Mowgli Dandamudi

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.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

EMPIAR, the Electron Microscopy Public Image Archive centered at EMBL-EBI, is a public resource for raw electron microscopy images related to EMDB, contains micrographs, particle sets and tilt-series.

Rapid chemo-electric signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain ¹ . The degenerin/epithelial sodium channel (DEG/ENaC) superfamily, which occurs throughout the animal kingdom, is unique in its diversity of gating stimuli, with some DEG/ENaCs gated by conventional ligands such as neuropeptides, and others gated by e.g. pH, mechanical force, or enzymatic activity ^2-5 . The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. We have therefore dissected the structural basis for neuropeptide binding and gating in a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus ⁶ , using cryo-electron microscopy. High-resolution structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes that mediate channel gating. Complementary mutagenesis experiments confirm the functional roles of particular amino acid residues implicated by the structures. Our results illuminate ligand-induced channel gating in DEG/ENaCs and offer a structural template for the experimental dissection of channel pharmacology and ion conduction in a characteristically metazoan ion channel superfamily.

FMRFamide and similar neuropeptides are important physiological modulators in most invertebrates, but the molecular basis of FMRFamide activity at its receptors is unknown. We therefore sought to identify the molecular determinants of FMRFamide potency in one of its native targets, the excitatory FMRFamide-gated sodium channel (FaNaC) from gastropod mollusks. Using molecular phylogenetics and electrophysiological measurement of function, we identified a broad FaNaC family that includes mollusk and annelid channels gated by FMRFamide, FVRIamides, and/or Wamides (or myoinhibitory peptides). A comparative analysis of this broader FaNaC family and other channels from the overarching DEG/ENaC superfamily, incorporating mutagenesis and experimental dissection of function, identified a pocket of amino acid residues that determines activation of FaNaCs by neuropeptides. Although this pocket has diverged in distantly related DEG/ENaC channels that are activated by other ligands, such as mammalian acid-sensing ion channels, we show that it nonetheless contains residues that determine enhancement of those channels by similar peptides. This study thus identifies amino acid residues that determine FMRFamide activity at FaNaCs and illuminates evolution of ligand recognition in one branch of the DEG/ENaC superfamily of ion channels.