Integrating WGBS with RNA/ChIP-seq: Cases for Plant Development and Cdx2 Binding Dynamics

Whole genome bisulfite sequencing (WGBS) technology, with its unique single-base resolution detection ability, can accurately capture DNA methylation modification sites in the whole genome, providing high-resolution and high-precision data support for the analysis of epigenetic regulation mechanism. RNA sequencing (RNA-seq) technology can realize systematic analysis and quantitative characterization of gene expression profiles at the transcriptome level by virtue of its Qualcomm quantity and high sensitivity.

The joint analysis strategy of WGBS and RNA-seq, by integrating epigenetic modification information and transcription expression data, constructs a cross-level regulatory association network of "DNA methylation-gene expression", which provides a multi-dimensional and systematic research perspective for analyzing the regulatory mechanism of biomolecules. This technology integration scheme has become an important tool in the frontier research of life science and has shown remarkable application value and research potential in the fields of tumor heterogeneity analysis and plant stress response mechanism research.

In this paper, WGBS combined with RNA-seq and ChIP-seq in revealing epigenetic regulation mechanism was expounded through two cases: AtSAMS regulating the development of Arabidopsis thaliana flower organs and the dynamic combination of Cdx2 in the development and steady state.

Reveal the Epigenetic Mechanism of AtSAMS Regulating Plants

Title: AtSAMS regulates floral organ development by DNA methylation andethylene signaling pathway

Publish Magazine: Plant Science

Impact Factors: 4.2

Publication Time: 2023.07.03

DOI: https://doi.org/10.1016/j.plantsci.2023.111767

The flower organ is the key structure of plant reproduction, and its development is strictly controlled by heredity. ABC model is a classic theory to explain the development of flower organs, in which functional genes A, B, C, and E regulate the formation of sepals, petals, stamens, and pistils respectively. However, the mechanism of expression regulation of these genes is not completely clear. As an important epigenetic modification, DNA methylation plays a key role in gene regulation, genome stability, and plant development. S-adenosylmethionine synthase (SAMS) is a key enzyme involved in S- adenosylmethionine biosynthesis, a universal methyl donor in methylation reaction, and a common precursor of ethylene, polyamine and other biosynthesis. Ethylene is an important plant hormone, which participates in many physiological processes of plants, including flower organ development. However, it is not clear how SAMS regulates flower organ development through methylation and ethylene signaling pathways.

Proposed working model for the AtSAMS regulation of floral organ in Arabidopsis (Hu et al., 2023)

The analysis results of this study revealed that the abnormal development of flower organs in plants with over-expression of AtSAMS in Arabidopsis thaliana was caused by DNA demethylation and ethylene signaling pathway. In SAMOE (AtSAMS overexpression plant), the whole genome DNA methylation level decreased and the ethylene content increased. In addition, the transcription level of the ACE gene is highly correlated with its methylation level, but the down-regulation of the b gene may not be related to demethylation but is caused by the ethylene signaling pathway independently SAMS-mediated methylation and ethylene signaling pathway may interact in the development of flower organs.

AtSAMS Overexpression Leads to Abnormal Floral Organs

SAMOE plants show a variety of floral organ abnormalities, including the transformation of sepals into pistils, the decrease of petals and stamens, etc., which are similar to the phenotypes of ABCE gene mutants or over-expressed strains.

• The transcription level of AtSAMS in the over-expressed plant (SAMOE) was detected. Actin was used as an internal reference, and the standardized level of wild type (WT) was set to 1. The results showed that the expression levels of AtSAMS in S1OE-10, S1OE-21, S2OE-5, and S2OE-6 were significantly higher than those in WT, indicating that the overexpression was successful.
• The proportion of abnormal flower organs in WT, S1OE, and S2OE was counted. More than 40% of flowers in SAMOE developed abnormally, while the proportion in WT was extremely low, indicating that over-expression of AtSAMS led to abnormal flower organs.
• Show the abnormal floral organ phenotype of SAMOE. WT flower has 4 sepals, 4 petals, 6 stamens, and 1 carpel. SAMOE flowers show many abnormalities, such as secondary flowers, sepals transformed into carpels, carpels increased, stamens of sepals decreased, carpels were not completely fused, sepals were leaflike, stamens transformed into carpels, and petals decreased or increased.
• The expression of the ABCE gene in WT and SAMOE was analyzed by qRT-PCR. The results showed that class A genes (AP1, AP2) and class B genes (AP3, PI) were down-regulated in SAMOE, while class C genes (AG) and class E genes (SEP1, SEP2, SEP3) were up-regulated, which conformed to the gene expression pattern in ABCE model.
• Compare the content of S-adenosylmethionine (SAM) in WT and SAMOE. The content of SAM in SAMOE was 14.46% (S1OE) and 13.38% (S2OE) higher than that in WT, which indicated that the over-expression of AtSAMS increased the synthesis of SAM.

AtSAMS overexpression displays abnormal floral organ phenotypes in Arabidopsis (Hu et al., 2023)

Low Methylation of Leads to Abnormal Flower Organs.

The results of WGBS showed that the whole genome DNA methylation level of SAMOE plants decreased significantly, especially in the environment of CHG and CHH sequences, which led to abnormal flower organs.

• Compare the average methylation rates of CG, CHG, and CHH sequences in WT and SAMOE. The methylation rates of CG, CHG, and CHH in WT are 38.82%, 20.03%, and 6.78% respectively. The methylation rates of SAMOE in these three sequence environments all decreased significantly, with S1OE decreasing by 24.17%, 61.16%, and 29.06%, and S2OE decreased by 18.80%, 51.28%, and 29.79%, respectively, indicating that there was genome-wide hypomethylation in SAMOE.
• The expression level of genes involved in DNA methylation in SAMOE (SAMOE vs WT) was analyzed. The results showed that several SAM-dependent methyltransferase coding genes (such as AT1G24480 and AT1G67990) were down-regulated, while the methyltransferase inhibitor SAH7 gene was up-regulated. At the same time, the expression of DRM2, NRPD1, DCL1/2/3, RDR1/2, AGO6 and other genes in RdDM pathway decreased, while the expression of DNA demethylase genes (ROS1, DME) increased, indicating that DNA hypomethylation in SAMOE may be caused by the decreased expression of SAM-dependent methyltransferase and RdDM pathway-related genes and the increased expression of DNA demethylase.

The methylation rate and the expression level of genes involved in DNA methylation of SAMOE (Hu et al., 2023)

Methylation Levels of The C and E Genes Changed

The results of WGBS and McrBC-qPCR showed that the changes in methylation levels of functional genes A, C, and E were significantly related to their expression levels, while the changes in expression of functional genes B had nothing to do with methylation.

• The phenotype of floral organs in WT plants treated with 20 mM DNA methylation inhibitor 5'-Aza. Compared with the control (mock), the plants treated with 5'-Aza showed dwarfing, twisted leaves, increased rosette leaves, clustered stems, and other phenotypes, and the flower organs also showed similar abnormalities as SAMOE, such as decreased number of petals, sepals and stamens, 6 petals, unfused carpels, and secondary flowers, which indicated that DNA demethylation could lead to abnormal flower organs.
• The methylation level of the ABCE gene promoter region after 5'-Aza treatment was analyzed by McrBC-qPCR. The results showed that class A genes (AP1, AP2) were hypermethylated, class C genes (AG) and class E genes (SEP1, SEP2, SEP3) hypomethylated, which was consistent with WGBS results.
• qRT-PCR was used to analyze the expression of the ABCE gene in inflorescences treated with mock and 5'-Aza. The results showed that class A and class B genes were down-regulated, while class C and class E genes were up-regulated, which was consistent with the expression pattern in SAMOE, further indicating that DNA demethylation led to abnormal flower organs by changing the expression of ABCE gene.

Effects of DNA dethylation on floral organs of Arabidopsis (Hu et al., 2023)

Changes in ABCE Gene Expression Level in SAMOE Plants

The results of RNA-seq and qRT-PCR showed that the expressions of A functional genes (AP1 and AP2) and B functional genes (AP3 and PI) were down-regulated, while the expressions of C functional genes (AG) and E functional genes (SEP1, SEP2, and SEP3) were up-regulated.

• The methylation level of the ABCE gene in WT and SAMOE was compared by WGBS-seq data. The results showed that class A genes (AP1, AP2) were hypermethylated, class C genes (AG) and class E genes (SEP1, SEP SEP3) were hypomethylated in SAMOE, while the methylation level of class B genes (AP3, PI) remained unchanged.
• McrBC-qPCR was used to analyze the methylation level of the ABCE gene promoter region in WT and SAMOE. The results are consistent with WGBS-seq, with AP1 and AP2 hypermethylated, AG, SEP1 and SEP3 hypomethylated.
• Thermogram of Differentially Expressed Genes (DEGs) in WT and SAMOE. The results showed that the expression level of most genes in SAMOE was lower than that of WT, among which there were 2027 dogs (down-regulated by 72.2%) in S1OE and 1040 dogs (down-regulated by 67%) in S2OE, indicating that there was global gene expression down-regulation in SAMOE.
• Analyze the expression level of the ABCE gene in SAMOE. AP1, AP2, AP3, and PI were down-regulated, while AG, SEP1, SEP2, and SEP3 were up-regulated, which was consistent with the results of qRT-PCR, indicating that the expression changes of the ABCE gene were related to methylation level (ACE gene) or not (B gene).

The methylation and expression level of ABCEgenes in SAMOE (Hu et al., 2023)

WGBS Combined with ChIP-seq Reveals the Dynamic Binding Mechanism of Cdx2

Title: Motif distribution and DNA methylationunderlie distinct Cdx2 binding duringdevelopment and homeostasis

Publish Magazine: Nature Communications

Impact Factors: 16.6

Publication Time: 2025.01.22

DOI: https://doi.org/10.1038/s41467-025-56187-0

Cdx2 is a key transcription factor and plays a decisive role in the development of intestinal epithelial cells in mice. It is expressed in both embryonic and adult intestinal epithelial cells, but its binding genomic sites are different in development and adulthood. DNA methylation is an epigenetic modification, which is usually associated with gene silencing. However, some transcription factors (such as Cdx2) may be more inclined to bind methylated DNA sequences. Transcription factors guide tissue development by combining developmental stage-specific targets and establishing an appropriate enhancer landscape. DNA sequence and chromatin modification will guide the genomic binding of transcription factors. However, little is known about how transcription factors navigate chromatin features to selectively bind to a small subset of all possible genomic target sites.

The results of this study reveal that Cdx2, as a pedigree-determining transcription factor that binds different targets in developing intestinal epithelial cells and adult intestinal epithelial cells, has a preferential affinity for an atypical motif containing CpG in vivo. The high frequency of motifs in embryonic Cdx2 targets and the methylation state of CpG during development enable Cdx2 to selectively bind and activate developmental enhancers and genes.

Dynamic Changes of Cdx2 Binding Sites

There are significant differences in the binding sites of Cdx2 between embryonic (E12.5 and E16.5) and adult mouse intestinal epithelial cells. In the embryonic stage, the binding sites were mostly distributed in the promoter region (24%), while in the adult stage, the binding sites were mainly distributed in the enhancer region (> 87%). This change of binding mode from promoter to enhancer reflects the functional changes of Cdx2 at different developmental stages. Cdx2 activates key developmental genes (such as Sox4 and Meis1) by binding promoter regions in the embryonic stage and maintains the steady-state function of tissues (such as Krt19 and Fabp2) by binding enhancer regions in the adult stage.

Evolution of Cdx2 binding across gene promoters and enhancers supportsdevelopmental and homeostatic functions in intestinal epithelium (Lorzadeh et al., 2025)

Cdx2 Binding Motif Preference

Cdx2 tends to bind atypical motifs (ATAAA+CpG) containing CpG in the embryonic stage, while it tends to bind classical motifs without CpG in the adult stage. This motif preference enables Cdx2 to selectively bind to different genomic sites at different developmental stages. The CpG bound by Cdx2 contains a motif, which is usually methylated in the embryonic stage, thus promoting the binding of Cdx2 and gene activation. In adulthood, demethylation of these sites prevented the binding of Cdx2, thus avoiding ectopic binding.

Heightened presence of CpG containing Cdx2 motif at its developmental bindirsites (Lorzadeh et al., 2025)

Synergistic Effect of Cdx2 Binding and Chromatin Modification

Cdx2 can recruit Ctcf and Hnf4a to different genomic sites by combining different motifs. In the embryonic stage, Cdx2 recruits Ctcf by combining the CpG motif to form super-enhancers, which activate development-related genes. In adulthood, Cdx2 recruits Hnf4a by combining classic motifs to form homeostatic enhancers, which maintain tissue function. Cdx2 binding site usually has high chromatin accessibility in the embryonic stage, but in the adult stage, it is necessary to regulate chromatin state through demethylation and other mechanisms, so as to realize gene expression regulation.

cdx2 facilitates establishment of adult homeostatic super-enhancers by directing Ctcf recruitment (Lorzadeh et al., 2025)

Effect of DNA Methylation on Cdx2 Binding

Through the change of DNA methylation induced by Eed gene knockout or GSK-3484862 inhibitor, it is found that this change can re-recruit Cdx2 to the target site of the developmental stage, thus activating the gene expression in the developmental stage. In adulthood, by inducing DNA methylation, Cdx2 can recombine at the target site of development, which indicates that the binding of Cdx2 is regulated by DNA methylation. This methylation-dependent binding enables Cdx2 to regulate gene expression by binding different genomic sites at different developmental stages and under steady-state conditions.

Loss of PRC2 activity causes gain of DNA methylation at enhancers leading toCdx2 recruitment (Lorzadeh et al., 2025)

Conclusion

The combination of WGBS with ChIP-seq and RNA-seq can analyze the gene expression regulatory network from multiple dimensions. WGBS can reveal DNA methylation patterns, ChIP-seq can locate protein-DNA interaction sites such as histone modification, and RNA-seq shows transcriptome dynamics. Through the combined application, the relationship between epigenetic modification, chromatin state, and gene expression can be established, and the mechanism of epigenetic regulation can be further explained. This combined technology has emerged in the research of development, diseases, etc. In the future, the integration with single-cell technology, will promote the development of apparent regulation research to a higher resolution and more dynamic direction, and open up a new path for life science research.

References:

1. Hu W, Hu S, Li S, et al. "AtSAMS regulates floral organ development by DNA methylation and ethylene signaling pathway." Plant Sci. 2023 334: 111767 https://doi.org/10.1016/j.plantsci.2023.111767
2. Lorzadeh A, Ye G, Sharma S, Jadhav U. "Motif distribution and DNA methylation underlie distinct Cdx2 binding during development and homeostasis." Nat Commun. 2025 16(1): 929 https://doi.org/10.1038/s41467-025-56187-0