Understanding the contributions of transcription factor DNA binding sites to transcriptional enhancers is a significant challenge. We developed Quantitative enhancer-FACS-Seq for highly parallel quantification of enhancer activities from a genomically integrated reporter in
During gene regulation, DNA accessibility is thought to limit the availability of transcription factor (TF) binding sites, while TFs can increase DNA accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events in the modulation of gene expression remain unknown for the vast majority of genes. We utilized deeply sequenced ATAC-Seq data and site-specific knock-in reporter genes to investigate the relationship between the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of Cebpa during macrophage-neutrophil differentiation. While the enhancers upregulate reporter expression during the earliest stages of differentiation, there is little corresponding increase in their total accessibility. Conversely, total accessibility peaks during the last stages of differentiation without any increase in enhancer activity. The accessibility of positions neighboring C/EBP-family TF binding sites, which indicates TF occupancy, does increase significantly during early differentiation, showing that the early upregulation of enhancer activity is driven by TF binding. These results imply that a generalized increase in DNA accessibility is not sufficient, and binding by enhancer-specific TFs is necessary, for the upregulation of gene expression. Additionally, high-coverage ATAC-Seq combined with time-series expression data can infer the sequence of regulatory events at binding-site resolution.
more » « less- Award ID(s):
- 1942471
- NSF-PAR ID:
- 10481435
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- G3: Genes, Genomes, Genetics
- ISSN:
- 2160-1836
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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We used capped analysis of gene expression with sequencing (CAGE-seq) to profile eRNA expression and enhancer activity during embryogenesis of a model echinoderm: the sea urchin, Strongylocentrotus purpuratus . We identified more than 18,000 enhancers that were active in mature oocytes and developing embryos and documented a burst of enhancer activation during cleavage and early blastula stages. We found that a large fraction (73.8%) of all enhancers active during the first 48 h of embryogenesis were hyperaccessible no later than the 128-cell stage and possibly even earlier. Most enhancers were located near gene bodies, and temporal patterns of eRNA expression tended to parallel those of nearby genes. Furthermore, enhancers near lineage-specific genes contained signatures of inputs from developmental gene regulatory networks deployed in those lineages. A large fraction (60%) of sea urchin enhancers previously shown to be active in transgenic reporter assays was associated with eRNA expression. Moreover, a large fraction (50%) of a representative subset of enhancers identified by eRNA profiling drove tissue-specific gene expression in isolation when tested by reporter assays. Our findings provide an atlas of developmental enhancers in a model sea urchin and support the utility of eRNA profiling as a tool for enhancer discovery and regulatory biology. The data generated in this study are available at Echinobase, the public database of information related to echinoderm genomics.more » « less
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Introduction Various sequencing based approaches are used to identify and characterize the activities of cis -regulatory elements in a genome-wide fashion. Some of these techniques rely on indirect markers such as histone modifications (ChIP-seq with histone antibodies) or chromatin accessibility (ATAC-seq, DNase-seq, FAIRE-seq), while other techniques use direct measures such as episomal assays measuring the enhancer properties of DNA sequences (STARR-seq) and direct measurement of the binding of transcription factors (ChIP-seq with transcription factor-specific antibodies). The activities of cis -regulatory elements such as enhancers, promoters, and repressors are determined by their sequence and secondary processes such as chromatin accessibility, DNA methylation, and bound histone markers. Methods Here, machine learning models are employed to evaluate the accuracy with which cis -regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis -regulatory activity that is reflective of sequence content versus secondary processes. Results and discussion Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis -regulatory element prediction.more » « less
