Biology subjects

Cochran, K.

Publications and source records attributed to Cochran, K..

9 recordsLinked to original sources

bioRxiv2025

A dual role for GLI3 signaling in neural crest development

Neural crest cells (NCCs) are a multipotent cell population that undergo specification, epithelial-to-mesenchymal transition, migration, and differentiation into a plethora of cell types. A wealth of studies across various embryonic model systems have established dogma as to the molecular mechanisms and signaling cascades that contribute to NCC development. While Wnt, FGF, and BMP signaling pathways have well-established and essential roles in several aspects of NCC development, the Hedgehog (HH) signaling pathway has received limited attention for any specific role in this process. Herein, we propose two distinct, temporal roles for the transcription factor GLI3 in NCC development. Gli3, and other members of the HH pathway, were robustly co-expressed with established NCC induction and specification markers in chick, mouse, and human embryonic stem cell derived NCCs. Early knock-down of GLI3 reduced expression of key markers of NCC specification and conditional knock-out of Gli3 post-specification specifically impaired the ability of cranial NCCs to differentiate into ectomesenchymal derivatives. Together, these results demonstrate dual, novel roles for GLI3 in early NCC specification and later in cranial NCC differentiation. Summary StatementGLI3 plays distinct temporal roles in neural crest specification and differentiation into ectomesenchymal derivatives.

developmental biology↗

bioRxiv2024

Directionality of Transcriptional Regulatory Elements

Divergent transcription is a critical marker of active transcriptional regulatory elements (TREs), including enhancers and promoters, in mammals. However, distal elements with unidirectional transcriptional patterns are often overlooked, leaving their identity and function poorly understood. Here, we performed a systematic comparison between divergent and unidirectional elements, revealing their distinct architectural and functional features. Our analysis also shows that unidirectional elements have younger sequence ages and are under weaker evolutionary constraints than divergent elements, indicating that they may represent a unique category of genomic regulatory function with more recent origins. Notably, we observed that some transcription factors, including CTCF, AP1, SP, and NFY, exhibit dual roles in modulating the directionality of TREs, either activating or repressing nascent transcription in a position-dependent manner. Overall, the elucidation of directionality enhances our understanding of the diverse architectural models, functional features, evolutionary dynamics, and regulatory logic of TREs.

molecular biology↗

bioRxiv2024

GENCODE: massively expanding the lncRNA catalog through capture long-read RNA sequencing

Accurate and complete gene annotations are indispensable for understanding how genome sequences encode biological functions. For more than twenty years, the GENCODE consortium has developed reference annotations for the human and mouse genomes, becoming a foundation for biomedical and genomics communities worldwide. Nevertheless, collections of important yet poorly-understood gene classes like long non-coding RNAs (lncRNAs) remain incomplete and scattered across multiple, uncoordinated catalogs. To address this, GENCODE has undertaken the most comprehensive lncRNA annotation effort to date. This is founded on the manually supervised computational annotation of full-length targeted long-read sequencing, on matched embryonic and adult tissues, of orthologous regions in human and mouse. Altogether 17,931 human genes (140,268 transcripts) and 22,784 mouse genes (136,169 transcripts) have been added to the GENCODE catalog representing a 2-fold and 6-fold growth in transcripts, respectively - the greatest increase in the number of annotated human genes since the sequencing of the human genome. Our targeted design assigned human-mouse orthologs at a rate beyond previous studies, tripling the number of human disease-associated lncRNAs that have mouse orthologs. Novel lncRNA genes consistently exhibit biological signals of functionality, and they greatly enhance the functional interpretability of the human genome. While poorly expressed in bulk RNA-Seq samples, many of them are highly expressed in specific cell populations, maybe even contributing to cell-type determination. The expanded GENCODE lncRNA annotations mark a critical step toward deciphering the human and mouse genomes.

genomics↗

bioRxiv2024

Dissecting the cis-regulatory syntax of transcription initiation with deep learning

Despite extensive characterization of mammalian Pol II transcription, the DNA sequence determinants of transcription initiation at a third of human promoters and most enhancers remain poorly understood. We trained and interpreted a neural network called ProCapNet that accurately models base-resolution initiation profiles from PRO-cap experiments using local DNA sequence. ProCapNet learns sequence motifs with distinct effects on initiation rates and TSS positioning and uncovers context-specific cryptic initiator elements intertwined within other TF motifs. ProCapNet annotates predictive motifs in nearly all actively transcribed regulatory elements across multiple cell-lines, revealing a shared cis-regulatory logic across promoters and enhancers and a highly epistatic sequence syntax of cooperative and competitive motif interactions. ProCapNet models of steady-state RAMPAGE profiles distill initiation signals on par with models trained directly on PRO-cap profiles. ProCapNet learns a largely cell-type-agnostic cis-regulatory code of initiation complementing sequence drivers of cell-type-specific chromatin state critical for accurate prediction of cell-type-specific transcription initiation.

genomics↗

bioRxiv2024

Notch dimerization provides robustness against environmental insults and is required for vascular integrity.

The Notch intracellular domain (NICD) regulates gene expression during development and homeostasis in a transcription factor complex that binds DNA either as monomer, or cooperatively as dimers. Mice expressing Notch dimerization-deficient (NDD) alleles of Notch1 and Notch2 have defects in multiple tissues that are sensitized to environmental insults. Here, we report that cardiac phenotypes and DSS (Dextran Sodium Sulfate) sensitivity in NDD mice can be ameliorated by housing mice under hypo-allergenic conditions (food/bedding). However, compound heterozygote NDD mice (N1RA/-; N2RA/-) in hypo-allergenic conditions subsequently develop severe hydrocephalus and hemorrhages. Further analysis revealed multiple vascular phenotypes in NDD mice including leakage, malformations of brain vasculature, and vasodilation in kidneys, leading to demise around P21. This mouse model is thus a hypomorphic allele useful to analyze vascular phenotypes and gene-environment interactions. The possibility of a non-canonical Notch signal regulating barrier formation in the gut, skin, and blood systems is discussed.

developmental biology↗

bioRxiv2023

Rewriting regulatory DNA to dissect and reprogram gene expression

Regulatory DNA sequences within enhancers and promoters bind transcription factors to encode cell type-specific patterns of gene expression. However, the regulatory effects and programmability of such DNA sequences remain difficult to map or predict because we have lacked scalable methods to precisely edit regulatory DNA and quantify the effects in an endogenous genomic context. Here we present an approach to measure the quantitative effects of hundreds of designed DNA sequence variants on gene expression, by combining pooled CRISPR prime editing with RNA fluorescence in situ hybridization and cell sorting (Variant-FlowFISH). We apply this method to mutagenize and rewrite regulatory DNA sequences in an enhancer and the promoter of PPIF in two immune cell lines. Of 672 variant-cell type pairs, we identify 497 that affect PPIF expression. These variants appear to act through a variety of mechanisms including disruption or optimization of existing transcription factor binding sites, as well as creation of de novo sites. Disrupting a single endogenous transcription factor binding site often led to large changes in expression (up to -40% in the enhancer, and -50% in the promoter). The same variant often had different effects across cell types and states, demonstrating a highly tunable regulatory landscape. We use these data to benchmark performance of sequence-based predictive models of gene regulation, and find that certain types of variants are not accurately predicted by existing models. Finally, we computationally design 185 small sequence variants ([≤]10 bp) and optimize them for specific effects on expression in silico. 84% of these rationally designed edits showed the intended direction of effect, and some had dramatic effects on expression (-100% to +202%). Variant-FlowFISH thus provides a powerful tool to map the effects of variants and transcription factor binding sites on gene expression, test and improve computational models of gene regulation, and reprogram regulatory DNA.

genetics↗

bioRxiv2022

Off the shelf: Investigating transfer of learning using commercially available virtual reality equipment

The evolution of virtual reality (VR) has created the opportunity for a relatively low-cost and accessible method to practice motor skills. Previous studies have demonstrated how motor skill practice in non-immersive virtual environments transfers to physical environments. Though minimal research has investigated motor learning and transfer within immersive VR, multiple experiments provide empirical evidence of positive transfer effects. To enhance the similarities between virtual and physical environments, most studies have used software engines and modified hardware. However, many learners and practitioners are currently using commercially available VR with the goal of enhancing real-world performance, though there is very little evidence to support the notion of positive transfer for these systems. Therefore, the purpose of this experiment was to investigate how motor skill practice using a commercially available VR system improved real-world performance and how that compared to physical practice. Participants (n = 68) were randomly selected into one of two groups: virtual reality (VR) practice (n = 33) or real-world (RW) practice (n = 35). The experiment took place over two consecutive days with a pretest, posttest, and practice phase occurring on both days. The pre- and post-testing phases were identical for both groups and consisted of putting a golf ball 10 times on a carpeted surface towards the center of a target. The practice phases consisted of 60 total putts per day in the respective environment (VR or RW). Participants continuously alternated golf putting holes from three different distances until they accrued 60 total putts. Participants in the RW group performed golf putts to three targets. Participants in the VR group also performed golf putts on three different miniature golf putting holes, using the commercially available Oculus Rift and the Cloudlands VR Minigolf game. The VR putting targets were designed to replicate the putting holes in the physical environment. Separate 2 (condition) x 4 (test phase) repeated measures ANOVAs were used to assess accuracy and club head kinematics. The results revealed a significant main effect for test phase, but not for condition. Post hoc analyses revealed both groups significantly improved their putting accuracy and club head kinematics at similar rates. The results from this study indicate that the transfer of learning that occurred from the commercially available VR practice was equally effective when compared to RW practice.

neuroscience↗

bioRxiv2021

Domain adaptive neural networks improvecross-species prediction of transcription factor binding

The intrinsic DNA sequence preferences and cell-type specific cooperative partners of transcription factors (TFs) are typically highly conserved. Hence, despite the rapid evolutionary turnover of individual TF binding sites, predictive sequence models of cell-type specific genomic occupancy of a TF in one species should generalize to closely matched cell types in a related species. To assess the viability of cross-species TF binding prediction, we train neural networks to discriminate ChIP-seq peak locations from genomic background and evaluate their performance within and across species. Cross-species predictive performance is consistently worse than within-species performance, which we show is caused in part by species-specific repeats. To account for this domain shift, we use an augmented network architecture to automatically discourage learning of training species-specific sequence features. This domain adaptation approach corrects for prediction errors on species-specific repeats and improves overall cross-species model performance. Our results demonstrate that cross-species TF binding prediction is feasible when models account for domain shifts driven by species-specific repeats.

bioinformatics↗

bioRxiv2020

From Farm to Pharmacy: Strawberry-Enabled Oral Delivery of Protein Drugs

Although oral drug delivery is preferred by patients, it is not possible for proteins because the gastrointestinal tract is not sufficiently permeable. To enable the non-toxic oral uptake of protein drugs, we investigated plant-based foods as intestinal permeation enhancers, hypothesizing that compounds found in food would be well-tolerated by the gastrointestinal tract. Following a screen of over 100 fruits, vegetables, herbs, and fungi, we identified strawberry as a potent enhancer of macromolecular permeability in vitro and in mice. Natural product chemistry techniques identified pelargonidin, an anthocyanidin, as the active compound. In mice, insulin was orally administered with pelargonidin to induce sustained pharmacodynamic effects with doses as low as 1 U/kg and bioactivity of over 100% relative to the current gold standard of subcutaneous injection. Pelargonidin-induced permeability was reversible within two hours of treatment, and one month of daily dosing did not adversely affect mice as determined by weight tracking, serum concentrations of inflammatory markers, and tight junction gene expression. Results underscore the utility of plant-based foods in biomedical applications and demonstrate pelargonidin as an especially potent enhancer for the oral delivery of biologics.

bioengineering↗