Biology subjects

Plajzer-Frick, I.

Publications and source records attributed to Plajzer-Frick, I..

9 recordsLinked to original sources

bioRxiv2024

Massively parallel reporter assays and mouse transgenic assays provide complementary information about neuronal enhancer activity

Genetic studies find hundreds of thousands of noncoding variants associated with psychiatric disorders. Massively parallel reporter assays (MPRAs) and in vivo transgenic mouse assays can be used to assay the impact of these variants. However, the relevance of MPRAs to in vivo function is unknown and transgenic assays suffer from low throughput. Here, we studied the utility of combining the two assays to study the impact of non-coding variants. We carried out an MPRA on over 50,000 sequences derived from enhancers validated in transgenic mouse assays and from multiple fetal neuronal ATAC-seq datasets. We also tested over 20,000 variants, including synthetic mutations in highly active neuronal enhancers and 177 common variants associated with psychiatric disorders. Variants with a high impact on MPRA activity were further tested in mice. We found a strong and specific correlation between MPRA and mouse neuronal enhancer activity including changes in neuronal enhancer activity in mouse embryos for variants with strong MPRA effects. Mouse assays also revealed pleiotropic variant effects that could not be observed in MPRA. Our work provides a large catalog of functional neuronal enhancers and variant effects and highlights the effectiveness of combining MPRAs and mouse transgenic assays.

genomics↗

bioRxiv2023

Cell Type- and Tissue-specific Enhancers in Craniofacial Development

The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development1-3. However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo. To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=138 HEIGHT=200 SRC="FIGDIR/small/546603v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@7595a9org.highwire.dtl.DTLVardef@1b48b81org.highwire.dtl.DTLVardef@32fe86org.highwire.dtl.DTLVardef@1e41fed_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2022

Uncovering Hidden Enhancers Through Unbiased In Vivo Testing

Transcriptional enhancers are a predominant class of noncoding regulatory elements that activate cell type-specific gene expression. Tissue-specific enhancer-associated chromatin signatures have proven useful to identify candidate enhancer elements at a genome-wide scale, but their sensitivity for the comprehensive detection of all enhancers active in a given tissue in vivo remains unclear. Here we show that a substantial proportion of in vivo enhancers are hidden from discovery by conventional chromatin profiling methods. In an initial comparison of over 1,200 in vivo validated tissue-specific enhancers with tissue-matched mouse developmental epigenome data, 14% (n=286) of active enhancers did not show canonical enhancer-associated chromatin signatures in the tissue in which they are active. To assess the prevalence of enhancers not detectable by conventional chromatin profiling approaches in more detail, we used a high throughput transgenic enhancer reporter assay to systematically screen over 1.3 Mb of mouse genomic sequence at two critical developmental loci, assessing a total of 281 consecutive 5kb regions for in vivo enhancer activity in mouse embryos. We observed reproducible enhancer-reporter activity in 88 tissue-specific elements, 26% of which did not show canonical enhancer-associated chromatin signatures in the corresponding tissues. Overall, we find these hidden enhancers are indistinguishable from marked enhancers based on levels of evolutionary conservation, enrichment of transcription factor families, and genomic positioning relative to putative target genes. In combination, our retrospective and prospective studies assessed only 0.1% of the mouse genome and identified 309 tissue-specific enhancers that are hidden from current chromatin-based enhancer identification approaches. Our findings suggest the existence of tens of thousands of active enhancers throughout the genome that remain undetected by current chromatin profiling approaches and are an unappreciated source of additional genome function of import in interpreting growing whole human genome sequencing data.

genomics↗

bioRxiv2021

Topologically Associating Domain Boundaries are Commonly Required for Normal Genome Function

Topologically associating domain (TAD) boundaries are thought to partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotype1-3, but the overall extent to which this occurs remains unknown. Here we show that TAD boundary deletions commonly disrupt normal genome function in vivo. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80kb in size) from the genome in mice. All deletions examined resulted in at least one detectable molecular or organismal phenotype, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. For 5 of 8 (62%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and suggest that noncoding deletions affecting TAD boundaries should be carefully considered for potential pathogenicity in clinical genetics screening.

genomics↗

bioRxiv2020

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

Gene deserts are defined as genomic regions devoid of protein coding genes and spanning more than 500 kilobases, collectively encompassing about 25% of the human genome. Approximately 30% of all gene deserts are enriched for conserved elements with cis-regulatory signatures. These are located predominantly near developmental transcription factors (TFs) but despite predicted critical functions, the transcriptional contributions and biological necessity of most gene deserts remain elusive. Here, we explore the cis-regulatory impact of a gene desert flanking the Shox2 gene, a TF indispensable for proximal limb, craniofacial and cardiac pacemaker development. Using a functional genomics approach in mouse embryos we identify the gene desert as a hub for numerous Shox2-overlapping enhancers arranged in a globular chromatin domain with tissue-specific features. In accordance, using endogenous CRISPR deletion, we demonstrate that the gene desert interval is essential for Shox2 transcriptional control in developing limbs, craniofacial compartments, and the heart. Phenotypically, gene desert ablation leads to pacemaker-related embryonic lethality due to Shox2 depletion in the cardiac sinus venosus. We show that this role is partially mediated through a distal gene desert enhancer, providing evidence for intra-gene desert regulatory robustness. Finally, we uncover a multi-layered functional role of the gene desert by revealing an additional requirement for stylopod morphogenesis, mediated through an array of proximal limb enhancers (PLEs). In summary, our study establishes the Shox2 gene desert as a fundamental genomic unit that controls pleiotropic gene expression through modular arrangement and coordinated dynamics of tissue-specific enhancers.

developmental biology↗

bioRxiv2020

The changing mouse embryo transcriptome at whole tissue and single-cell resolution

In mammalian embryogenesis differential gene expression gradually builds the identity and complexity of each tissue and organ system. We systematically quantified mouse polyA-RNA from embryo day E10.5 to birth, sampling 17 whole tissues, enhanced with single-cell measurements for the developing limb. The resulting developmental transcriptome is globally structured by dynamic cytodifferentiation, body-axis and cell-proliferation gene sets, characterized by their promoters transcription factor (TF) motif codes. We decomposed the tissue-level transcriptome using scRNA-seq and found that neurogenesis and haematopoiesis dominate at both the gene and cellular levels, jointly accounting for 1/3 of differential gene expression and over 40% of identified cell types. Integrating promoter sequence motifs with companion ENCODE epigenomic profiles identified a promoter de-repression mechanism unique to neuronal expression clusters and attributable to known and novel repressors. Focusing on the developing limb, scRNA-seq identified 25 known and candidate novel cell types, including progenitor and differentiating states with computationally inferred lineage relationships. We extracted cell type TF networks and complementary sets of candidate enhancer elements by de-convolving whole-tissue IDEAS epigenome chromatin state models. These ENCODE reference data, computed network components and IDEAS chromatin segmentations, are companion resources to the matching epigenomic developmental matrix, available for researchers to further mine and integrate.

developmental biology↗

bioRxiv2019

Genome-Wide Fetalization of Enhancer Architecture in Heart Disease

Heart disease is associated with re-expression of key transcription factors normally active only during prenatal development of the heart. However, the impact of this reactivation on the genome-wide regulatory landscape in heart disease has remained obscure. Here we show that pervasive epigenomic changes occur in heart disease, with thousands of regulatory sequences reacquiring fetal-like chromatin signatures. We used RNA-seq and ChIP-seq targeting a histone modification associated with active transcriptional enhancers to generate genome-wide enhancer maps from left ventricle tissue from 18 healthy controls and 18 individuals with idiopathic dilated cardiomyopathy (DCM). Healthy individuals had a highly reproducible epigenomic landscape, consisting of more than 31,000 predicted heart enhancers. In contrast, we observed reproducible disease-associated gains or losses of activity at more than 7,500 predicted heart enhancers. Next, we profiled human fetal heart tissue by ChIP-seq and RNA-seq. Comparison with adult tissues revealed that the heart disease epigenome and transcrip-tome both shift toward a fetal-like state, with 3,400 individual enhancers sharing fetal regulatory properties. Our results demonstrate widespread epigenomic changes in DCM, and we provide a comprehensive data resource (http://heart.lbl.gov) for the mechanistic exploration of heart disease etiology.

genomics↗

bioRxiv2018

A cross-organism framework for supervised enhancer prediction with epigenetic pattern recognition and targeted validation

Enhancers are important noncoding elements, but they have been traditionally hard to characterize experimentally. Only a few mammalian enhancers have been validated, making it difficult to train statistical models for their identification properly. Instead, postulated patterns of genomic features have been used heuristically for identification. The development of massively parallel assays allows for the characterization of large numbers of enhancers for the first time. Here, we developed a framework that uses Drosophila STARR-seq data to create shape-matching filters based on enhancer-associated meta-profiles of epigenetic features. We combined these features with supervised machine learning algorithms (e.g., support vector machines) to predict enhancers. We demonstrated that our model could be applied to predict enhancers in mammalian species (i.e., mouse and human). We comprehensively validated the predictions using a combination of in vivo and in vitro approaches, involving transgenic assays in mouse and transduction-based reporter assays in human cell lines. Overall, the validations involved 153 enhancers in 6 mouse tissues and 4 human cell lines. The results confirmed that our model can accurately predict enhancers in different species without re-parameterization. Finally, we examined the transcription-factor binding patterns at predicted enhancers and promoters in human cell lines. We demonstrated that these patterns enable the construction of a secondary model effectively discriminating between enhancers and promoters.

bioinformatics↗

bioRxiv2017

Systematic mapping of chromatin state landscapes during mouse development

Embryogenesis requires epigenetic information that allows each cell to respond appropriately to developmental cues. Histone modifications are core components of a cells epigenome, giving rise to chromatin states that modulate genome function. Here, we systematically profile histone modifications in a diverse panel of mouse tissues at 8 developmental stages from 10.5 days post conception until birth, performing a total of 1,128 ChIP-seq assays across 72 distinct tissue-stages. We combine these histone modification profiles into a unified set of chromatin state annotations, and track their activity across developmental time and space. Through integrative analysis we identify dynamic enhancers, reveal key transcriptional regulators, and characterize the role of chromatin-based repression in developmental gene regulation. We also leverage these data to link enhancers to putative target genes, revealing connections between coding and non-coding sequence variation in disease etiology. Our study provides a compendium of resources for biomedical researchers, and achieves the most comprehensive view of embryonic chromatin states to date.

genomics↗