Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 May 17:38:101733.
doi: 10.1016/j.bbrep.2024.101733. eCollection 2024 Jul.

Epigenome editing revealed the role of DNA methylation of T-DMR/CpG island shore on Runx2 transcription

Yutaro Kawa  1 , Miyuki Shindo  2 , Jun Ohgane  3 , Masafumi Inui  1
Affiliations

Epigenome editing revealed the role of DNA methylation of T-DMR/CpG island shore on Runx2 transcription

Yutaro Kawa et al. Biochem Biophys Rep. .

Abstract

RUNX2 is a transcription factor crucial for bone formation. Mutant mice with varying levels of Runx2 expression display dosage-dependent skeletal abnormalities, underscoring the importance of Runx2 dosage control in skeletal formation. RUNX2 activity is regulated by several molecular mechanisms, including epigenetic modification such as DNA methylation. In this study, we investigated whether targeted repressive epigenome editing including hypermethylation to the Runx2-DMR/CpG island shore could influence Runx2 expression using Cas9-based epigenome-editing tools. Through the transient introduction of CRISPRoff-v2.1 and gRNAs targeting Runx2-DMR into MC3T3-E1 cells, we successfully induced hypermethylation of the region and concurrently reduced Runx2 expression during osteoblast differentiation. Although the epigenome editing of Runx2-DMR did not impact the expression of RUNX2 downstream target genes, these results indicate a causal relationship between the epigenetic status of the Runx2-DMR and Runx2 transcription. Additionally, we observed that hypermethylation of the Runx2-DMR persisted for at least 24 days under growth conditions but decreased during osteogenic differentiation, highlighting an endogenous DNA demethylation activity targeting the Runx2-DMR during the differentiation process. In summary, our study underscore the usefulness of the epigenome editing technology to evaluate the function of endogenous genetic elements and revealed that the Runx2-DMR methylation is actively regulated during osteoblast differentiation, subsequently could influence Runx2 expression.

Keywords: CpG island shore; Differentially methylated region; Epigenome editing; Osteoblast; Runx2.

PubMed Disclaimer

Conflict of interest statement

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masafumi Inui reports financial support was provided by 10.13039/501100001691Japan Society for the Promotion of Science. Jun Ohgane reports financial support was provided by 10.13039/501100001691Japan Society for the Promotion of Science. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
DNA methylation status of Runx2-DMR in MC3T3-E1 cell (A) Conceptual diagram representing the DNA methylation variations of T-DMRs in CpG island shores. The upper red line illustrates the DNA methylation levels of each region. Circles in the lower part represent CpG sites, with black and white indicating methylated and unmethylated CpG, respectively. (B) Methylation analysis of Runx2-DMR in untreated MC3T3-E1 cells. The upper panel schematically illustrates Runx2-DMR where Left/Middle/Right1/Right2 regions were depicted by black bars and CpG sites were indicated by orange circles. Numbers at the bottom of the line indicate the distance from the Runx2-I TSS. The lower panel presents the result of the bisulfite sequence. Numbers at the bottom indicate the ratio of methylated CpG in each region in a single experiment shown in this Figure. Similar result was reproduced in one more independent experiment of which the result is shown in Fig. S1 (n = 2). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2
Fig. 2
Epigenome editing of Runx2-DMR using G418 selection (A) A schematic drawing illustrating the positions of gRNAs targeting Runx2-DMR, which are shown in black arrows at the bottom. The PAM sequences are on the arrowhead sides. (B) The time course of the DNA methylation analysis of the Runx2-DMR using G418 selection. (C) The result of DNA methylation analysis. gRNAs introduced in each sample are shown on the top. The analyzed regions are denoted as L: Left, M: Middle, R1: Right1, R2: Right2. Numbers at the bottom indicate the ratio of methylated CpG in each region in a single experiment shown in this Figure. Similar result was reproduced in one more independent experiment of which the result is shown in Fig. S3 (n = 2).
Fig. 3
Fig. 3
Optimization of Runx2-DMR epigenome editing using FACS (A) The time course of the DNA methylation analysis of the Runx2-DMR using FACS sorting. (B, C) DNA methylation rates of the Right1 and Right2 regions of the cells selected through various conditions. Five conditions for selection are shown at the bottom. NC: no treatment. (D) The bisulfite sequence results of selection condition 5. M: Middle, R1: Right1, and R2: Right2 regions. The DNA methylation analyses were performed once for condition 1–4 (n = 1) for screening, and twice for condition 5 (n = 2) to confirm relatively high methylation state by this method. Essentially the same results were obtained in a reproductive experiment and the result is shown in Fig. S4.
Fig. 4
Fig. 4
Effects of Runx2-DMR epigenome editing on gene expression and osteoblast differentiation (A) The time course of the epigenome editing and differentiation analysis. (B, C) The DNA methylation analysis for the control (“empty”) and gRNA1,2,3-transfected cells (“pooled”) at Differentiation Day 0, Differentiation Day 14, and Day 24 without differentiation. The analyzed regions are denoted as M: Middle, R1: Right1, R2: Right2. Numbers at the bottom indicate the ratio of methylated CpG in each region in a single experiment shown in this Figure. Similar result was reproduced in one more independent experiment of which the result is shown in Fig. S5 (n = 2). (D) The gene expression analysis for control (“empty”) and gRNA1,2,3-transfected cells (“pooled”) at Differentiation Day 0 and Differentiation Day 14. The expression levels of each mRNA were normalized by that of GAPDH. The data were shown as mean ± SE and analyzed by Student's t-test. *p < 0.05, **p < 0.01. (E, F) The alizarin red S staining and its quantification for the control (“empty”) and gRNA1,2,3-transfected cells (“pooled”) at Differentiation Day 14. Triplicated wells for each condition showed similar staining levels and the representative images are shown. The quantification data were statistically analyzed using Student's t-test. Mean ± SE. *p < 0.05, **p < 0.01. experiments were performed twice and essentially the same results were obtained. The figure shows representative results. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Komori T. Regulation of proliferation, differentiation and functions of osteoblasts by runx2. Int. J. Mol. Sci. 2019;20:1694. - PMC - PubMed
    1. Komori T., Yagi H., Nomura S., et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell. 1997;89:755–764. - PubMed
    1. Otto F., Thornell A.P., Crompton T., et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell. 1997;89:765–771. - PubMed
    1. Mundlos S., Otto F., Mundlos C., et al. Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia hereditary skeletal disorders comprise a large group. Cell. 1997;89:773–779. - PubMed
    1. Liu W., Toyosawa S., Furuichi T., et al. Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures. J. Cell Biol. 2001;155:157–166. - PMC - PubMed