Biology subjects

Yost, H. J.

Publications and source records attributed to Yost, H. J..

8 recordsLinked to original sources

bioRxiv2024

Nuclear Histone H3 Post-translational Modification Profiling in Whole Cells using Spectral Flow Cytometry

Histone modifications play essential roles in regulating chromatin accessibility and downstream transcription, serving as critical determinants of cell identity and function. However, the diversity of histone post-translational modifications (PTMs) and their tendency to be studied in isolation limits our understanding of their coordinated roles in shaping cellular states. Conventional flow cytometry methods for histone PTM assessment suffer from low multiplexing capacity and typically require nuclear isolation, resulting in significant sample loss and an incomplete picture of the overall cell state. Here, we present EpiFlow, a spectral flow cytometry protocol designed for multiparametric analysis of histone PTMs within whole cells. By utilizing a 96-well plate format and preserving the cell entirely, EpiFlow improves throughput and efficiency while retaining sample integrity. This method resolves subtle variations in histone PTMs within neural progenitor cells, capturing distinct chromatin states across the cell cycle and correlating them with several markers. Furthermore, we provide an open-access GitHub repository containing detailed protocols and analysis workflows, ensuring reproducibility and accessibility of this approach. EpiFlow offers a robust framework for exploring chromatin dynamics, with broad implications for advancing fundamental research and therapeutic research.

developmental biology↗

bioRxiv2024

Automated Identification of Germline de novo Mutations in Family Trios: A Consensus-Based Informatic Approach

Accurate identification of germline de novo variants (DNVs) remains a challenging problem despite rapid advances in sequencing technologies as well as methods for the analysis of the data they generate, with putative solutions often involving ad hoc filters and visual inspection of identified variants. Here, we present a purely informatic method for the identification of DNVs by analyzing short-read genome sequencing data from proband-parent trios. Our method evaluates variant calls generated by three genome sequence analysis pipelines utilizing different algorithms--GATK HaplotypeCaller, DeepTrio and Velsera GRAF--exploring the assumption that a requirement of consensus can serve as an effective filter for high- quality DNVs. We assessed the efficacy of our method by testing DNVs identified using a previously established, highly accurate classification procedure that partially relied on manual inspection and used Sanger sequencing to validate a DNV subset comprising less confident calls. The results show that our method is highly precise and that applying a force-calling procedure to putative variants further removes false-positive calls, increasing precision of the workflow to 99.6%. Our method also identified novel DNVs, 87% of which were validated, indicating it offers a higher recall rate without compromising accuracy. We have implemented this method as an automated bioinformatics workflow suitable for large- scale analyses without need for manual intervention.

bioinformatics↗

bioRxiv2020

SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries

CRISPR-Cas9 sgRNA libraries have transformed functional genetic screening and have enabled innovative CRISPR-based methods, such as the visualization of chromatin dynamics in living cells. These libraries have the potential to be applied to a vast number of biological systems and aid in the development of new technologies, but their synthesis is hindered by the cost, time requirements, and technical difficulty of current sgRNA library generation methods. Here, we describe SLALOM--a rapid enzymatic method for generating robust, variant-matched sgRNA libraries from any source of DNA in under 3 hours. This method utilizes a custom sgRNA scaffold sequence and a novel method for detaching oligonucleotides from solid supports using a strand displacing polymerase. Using this method, we have constructed libraries targeting the E. coli genome and the transcriptome of developing zebrafish hearts, demonstrating its potential to expand the reach of CRISPR technology and facilitate methods requiring custom sgRNA libraries.

molecular biology↗

bioRxiv2020

Syndecan 2 regulates hematopoietic lineages and infection resolution in zebrafish

Syndecan 2 (Sdc2) is a transmembrane cell-surface heparan sulfate proteoglycan (HSPG) that has been implicated in the regulation of cell-cell signaling pathways and cell-matrix interactions. Surprisingly, homozygous recessive maternal zygotic (MZ) sdc2 null mutants in zebrafish appear to have normal development, normal morphology and are viable and fertile in adulthood. Whole transcriptome RNA sequencing, FACS analyses, and imaging of transgenic reporter lines that distinguish specific hematopoietic lineages revealed that sdc2 mutants have defects in the specification and proportions of red blood cells and neutrophils that initiate during embryonic hematopoiesis and likely persist through adulthood. During bacterial infections, MZsdc2 mutants have markedly reduced neutrophil recruitment and significantly higher death rates. Hematopoietic stem/progenitor cell (HSPC) numbers are also significantly reduced in MZsdc2 mutants. In zebrafish, cells that bud off of the ventral region of somites are thought to give rise to the reticular stromal cells of the caudal hematopoietic tissue (CHT) stem cell niche. In MZsdc2 mutants, these budding cells have abberant blebbing morphology associated with widespread apoptosis during induction of HSPCs and with changes in the vascularization and stromal cell structure of the CHT stem cell niche. This suggests that loss of sdc2 disrupts the earliest events of definitive hematopoiesis. Our findings of hematopoietic defects, nascent immune system alterations and inability to resolve infection in sdc2 mutants sets the stage for examining the roles of HSPG genes in a wide range of hematopoietic and immune defects in humans. Key PointsO_LISyndecan 2 regulates the formation of Hematopoietic Stem/Progenitor Cells, differentiation into hematopoietic populations and the CHT architecture C_LIO_LISyndecan 2 mutants are significantly more susceptible to bacterial infection C_LI

developmental biology↗

bioRxiv2018

Inhibition of Notch signaling rescues cardiovascular development in Kabuki Syndrome

Kabuki Syndrome patients have a spectrum of congenital disorders, including congenital heart defects, the primary determinant of mortality. Seventy percent of Kabuki Syndrome patients have mutations in the histone methyl-transferase KMT2D. However, the underlying mechanisms that drive these congenital disorders are unknown. Here, we generated and characterized a zebrafish kmt2d null mutant that recapitulates the cardinal phenotypic features of Kabuki Syndrome, including microcephaly, palate defects, abnormal ear development and cardiac defects. The cardiovascular defects consist of abnormal aortic arches and hypoplastic ventricle, driven by previously unknown aberrant endocardial and endothelial vasculogenesis. We identify a regulatory link between the Notch pathway and Kmt2d during vasculogenesis and show that pharmacological inhibition of Notch signaling rescues the cardiovascular phenotype in zebrafish Kabuki Syndrome. Taken together these findings demonstrate that Kmt2d regulates vasculogenesis, provide evidence for interactions between Kmt2d and Notch signaling in Kabuki Syndrome, and suggest future directions for clinical research.

developmental biology↗

bioRxiv2018

Deriving Cardiomyocytes from Human Amniocytes

Many forms of congenital heart disease (CHD) have high morbidity-mortality rates and require challenging surgeries. Human amniocytes have important stem cell characteristics and could potentially provide patient-specific tissue for repairs of some types of CHDs. We report that amniocytes express features of poised cardiomyocytes. However, a variety of direct reprogramming approaches failed to convert their fetal and transcriptionally repressed state into bona fide cardiomyocytes. Induced-pluripotent stem cell (iPSC) reprogramming removes repression and converts amniocytes to a baseline pluripotent state. Based on molecular and electrophysiological signatures, iPSC reprogrammed amniocytes can be induced to differentiate into functionally immature, predominantly ventricular cardiomyocytes and a heterogeneous mixture of vascular and unspecified epithelial cells. Developmental time course analyses and pattern clustering of amniocyte-derived cardiomyocytes identifies numerous temporal co-regulators of cardiac induction and maturation as well as distinct sarcomeric and ion channel gene signatures. Normal fetal cardiomyocytes are derived by overcoming complex forms of transcriptional repression that suppress direct transdifferentiation of human amniocytes. These results suggest the possibility of using amniocytes as a source of patient-specific ventricular cardiomyocytes for cell therapies.\n\nSUMMARY STATEMENTAmniocytes are a possible source of patient-specific cardiomyocytes for newborns with congenital heart disease. Genome-wide DNA methylation patterns and transcriptional repressors preclude direct differentiation, but pluripotent reprogramming provides cardiomyocytes for dissecting genetic pathways contributing to this disease.

developmental biology↗

bioRxiv2018

Loss of embryonic neural crest cardiomyocytes causes adult hypertrophic cardiomyopathy

Neural crest cells migrate to the embryonic heart and transform into a small number of cardiomyocytes, but their functions in the developing and adult heart are unknown. Here, we map the fates of neural crest derived cardiomyocytes (NC-Cms) and genetically ablate them in embryogenesis in zebrafish. Specific NC-Cm ablation results in aberrant trabeculation patterns and altered Notch signaling, but is not detrimental to the development of the fish or early heart function. Strikingly, embryonic NC-Cm ablation results in adult fish that show severely hypertrabeculated hearts, altered cardiomyocyte size, diminished adult heart capacity and consequently poor physiological response to cardiac stress tests. Thus, we identify a novel developmental mechanism and genetic pathway that predisposes adults to hypertrophic cardiomyopathy and provides the first zebrafish model of adult-onset heart failure.

developmental biology↗

bioRxiv2017

Nkx2.5-dependent alterations of the embryonic heart DNA methylome identify novel cis-regulatory elements in cardiac development

Transcription factor Nkx2.5 is frequently mutated in congenital heart disease, but the mechanisms by which Nkx2.5 regulates heart development are poorly understood. By generating comprehensive DNA methylome maps from zebrafish embryonic hearts in nxk2.5 mutants and siblings, we discovered that Nkx2.5 regulates DNA methylation patterns during cardiac morphogenesis. We identified hundreds of Nkx-dependent heart-specific Differentially Methylated Regions (nhDMRs). A majority of the nhDMRs were hypomethylated in nkx2.5-/- hearts, correlating with changes in the mutant transcriptome, suggesting Nkx2.5 functions largely as a repressor. Distinct Nkx DNA-binding motifs were significantly enriched in subclasses of nhDMRs. Furthermore, nhDMRs were significantly associated with histone H3K4me1 and H3K27ac post-translational modifications, suggesting Nkx2.5 regulates gene expression by differential methylation of cis-regulatory elements. Using transgenics, we validated several nhDMRs with enhancer activities in the heart. We propose a novel role of Nkx2.5 mediated DNA methylation is integral in activating and repressing Nkx2.5 target genes during heart development.

developmental biology↗