Genome-Wide Prediction and Validation of Intergenic Enhancers in Arabidopsis Using Open Chromatin Signatures

Enhancers are important regulators of gene expression in eukaryotes. Enhancers function independently of their distance and orientation to the promoters of target genes. Thus, enhancers have been difficult to identify. Only a few enhancers, especially distant intergenic enhancers, have been identified in plants. We developed an enhancer prediction system based exclusively on the DNase I hypersensitive sites (DHSs) in the Arabidopsis thaliana genome. A set of 10,044 DHSs located in intergenic regions, which are away from any gene promoters, were predicted to be putative enhancers. We examined the functions of 14 predicted enhancers using the β-glucuronidase gene reporter. Ten of the 14 (71%) candidates were validated by the reporter assay. We also designed 10 constructs using intergenic sequences that are not associated with DHSs, and none of these constructs showed enhancer activities in reporter assays. In addition, the tissue specificity of the putative enhancers can be precisely predicted based on DNase I hypersensitivity data sets developed from different plant tissues. These results suggest that the open chromatin signature-based enhancer prediction system developed in Arabidopsis may serve as a universal system for enhancer identification in plants.     

Cross-species enhancer prediction using machine learning

Cis-regulatory elements (CREs) are non-coding parts of the genome that play a critical role in gene expression regulation. Enhancers, as an important example of CREs, interact with genes to influence complex traits like disease, heat tolerance and growth rate. Much of what is known about enhancers come from studies of humans and a few model organisms like mouse, with little known about other mammalian species. Previous studies have attempted to identify enhancers in less studied mammals using comparative genomics but with limited success. Recently, Machine Learning (ML) techniques have shown promising results to predict enhancer regions. Here, we investigated the ability of ML methods to identify enhancers in three non-model mammalian species (cattle, pig and dog) using human and mouse enhancer data from VISTA and publicly available ChIP-seq. We tested nine models, using four different representations of the DNA sequences in cross-species prediction using both the VISTA dataset and species-specific ChIP-seq data. We identified between 809,399 and 877,278 enhancer-like regions (ELRs) in the study species (11.6–13.7% of each genome). These predictions were close to the ~8% proportion of ELRs that covered the human genome. We propose that our ML methods have predictive ability for identifying enhancers in non-model mammalian species. We have provided a list of high confidence enhancers at https://github.com/DaviesCentreInformatics/Cross-species-enhancer-prediction and believe these enhancers will be of great use to the community.     

ETph: enhancers and their targets in pig and human database

Enhancers, as the genomic non-coding sequences, play a key role in the activation of gene expression. They have been widely identified in the human genome. Pig is an important biomedical model for human health. Few studies have been performed to explore the enhancers in the pig genome. The human enhancer information may be useful to identify enhancers in the pig genome. In addition, the genetic background of pig traits could be useful to annotate human enhancers and diseases. Thus, in order to further study enhancers and their potential roles in human and pig, we developed a public database, ETph (Enhancers and their Targets in pig and human). ETph integrates the information on human enhancers, pig putative enhancers, target genes, pig QTL terms, human diseases, GO terms and the KEGG pathway. A total of 25 182 enhancers were identified in the pig genome using the human homology sequence information. Among them, 6232 high-confidence enhancers were used to build the ETph. ETph provides a convenient platform to search, browse and download data. Moreover, a web-based analytical tool was designed to visualize networks and topology graphs among pig putative enhancers, target genes, pig QTL traits and human diseases. ETph might provide a useful tool for researchers to investigate the genetic background of pig traits and human diseases. ETph is freely accessible at http://klab.sjtu.edu.cn/enhancer/ .     

Inducible CRISPRa screen identifies putative enhancers

Enhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here, we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox)-inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific guide RNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible, and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.     

Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals

Sox2 expression marks neural and sensory primordia at various stages of development. A 50 kb genomic region of chicken Sox2 was isolated and scanned for enhancer activity utilizing embryo electroporation, resulting in identification of a battery of enhancers. Although Sox2 expression in the early embryonic CNS appears uniform, it is actually pieced together by five separate enhancers with distinct spatio-temporal specificities, including the one activated by the neural induction signals emanating from Hensen's node. Enhancers for Sox2 expression in the lens and nasal/otic placodes and in the neural crest were also determined. These functionally identified Sox2 enhancers exactly correspond to the extragenic sequence blocks conspicuously conserved between chicken and mammals, which are not discernible by sequence comparison among mammals.