Biology subjects

Whalen, S.

Publications and source records attributed to Whalen, S..

14 recordsLinked to original sources

bioRxiv2025

Comparative evaluation of genomic footprinting algorithms for predicting transcription factor binding sites in single-cell data

Transcription factors (TFs) have millions of potential binding sites across the human genome, but only a fraction are bound in a given context. Genomic footprinting aims to identify context-specific binding sites by detecting patterns in open chromatin data. While powerful, these approaches face technical challenges, especially in single-cell applications. We developed a benchmarking framework for cell-type specific footprinting and used it to evaluate the consistency, reproducibility, and equivalency of three leading methods across data quality scenarios and as a function of cell-type similarity. Peak-level read coverage emerged as the strongest predictor of stable footprints. Motivated by limited reproducibility across tools, we built an ensemble model that improved concordance with ChIP-seq. To encourage broader adoption and continued tool development, we provide practical guidelines for robust genomic footprinting in single-cell datasets and a roadmap for extracting deeper insights about how gene regulatory networks vary across cell types in complex tissues.

genomics↗

bioRxiv2024

MMP21 behaves as a fluid flow transported morphogen to impart laterality during development

Heterotaxy (HTX) is frequently caused by deleterious variants in the gene encoding Matrix metallopeptidase 21 (MMP21). However, the underlying pathomechanism has not been ascertained. In this study, we report on a novel HTX-associated MMP21 knockout allele in humans and investigate the peptidases role during laterality development using Xenopus embryos as animal model. The targeted inactivation of mmp21 in f0 mutant Xenopus successfully phenocopied the human HTX condition, yet the cilia-driven leftward fluid flow, which initiates asymmetric gene activity at the left-right organizer (LRO), was unaltered in mmp21 null frogs. Instead, our analysis of downstream events revealed that flow response, the left-sided repression of dand5, could not take place. Remarkably, gain-of-function experiments demonstrated that Mmp21 spreads over LRO cells and triggers flow response. Additionally, Mmp21 functions upstream of Cirop, another metallopeptidase, which we found specifically localized to LRO cilia. Thus, our findings suggest that Mmp21 may be the long-sought morphogen, which is actively transported by the leftward fluid flow to Cirop-laden cilia, in order to specify the left side of the embryo.

developmental biology↗

bioRxiv2024

Deep-learning prediction of gene expression from personal genomes

Models that predict RNA levels from DNA sequences show tremendous promise for decoding tissue-specific gene regulatory mechanisms1-5, revealing the genetic architecture of traits6-10, and interpreting noncoding genetic variation10,11. Existing methods take two different approaches: 1) associating expression with linear combinations of common genetic variants (training across individuals on single genes)12,13, or 2) learning genome-wide sequence-to-expression rules with neural networks (training across loci using a reference genome)11,14,15. Since limitations of both strategies have been highlighted recently16-20, we sought to combine the sequence context provided by deep learning with the information provided by cross-individual training. We utilized fine-tuning to develop Performer, a model with accuracy approaching the cis-heritability of most genes. Performer prioritizes genetic variants across the allele frequency spectrum that disrupt motifs, fall in annotated regulatory elements, and have functional evidence for modulating gene expression. While obstacles remain in personalized expression prediction, our findings establish deep learning as a viable strategy.

genetics↗

bioRxiv2023

Massively parallel characterization of psychiatric disorder-associated and cell-type-specific regulatory elements in the developing human cortex

Nucleotide changes in gene regulatory elements are important determinants of neuronal development and disease. Using massively parallel reporter assays in primary human cells from mid-gestation cortex and cerebral organoids, we interrogated the cis-regulatory activity of 102,767 sequences, including differentially accessible cell-type specific regions in the developing cortex and single-nucleotide variants associated with psychiatric disorders. In primary cells, we identified 46,802 active enhancer sequences and 164 disorder-associated variants that significantly alter enhancer activity. Activity was comparable in organoids and primary cells, suggesting that organoids provide an adequate model for the developing cortex. Using deep learning, we decoded the sequence basis and upstream regulators of enhancer activity. This work establishes a comprehensive catalog of functional gene regulatory elements and variants in human neuronal development. One Sentence SummaryWe identify 46,802 enhancers and 164 psychiatric disorder variants with regulatory effects in the developing cortex and organoids.

genomics↗

bioRxiv2022

Three-dimensional genome re-wiring in loci with Human Accelerated Regions

Human Accelerated Regions (HARs) are conserved genomic loci that evolved at an accelerated rate in the human lineage and may underlie human-specific traits. We generated HARs and chimpanzee accelerated regions with the largest alignment of mammalian genomes to date. To facilitate exploration of accelerated evolution in other lineages, we implemented an open-source Nextflow pipeline that runs on any computing platform. Combining deep-learning with chromatin capture experiments in human and chimpanzee neural progenitor cells, we discovered a significant enrichment of HARs in topologically associating domains (TADs) containing human-specific genomic variants that change three-dimensional (3D) genome organization. Differential gene expression between humans and chimpanzees at these loci in multiple cell types suggests rewiring of regulatory interactions between HARs and neurodevelopmental genes. Thus, comparative genomics together with models of 3D genome folding revealed enhancer hijacking as an explanation for the rapid evolution of HARs. One-Sentence SummaryHuman-specific changes to 3D genome organization may have contributed to rapid evolution of mammalian-conserved loci in the human genome.

evolutionary biology↗

bioRxiv2022

Single Cell Epigenetics Reveal Cell-Cell Communication Networks in Normal and Abnormal Cardiac Morphogenesis

Communication between myriad cell types during organ formation underlies proper morphogenesis1. In cardiac development, reciprocal signaling between mesoderm progenitors and neural crest cells is essential, and its disruption leads to congenital heart malformations, the most common human birth defect. However, mechanistic interrogation of temporal gene networks and cis regulatory elements in this crosstalk is limited2,3. Here, we integrated single cell chromatin accessibility and transcriptomics to establish an unbiased and temporal epigenomic map of the embryonic mouse heart over multiple stages and developed machine learning models to predict enhancers for heart and neural crest. We leveraged these advances to determine the consequences of dysregulated signaling at single cell resolution caused by deletion of TBX1, a transcription factor that causes morphogenetic defects of the cardiac outflow tract in humans and functions non-cell autonomously in cardiac mesodermal progenitors to direct pharyngeal neural crest differentiation4-6. Loss of Tbx1 in mice led to broad closure of chromatin regions enriched in cardiac progenitor transcription factor motifs within a narrow subset of cardiac mesodermal progenitors and correlated with diminished expression of numerous members of the fibroblast growth factor, retinoic acid, Notch and Semaphorin pathways. In affected progenitors, ectopic accessibility and expression of posterior heart field factors in the anterior heart field suggested impaired axial patterning. In response, a subset of cardiac neural crest cells displayed epigenomic and transcriptional defects, indicating a failure of differentiation corresponding to dysregulation of the anterior-posterior gradient of pharyngeal Hox gene expression. This study demonstrates that single-cell genomics and machine learning can generate a mechanistic model for how disruptions in cell communication selectively affect spatiotemporally dynamic regulatory networks in cardiogenesis.

developmental biology↗

bioRxiv2021

Autism risk gene POGZ promotes chromatin accessibility and expression of clustered synaptic genes

De novo mutations in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Here we find that in the developing mouse and human brain POGZ binds predominantly euchromatic loci and these are enriched for human neurodevelopmental disorder genes and transposable elements. We profile chromatin accessibility and gene expression in Pogz-/- mice and find that POGZ promotes chromatin accessibility of candidate regulatory elements (REs) and the expression of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with HP1{gamma} and ADNP, another high-confidence ASD risk gene. In Pogz+/- mice, Adnp expression is reduced. We postulate that reduced POGZ dosage disrupts cortical function through alterations in the POGZ-ADNP balance which modifies neuronal gene expression.

neuroscience↗

bioRxiv2020

An atlas of lamina-associated chromatin across thirteen human cell types reveals cell-type-specific and multiple subtypes of peripheral heterochromatin

Association of chromatin with lamin proteins at the nuclear periphery has emerged as a potential mechanism to coordinate cell type-specific gene expression and maintain cellular identity via gene silencing. Unlike many histone modifications and chromatin-associated proteins, lamin-associated domains (LADs) have yet to be mapped genome-wide in a diverse panel of human cell types, which has limited our understanding of the role peripheral chromatin plays in development and disease. To address this gap, we mapped LAMIN B1 (LB1) across twelve human cell types encompassing pluripotent stem cells, intermediate progenitors, and differentiated cells from all three germ layers. Integrative analyses of this atlas of peripheral chromatin with publicly available genomic data, as well as gene expression and repressive histone maps generated for this study, revealed that in all twelve cellular contexts lamin-associated chromatin is organized into at least two subtypes defined by differences in LB1 occupancy, gene expression, chromatin accessibility, transposable elements, replication timing, and radial positioning. Most genes gain or lose LB1 occupancy consistent with their cell type along developmental trajectories; however, we also identified examples where the enhancer, but not the gene body and promoter, change LAD state. Imaging of fluorescently labeled DNA in single cells validated these transitions and showed intermediate radial positioning of LADs that are gene dense, relatively accessible, and dynamic across development. This atlas represents the largest resource to date for peripheral chromatin organization studies.

developmental biology↗

bioRxiv2019

A Chromatin Accessibility Atlas of the Developing Human Telencephalon

Gene expression differs between cell types and regions within complex tissues such as the developing brain. To discover regulatory elements underlying this specificity, we generated genome-wide maps of chromatin accessibility in eleven anatomically-defined regions of the developing human telencephalon, including upper and deep layers of the prefrontal cortex. We predicted a subset of open chromatin regions (18%) that are most likely to be active enhancers, many of which are dynamic with 26% differing between early and late mid-gestation and 28% present in only one brain region. These region-specific predicted regulatory elements (pREs) are enriched proximal to genes with expression differences across regions and developmental stages and harbor distinct sequence motifs that suggest potential upstream regulators of regional and temporal transcription. We leverage this atlas to identify regulators of genes associated with autism spectrum disorder (ASD) including an enhancer of BCL11A, validated in mouse, and two functional de novo mutations in individuals with ASD in an enhancer of SLC6A1, validated in neuroblastoma cells. These applications demonstrate the utility of this atlas for decoding neurodevelopmental gene regulation in health and disease.\n\nSummaryTo discover regulatory elements driving the specificity of gene expression in different cell types and regions of the developing human brain, we generated an atlas of open chromatin from eleven dissected regions of the mid-gestation human telencephalon, including upper and deep layers of the prefrontal cortex. We identified a subset of open chromatin regions (OCRs), termed predicted regulatory elements (pREs), that are likely to function as developmental brain enhancers. pREs showed regional differences in chromatin accessibility, including many specific to one brain region, and were correlated with gene expression differences across the same regions and gestational ages. pREs allowed us to map neurodevelopmental disorder risk genes to developing telencephalic regions, and we identified three functional de novo noncoding variants in pREs that alter enhancer function. In addition, transgenic experiments in mouse validated enhancer activity for a pRE proximal to BCL11A, showing how this atlas serves as a resource for decoding neurodevelopmental gene regulation in health and disease.

neuroscience↗

bioRxiv2018

Leveraging genetic variants for personalized and allele-specific sgRNA design

The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. However, current tools only design sgRNAs for a reference genome, not taking into account individual variants which may generate, remove, or modify CRISPR/Cas sgRNA sites. This may cause mismatches between designed sgRNAs and the individual genome they are intended to target, leading to decreased experimental performance. Here we describe AlleleAnalyzer, a tool for designing personalized and allele-specific sgRNAs for genome editing. We leverage >2,500 human genomes to identify optimized pairs of sgRNAs that can be used for human therapeutic editing in large populations in the future.

bioinformatics↗

bioRxiv2018

Most regulatory interactions are not in linkage disequilibrium

Linkage disequilibrium (LD) and genomic proximity are commonly used to map non-coding variants to genes, despite increasing examples of causal variants outside the LD block of the gene they regulate. We compared chromatin contacts in 22 cell types to LD across billions of pairs of loci in the human genome and found no concordance, even at genomic distances below 25 kilobases where both tend to be high. Gene expression and ontology data suggest that chromatin contacts identify regulatory variants more reliably than do LD and genomic proximity. We conclude that the genomic architectures of genetic and physical interactions are independent, with important implications for gene regulatory evolution and precision medicine.

genomics↗

bioRxiv2018

Massively parallel dissection of human accelerated regions in human and chimpanzee neural progenitors

Using machine learning (ML), we interrogated the function of all human-chimpanzee variants in 2,645 Human Accelerated Regions (HARs), some of the fastest evolving regions of the human genome. We predicted that 43% of HARs have variants with large opposing effects on chromatin state and 14% on neurodevelopmental enhancer activity. This pattern, consistent with compensatory evolution, was confirmed using massively parallel reporter assays in human and chimpanzee neural progenitor cells. The species-specific enhancer activity of assayed HARs was accurately predicted from the presence and absence of transcription factor footprints in each species. Despite these striking cis effects, activity of a given HAR sequence was nearly identical in human and chimpanzee cells. These findings suggest that HARs did not evolve to compensate for changes in the trans environment but instead altered their ability to bind factors present in both species. Thus, ML prioritized variants with functional effects on human neurodevelopment and revealed an unexpected reason why HARs may have evolved so rapidly.

evolutionary biology↗

bioRxiv2017

The Epstein-Barr virus episome maneuvers between nuclear chromatin compartments during reactivation

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposis sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts localize on the EBV side to OriP, the latent origin of replication. This genetic element, and the EBNA1 protein that binds there, are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts localize on the human side to gene-poor regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of a myriad of interchromosomal associations also suggests a role for this type of long - range association in gene regulation.\n\nIMPORTANCEThe human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double stranded DNA virus, Epstein-Barr virus (EBV), uses similar mechanisms as the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and active compartments during reactivation. This study is advances our knowledge of the EBV life cycle, adding three-dimensional re-localization as a novel component to the molecular events that occur during reactivation. Furthermore, the data adds to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.

microbiology↗

bioRxiv2017

A framework to identify modifier genes in patients with Phelan-McDermid syndrome

Phelan-McDermid syndrome (PMS) is characterized by a variety of clinical symptoms with heterogeneous degrees of severity, including intellectual disability, speech impairment, and autism spectrum disorders (ASD). It results from a deletion of the 22q13 locus that in most cases includes the SHANK3 gene. SHANK3 is considered a major gene for PMS, but the factors modulating the severity of the syndrome remain largely unknown. In this study, we investigated 85 PMS patients with different 22q13 rearrangements (78 deletions, 7 duplications). We first explored their clinical features and provide evidence for frequent corpus callosum abnormalities. We then mapped candidate genomic regions at the 22q13 locus associated with risk of clinical features, and suggest a second locus associated with absence of speech. Finally, in some cases, we identified additional rearrangements at loci associated with ASD, potentially modulating the severity of the syndrome. We also report the first SHANK3 deletion transmitted to five affected daughters by a mother without intellectual disability nor ASD, suggesting that some individuals could compensate for such mutations. In summary, we shed light on the genotype-phenotype relationship of PMS, a step towards the identification of compensatory mechanisms for a better prognosis and possibly treatments of patients with neurodevelopmental disorders.

genetics↗