximal promoter region [4, 21], which are in close proximity to each other . Thus, our data suggest that the autoregulatory loop may not form well due to incomplete demethylation at the proximal promoter. The presence of other transcription factors that are expressed in SPI1-expressing hematopoietic cells could also be necessary to induce higher endogenous expression of SPI1.
We used HEK-293T cells in this study because they are from a cell line that does not express RUNX1. The propensity of these cells to undergo transfection, their availability, and the possibility of avoiding passage biasness during analysis also make them easy to use. Since our results are derived from nonhematopoietic cells by using an artificial overexpression system, it would be preferable for our results to be evaluated further using hematopoietic cell lines in which RUNX1 and SPI1 are endogenously expressed. However, DNA demethylation at SPI1 regulatory regions already occurs at the hematopoietic stem cell stage at which RUNX1 is expressed. As the next step, it may be necessary to perform such evaluation in hematopoietic cell lines under specific conditions or with gene manipulation.
To our knowledge, this is the first study evaluating the potential role of RUNX1 as a demethylating inducer. Our results provide a hint about how demethylation occurs at SPI1 regulatory regions.
Cell culture and RUNX1 over expression
HEK-293T cells, a human embryonic cell line provided by Riken Cell Bank (RCB), were cultured in High-Glucose Dulbecco’s Modified Eagle Medium (DMEM, Wako, Japan) supplemented with 10% fetal bovine serum (Lonza, Basel, Switzerland) and 2 mM penicillin–streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO2.
RUNX1 overexpression in HEK-293T cells was carried out by using the lentivirus transduction method. The lentivirus for RUNX1 overexpression and its control MCS were prepared in a mixture of packaging constructs, pCMV-VSV-G-RSV-Rev and pCAG-HIVgp (provided by RIKEN BRC) in accordance with a previously reported protocol . HEK-293T cells (1 × 106 cells per well) were seeded in a poly-d-lysine-coated 12-well plate, followed by transduction on the second day by adding 8 µg/ml polybrene and 10 multiplicity of infection (MOI) of lentivirus in each well. One day after the transduction, the medium was replaced with fresh DMEM medium containing 1 µg/ml puromycin (Thermo Fisher Scientific, Waltham, MA, USA) for the selection of transduced cells. Cells were harvested 7 days after the transduction. Wild-type HEK-293T cells were also kept as a control.
List of primers used for bisulfite PCR and qRT-PCR
SPI1 proximal promoter (Fwd)
SPI1 proximal promoter (Rv)
SPI1 −17-kb URE (Fwd)
SPI1 −17-kb URE (Rv)
Cell cycle arrest
HEK-293T cells (2 × 106 cells per well) were plated into a six-well plate, treated with different concentrations of mitomycin-C (Sigma-Aldrich, St. Louis, MO), and incubated for 4 h at 37 °C in 5% CO2. To check cell cycle arrest (at the G1 phase), the proliferation assay was performed by following the Click-iT® EdU Cytometry Cell Proliferation Assay protocol (Thermo Fisher Scientific, Waltham, MA, USA); for assessment, FACS was used. After the confirmation of cell cycle arrest at 50 µg/ml mitomycin-C, the cells were plated and transduced with RUNX1 lentivirus on the second day by following the above-mentioned protocol. Wild-type 293T cells were also treated with 50 µg/ml mitomycin-C and transduced with MCS, which were kept as a control.
Total RNA was isolated by using the Nucleospin® RNA kit protocol (Macherey–Nagel, Germany). Five hundred nanograms of total RNA was amplified using the Ambion total RNA amplification kit (Ambion, Carlsbad, CA), followed by hybridization of the synthesized cRNA with the Human HT-12 v4 Expression BeadChip kit (Illumina, San Diego, CA, USA), in accordance with the manufacturer’s protocol. Scanning of the chip was performed using Illumina BeadScan and BeadStudio (version 3.1). Data were processed with a package from Bioconductor (lumi) [23, 24] using the free software environment R (http://www.r-project.org/). The microarray data has been registered in gene expression omnibus (https://www.ncbi.nlm.nih.gov/geo/) in NCBI (GSE95308).
Total RNA was isolated using the Nucleospin® RNA kit protocol (Macherey–Nagel, Germany). Reverse transcription of total RNA was performed using the Prime Script RT kit (Takara, Takara Bio, Japan); qRT-PCR was performed with the ABI PRISM® 7500 sequence detection system (Applied Biosystems, USA) using SYBR Premix Ex Taq™ II (Tli RNaseH plus, Takara Bio, Japan) and gene-specific primers for SPI1 and GAPDH (Table 1). PCR cycling conditions consisted of initial denaturation at 95 °C for 10 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. Data was analyzed using the 2−ΔΔCt method . Human peripheral blood CD14+ monocytes (Lonza, Basel, Switzerland) were kept as a positive control for the comparison of SPI1 expression.
All of the statistical analyses for DNA methylation status and calculation of methylation percentage were performed using the online quantification tool QUMA. The significance of differences between the transduced and untransduced cells was determined by using unpaired, two-tailed, and Student’s tests (t test), where p < 0.05 was considered significant.
upstream regulatory element
acute myeloid leukemia
multiple cloning sites
multiplicity of infection
Conceived and designed the experiments: SG, TS, and HS. Performed the experiments: SG, HN, MK, SM, YS. Analyzed the data: SG, TS, HS, and JRL. Contributed reagents/materials/analysis tools: SG, TS, JRL, HN, MK, SM, YS. Wrote the paper: SG, HS. All authors read and approved the final manuscript.
The authors wish to thank RIKEN-CLST for all the facilities.
The authors declare that they have no competing interests.
Availability of data and materials
All the data generated during this study are included in this article. The microarray data generated in this article is available in the gene expression omnibus of NCBI [GSE95308, https://www.ncbi.nlm.nih.gov/geo/].
This work was supported by a research grant from MEXT for RIKEN Center for Life Science Technologies. The funding agency has no role in the design of study, data collection, data interpretation, decision to publish, or preparation of the manuscript.
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