Emilia Skafida

Identifying cis- regulatory elements typically relies on trial and error to test the activity of DNA fragments using reporter constructs. This approach is particularly challenging in large eukaryotic genomes, where cis- regulatory elements can be dispersed over long distances, separated by large stretches of non-functional DNA. Here, we generate two types of resources that can be used to narrow the search for such elements in the 3.6 Gbp genome of Parhyale hawaiensis . First, we use bulk ATAC-seq to uncover genome-wide patterns of chromatin accessibility in Parhyale embryonic and adult tissues, and single-nucleus ATAC-seq to identify regions of open chromatin in diverse cell types. Second, by sequencing the genomes of three congeneric species – P. darvishi , P. aquilina , and P. plumicornis – we identify islands of sequence conservation across the genome, likely corresponding to functionally constrained DNA. We find that low-coverage (10–15×) short-read genome sequencing, without genome assembly, is sufficient to provide reliable maps of sequence conservation. This approach cuts the cost and labour required to generate these maps, making the identification of cis- regulatory elements more widely accessible. We demonstrate the utility of these resources by identifying cis- regulatory elements that drive robust expression of fluorescent reporters ubiquitously and in specific cell types.

Identifying non-coding regulatory elements in the genome poses a challenge in most organisms. Classical methods rely on trial and error to test the regulatory activities of DNA fragments using reporter constructs. In large eukaryotic genomes, where cis-regulatory elements can spread over long distances, separated by large stretches of non-functional DNA, this trial and error approach is particularly challenging. Here, we generate two types of resources that can be used to narrow the search for such cis-regulatory elements in the 3.6 Gbp genome of Parhyale hawaiensis (comparable in size to the human genome). First, we use bulk ATAC-seq to uncover genome-wide patterns of chromatin accessibility in embryonic and adult tissues of Parhyale (whole embryos and legs), and single-nucleus ATAC-seq to identify regions of open chromatin in diverse cell types recovered from adult legs, including epidermal, neuronal, muscle and blood cells. Second, by sequencing the genomes of three congeneric species of Parhyale hawaiensis – P. darvishi, P. aquilina and P. plumicornis – we identify islands of sequence conservation across the genome, corresponding to DNA elements that are functionally constrained during evolution. We present an approach by which low-coverage (10-15x) short-read genome sequencing, without genome assembly, is sufficient to provide reliable maps of sequence conservation. This approach cuts the cost and labour required to generate these maps, making the identification of cis-regulatory elements more widely accessible. We demonstrate the utility of these resources by identifying cis-regulatory elements that drive robust expression of fluorescent reporters ubiquitously and in specific cell types.

Identifying non-coding regulatory elements in the genome poses a challenge in most organisms. Classical methods rely on trial and error to test the regulatory activities of DNA fragments using reporter constructs. In large eukaryotic genomes, where cis- regulatory elements can spread over long distances, separated by large stretches of non-functional DNA, this trial and error approach is particularly challenging. Here, we generate two types of resources that can be used to narrow the search for such cis- regulatory elements in the 3.6 Gbp genome of Parhyale hawaiensis (comparable in size to the human genome). First, we use bulk ATAC-seq to uncover genome-wide patterns of chromatin accessibility in embryonic and adult tissues of Parhyale (whole embryos and legs), and single-nucleus ATAC-seq to identify regions of open chromatin in diverse cell types recovered from adult legs, including epidermal, neuronal, muscle and blood cells. Second, by sequencing the genomes of three congeneric species of Parhyale hawaiensis – P. darvishi , P. aquilina and P. plumicornis – we identify islands of sequence conservation across the genome, corresponding to DNA elements that are functionally constrained during evolution. We present an approach by which low-coverage (10-15x) short-read genome sequencing, without genome assembly, is sufficient to provide reliable maps of sequence conservation. This approach cuts the cost and labour required to generate these maps, making the identification of cis- regulatory elements more widely accessible. We demonstrate the utility of these resources by identifying cis- regulatory elements that drive robust expression of fluorescent reporters ubiquitously and in specific cell types.

Drosophila Hey is a basic helix–loop–helix–orange (bHLH-O) protein with an important role in the establishment of distinct identities of postmitotic cells. We have previously identified Hey as a transcriptional target and effector of Notch signalling during the asymmetric division of neuronal progenitors, generating neurons of two types, and we have shown that Notch-dependent expression of Hey also marks a subpopulation of the newborn enteroendocrine (EE) cells in the midgut primordium of the embryo. Here, we investigate the transcriptional regulation of Hey in neuronal and intestinal tissues. We isolated two genomic regions upstream of the promoter (HeyUP) and in the second intron (HeyIN2) of the Hey gene, based on the presence of binding motifs for Su(H), the transcription factor that mediates Notch activity. We found that both regions can direct the overlapping expression patterns of reporter transgenes recapitulating endogenous Hey expression. Moreover, we showed that while HeyIN2 represents a Notch-dependent enhancer, HeyUP confers both Notch-dependent and independent transcriptional regulation. We induced mutations that removed the Su(H) binding motifs in either region and then studied the enhancer functionality in the respective Hey mutant lines. Our results provide direct evidence that although both enhancers support Notch-dependent regulation of the Hey gene, their role is redundant, as a Hey loss-of-function lethal phenotype is observed only after deletion of all their Su(H) binding motifs by CRISPR/Cas9.