Arachis pintoi (Fabaceae) is a common relative of the cultivated peanut, and folds its four leaflets up to look like one at night. The adaptive significance of this behavior (foliar nyctinasty) is unknown. To test the hypothesis that leaflet folding alone can deter herbivores, a leaf preference experiment was performed on Chromacris trogon grasshoppers. Small oval cutouts were made from leaves of the grasshopper’s preferred food source, Iochroma arborescens (Solanaceae), and were combined with small pieces of tape and dry grass to construct artificial leaves resembling the day and night form of A. pintoi. In the experiment, groups of three grasshoppers were starved for 24 hours and then placed in petri dishes containing one closed and one open artificial leaf. After 30 six-hour trials, the average herbivory of open leaves was 12.3%, while closed leaves was 5.2% (p = 0.00145), indicating a significant preference for open leaves. This suggests that the folded configuration of A. pintoi leaves can be a defense against herbivory.

Each language has standard books describing that language's grammatical rules. Biologists have searched for similar, albeit more complex, principles relating enhancer sequence to gene expression. Here, we review the literature on enhancer grammar. We introduce dependency grammar, a model where enhancers encode information based on dependencies between enhancer features shaped by mechanistic, evolutionary, and biological constraints. Classifying enhancers based on the types of dependencies may identify unifying principles relating enhancer sequence to gene expression. Such rules would allow us to read the instructions for development within genomes and pinpoint causal enhancer variants underlying disease and evolutionary changes.

Electroporation is a technique to introduce DNA constructs into cells using electric current. Here, we present a protocol to electroporate DNA plasmids into Ciona robusta embryos at the 1-cell stage. We describe steps for setting up and conducting electroporation. We then detail procedures for collecting, fixing, and mounting embryos and counting expression. This protocol can be used to study the expression of enhancers via reporter assays, manipulating cells using genes or modified genes such as dominant negatives, and genome editing. For complete details on the use and execution of this protocol, please refer to Song, et al.¹.

Due to many historical injustices, communities of color have often felt misused by medicine at large. There is a cycle of distrust and general unpleasantness with healthcare providers. Unfortunately, the injustices haven’t stopped, as many people of color feel that those in the medical profession lack one of the basic qualities that are needed in healthcare: compassion. In many research studies about compassion, healthcare professionals and other experts define compassion. However, the people we should be asking are not the providers themselves, but the patients, as they are the ones that will know if they receive compassionate care. Focusing research like this on communities of color, especially since there is already distrust, is important. Although research on compassion has increased, there is little data on how under-resourced, culturally, and ethnically diverse communities define compassion, which can help mitigate the health disparities plaguing these communities. This study will help delineate how compassionate the healthcare industry is, and if it is not, what work can be done to make it more compassionate. A purposely made survey was created with a focus group of Black community leaders, non-profit founders, and physicians from San Diego. This survey is composed of questions that target experiences and attitudes towards physicians and healthcare providers for people from the Black community to expand on. Therefore, the Black community, across all socioeconomic groups, can be directly asked what compassion personally means to them, allowing us to finally understand ‘What Does Compassion Mean to the Black Community of San Diego?’

The notochord is a defining feature of all chordates. The transcription factors Zic and ETS regulate enhancer activity within the notochord. We conduct high-throughput screens of genomic elements within developing Ciona embryos to understand how Zic and ETS sites encode notochord activity. Our screen discovers an enhancer located near Lama, a gene critical for notochord development. Reversing the orientation of an ETS site within this enhancer abolishes expression, indicating that enhancer grammar is critical for notochord activity. Similarly organized clusters of Zic and ETS sites occur within mouse and human Lama1 introns. Within a Brachyury (Bra) enhancer, FoxA and Bra, in combination with Zic and ETS binding sites, are necessary and sufficient for notochord expression. This binding site logic also occurs within other Ciona and vertebrate Bra enhancers. Collectively, this study uncovers the importance of grammar within notochord enhancers and discovers signatures of enhancer logic and grammar conserved across chordates.

Terminal regions of Drosophila embryos are patterned by signaling through ERK, which is genetically deregulated in multiple human diseases. Quantitative studies of terminal patterning have been recently used to investigate gain-of-function variants of human MEK1, encoding the MEK kinase that directly activates ERK by dual phosphorylation. Unexpectedly, several mutations reduced ERK activation by extracellular signals, possibly through a negative feedback triggered by signal-independent activity of the mutant variants. Here we present experimental evidence supporting this model. Using a MEK variant that combines a mutation within the negative regulatory region with alanine substitutions in the activation loop, we prove that pathogenic variants indeed acquire signal-independent kinase activity. We also demonstrate that signal-dependent activation of these variants is independent of kinase suppressor of Ras, a conserved adaptor that is indispensable for activation of normal MEK. Finally, we show that attenuation of ERK activation by extracellular signals stems from transcriptional induction of Mkp3, a dual specificity phosphatase that deactivates ERK by dephosphorylation. These findings in the Drosophila embryo highlight its power for investigating diverse effects of human disease mutations.

Enhancers control the location and timing of gene expression and contain the majority of variants associated with disease^1-3. The ZRS is arguably the most well-studied vertebrate enhancer and mediates the expression of Shh in the developing limb⁴. Thirty-one human single-nucleotide variants (SNVs) within the ZRS are associated with polydactyly^4-6. However, how this enhancer encodes tissue-specific activity, and the mechanisms by which SNVs alter the number of digits, are poorly understood. Here we show that the ETS sites within the ZRS are low affinity, and identify a functional ETS site, ETS-A, with extremely low affinity. Two human SNVs and a synthetic variant optimize the binding affinity of ETS-A subtly from 15% to around 25% relative to the strongest ETS binding sequence, and cause polydactyly with the same penetrance and severity. A greater increase in affinity results in phenotypes that are more penetrant and more severe. Affinity-optimizing SNVs in other ETS sites in the ZRS, as well as in ETS, interferon regulatory factor (IRF), HOX and activator protein 1 (AP-1) sites within a wide variety of enhancers, cause gain-of-function gene expression. The prevalence of binding sites with suboptimal affinity in enhancers creates a vulnerability in genomes whereby SNVs that optimize affinity, even slightly, can be pathogenic. Searching for affinity-optimizing SNVs in genomes could provide a mechanistic approach to identify causal variants that underlie enhanceropathies.

Transcriptional enhancers direct precise gene expression patterns during development and harbor the majority of variants associated with phenotypic diversity, evolutionary adaptations, and disease. Pinpointing which enhancer variants contribute to changes in gene expression and phenotypes is a major challenge. Here, we find that suboptimal or low-affinity binding sites are necessary for precise gene expression during heart development. Single-nucleotide variants (SNVs) can optimize the affinity of ETS binding sites, causing gain-of-function (GOF) gene expression, cell migration defects, and phenotypes as severe as extra beating hearts in the marine chordate Ciona robusta. In human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a SNV within a human GATA4 enhancer increases ETS binding affinity and causes GOF enhancer activity. The prevalence of suboptimal-affinity sites within enhancers creates a vulnerability whereby affinity-optimizing SNVs can lead to GOF gene expression, changes in cellular identity, and organismal-level phenotypes that could contribute to the evolution of novel traits or diseases.