The Mystery of Cis-Regulatory Elements Function during Mouse Fetal Development: What Genes are Involved?
Analyzing layers of epigenetic regulation such as chromatin accessibility and histone modification patterning has supported the identification of candidate cis-regulatory elements (cCREs, which include enhancers) that contribute to gene expression control during the fetal development of mice through the dynamic binding of a range of transcription factors (Gorkin et al. and He et al.). A recent Nature Structural & Molecular Biology article led by Miao Yu (Fudan University/Ludwig Institute for Cancer Research), Ming Hu (Cleveland Clinic Foundation), and Bing Ren (University of California, San Diego) aimed to delineate the roles of these cCREs in tissue-specific and developmentally-regulated gene expression during mouse fetal development by evaluating 3D chromatin contacts, integrating layers of epigenetic and transcriptional information, and identifying the distant target genes of cCREs (Yu, Zemke, and Chen et al.). Identifying these enhancers remains challenging as defining functional enhancers represents a difficult task, and deleting individual enhancers does not always significantly alter target gene expression. Additionally, this exciting study also provided insight into how developmentally associated cCREs can contribute to the development of diseases (such as schizophrenia) that impact adults.
Mapping Chromatin Contacts, Integrating Epigenetic Data, and Identifying cCRE-Target Genes
Yu, Zemke, and Chen et al. mapped approximately 250,000 long-range chromatin contacts between nearly 15,000 protein-coding gene promoters and distal interacting potential regulatory elements (or cCREs) in multiple tissues and across developmental stages (for one tissue) in developing mouse embryos. They performed the mapping by applying "proximity ligation-assisted ChIP-seq" or PLAC-seq (Fang et al.) – a high-throughput chromatin conformation capture-based technique – using an H3K4me3-specific antibody, which should detect levels of this permissive chromatin modification active and poised gene promoter regions. These data revealed that long-range chromatin interactions between promoters and cCREs displayed tissue-to-tissue variability and developmental dynamics, with tissue-specific promoter-interacting cCREs enriched for accessible chromatin and enhancer-associated histone modifications. In addition, the authors employed the ChIP-seq technique to reveal a prominent role of the CCCTC-binding factor (CTCF) - which forms topologically associating domains and CTCF-CTCF loops through a cohesin-driven loop extrusion process (Sanborn et al.) - in facilitating contacts between promoters and cCREs during mouse fetal development.
The authors next integrated their 3D chromatin contact data with previously published histone modification/chromatin accessibility profiles (Gorkin et al.) and transcriptomic profiles (He et al.) from matching mouse tissue samples to examine relationships between long-range chromatin interactions, chromatin states, chromatin accessibility, CTCF binding, and gene expression during mouse fetal development. In summary, the enormous datasets provided evidence for a strong correlation between chromatin contacts and the chromatin state at distal cCREs and gene expression patterns at predicted target genes. These results also supported the prediction of approximately 15,000 cCRE target genes (identifying around 17,000 enhancer-gene pairs) and the exploration of tissue- and developmental stage-specific gene expression programs.
While integrating epigenetic and transcriptomic datasets from different modalities permitted the authors of this study to draw certain conclusions, techniques that support joint epigenetic and transcriptomic analysis in the same cell represent a means of ensuring the robust nature of any correlations observed. Advances in this area include single-nucleus methyl-3C sequencing (snm3C-seq3) (Lee et al. and Tian et al.), as reported in a previous series of blogs from Epigenome Technologies, and parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing or "Paired-Tag" from Epigenome Technologies. Paired-Tag generates joint epigenetic and gene expression profiles at the single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP-seq assays. Applying Paired-Tag may enable researchers to take huge leaps forward in our understanding of fetal development and significantly improve disease management, as discussed in this study of mouse fetal development and human disease, as discussed below.
Finally, and perhaps most interestingly, Yu, Zemke, and Chen et al. demonstrated that their 3D chromatin contact dataset generated from fetal mouse samples may support the functional annotation of non-coding risk variants in the human genome and prioritize gene targets in human adult disease. They leveraged their list of enhancer-gene pairs to assign non-coding genetic variants associated with human diseases and traits; their analysis of schizophrenia-associated non-coding variants revealed the exclusive expression of disease-associated genes in fetal mouse brains that had remained unidentified in previous studies evaluating non-fetal human data. These fascinating findings reveal how multi-omic studies in the developing fetal mouse may provide deep insight into human disease.
What is Next for Cis-Regulatory Elements?
While this exciting study has provided a new understanding of cCRE function during mouse fetal development, the authors acknowledge limitations that need to be addressed in subsequent studies. New research avenues may involve validating enhancer function via their deletion in relevant cell types/tissues coupled with gene expression profiling (Agarwal and Inoue et al.) and a shift in focus from bulk tissue analyses to the single-cell level by implementing appropriate technologies. The latter approach would offer a high-resolution picture of the regulatory networks involved and may reveal previously unrecognized layers of complexity in fetal development.
Said single-cell technologies include Paired-tag - a complementary analytic platform that creates joint epigenetic and gene expression profiles at single-cell resolution and detects histone modifications and RNA transcripts in individual nuclei. This huge advance was first developed by a team guided by Bing Ren, one of the lead authors of this new study; now, Epigenome Technologies provides optimized Paired-Tag kits and services to researchers in the epigenetics field under an exclusive license from the Ludwig Institute for Cancer Research.
See Nature Structural & Molecular Biology, December 2024 for all the details on how the 3D mapping of chromatin contacts helped define cCRE function during mouse fetal development and provide insight into human disease.
By Stuart. P Atkinson
Comments