Biology subjects

Gerhardinger, C.

Publications and source records attributed to Gerhardinger, C..

12 recordsLinked to original sources

bioRxiv2023

Transcriptional dynamics orchestrating the development and integration of neurons born in the adult hippocampus

The adult hippocampus generates new granule cells (aGCs) that exhibit distinct functional capabilities along development, conveying a unique form of plasticity to the preexisting circuits. While early differentiation of adult radial glia-like neural stem cells (RGL) has been studied extensively, the molecular mechanisms guiding the maturation of postmitotic neurons remain unknown. Here, we used a precise birthdating strategy to follow newborn aGCs along differentiation using single-nuclei RNA sequencing (snRNA-seq). Transcriptional profiling revealed a continuous trajectory from RGLs to mature aGCs, with multiple sequential immature stages bearing increasing levels of effector genes supporting growth, excitability and synaptogenesis. Remarkably, four discrete cellular states were defined by the expression of distinct sets of transcription factors (TFs): quiescent neural stem cells, proliferative progenitors, postmitotic immature aGCs, and mature aGCs. The transition from immature to mature aCGs involved a transcriptional switch that shutdown molecular cascades promoting cell growth, such as the SoxC family of TFs, to activate programs controlling neuronal homeostasis. Indeed, aGCs overexpressing Sox4 or Sox11 remained stalled at the immature state. Our results unveil precise molecular mechanisms driving adult neural stem cells through the pathway of neuronal differentiation.

neuroscience↗

bioRxiv2022

Single-cell multiomics atlas of organoid development uncovers longitudinal molecular programs of cellular diversification of the human cerebral cortex

Realizing the full utility of brain organoids as experimental systems to study human cortical development requires understanding whether organoids replicate the cellular and molecular events of this complex process precisely, reproducibly, and with fidelity to the embryo. Here we present a comprehensive single-cell transcriptomic, epigenetic, and spatial atlas of human cortical organoid development, comprising over 610,000 cells, spanning initial generation of neural progenitors through production of differentiated neuronal and glial subtypes. We define the lineage relationships and longitudinal molecular trajectories of cortical cell types during development in organoids, and show that developmental processes of cellular diversification in organoids correlate closely to endogenous ones, irrespective of metabolic state. Using this data, we identify genes with predicted human-specific roles in lineage establishment, and discover a developmental origin for the transcriptional diversity of human callosal projection neurons, a population that has undergone dramatic expansion and diversification during human evolution. Our work provides a comprehensive, single-cell molecular map of human corticogenesis in vitro, identifying developmental trajectories and molecular mechanisms associated with human cellular diversification.

neuroscience↗

bioRxiv2021

Human-specific enrichment of schizophrenia risk-genes in callosal neurons of the developing neocortex

Human genetic studies have provided a wealth of information on genetic risk factors associated with neuropsychiatric diseases. However, whether different brain cell types are differentially affected in disease states and when in their development and maturation alterations occur is still poorly understood. Here we generated a longitudinal transcriptional map of excitatory projection neuron (PN) and inhibitory interneuron (IN) subtypes of the cerebral cortex, across a timeline of mouse embryonic and postnatal development, as well as fetal human cortex and human cortical organoids. We found that three types of gene signatures uniquely defined each cortical neuronal subtype: dynamic (developmental), adult (terminal), and constitutive (stable), with individual neuronal subtypes varying in the degree of similarity of their signatures between species. In particular, human callosal projection neurons (CPN) displayed the greatest species divergence, with molecular signatures highly enriched for non-coding, human-specific RNAs. Evaluating the association of neuronal class-specific signatures with neuropsychiatric disease risk genes using linkage disequilibrium score regression showed that schizophrenia risk genes were enriched in CPN identity signatures from human but not mouse cortex. Human cortical organoids confirmed the association with excitatory projection neurons. The data indicate that risk gene enrichment is both species- and cell type-specific. Our study reveals molecular determinants of cortical neuron diversification and identifies human callosal projection neurons as the most species-divergent population and a potentially vulnerable neuronal class in schizophrenia.

developmental biology↗

bioRxiv2021

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

molecular biology↗

bioRxiv2020

Resolving mechanisms of immune-mediated disease in primary CD4 T cells

Deriving mechanisms of immune-mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by linkage disequilibrium. To determine whether causal variants could be identified via their functional effects, we adapted a massively-parallel reporter assay for use in primary CD4 T-cells, key effectors of many immune-mediated diseases. Using the results to guide further study, we provide a generalisable framework for resolving disease mechanisms from non-coding associations - illustrated by a locus linked to 6 immune-mediated diseases, where the lead functional variant causally disrupts a super-enhancer within an NF-{kappa}B-driven regulatory circuit, triggering unrestrained T-cell activation.

genetics↗

bioRxiv2019

Analysis of regulatory element evolution between human and mouse reveals a lack of cis-trans compensation

Gene expression differences between species are driven by both cis and trans effects. Whereas cis effects are caused by genetic variants in close proximity to the target gene, trans effects are due to distal genetic variants that affect diffusible elements such as transcription factors. Previous studies have mostly assessed the impact of cis and trans effects at the gene level. However, how cis and trans effects differentially impact regulatory elements such as enhancers and promoters remains poorly understood. Here, we used massively parallel reporter assays to directly measure cis and trans effects between human and mouse embryonic stem cells at thousands of individual regulatory elements. Our approach revealed that cis effects are widespread across regulatory elements, and the strongest cis effects are associated with the disruption of motifs recognized by strong transcriptional activators. Conversely, we found that trans effects are rare but stronger in enhancers than promoters, and can be attributed to a subset of transcription factors that are differentially expressed between human and mouse. While previous studies have found extensive co-occurrence of cis and trans effects in opposite directions that stabilize gene expression throughout evolution, we find that cis-trans compensation is uncommon within individual regulatory elements. Thus, our results are consistent with a model wherein compensatory cis-trans effects at the gene level are explained by cis and trans effects that separately impact several regulatory elements rather than cis-trans effects that occur simultaneously within a single regulatory element. Together, these results indicate that studying the evolution of individual regulatory elements is pivotal to understand the tempo and mode of gene expression evolution.

genomics↗

bioRxiv2019

The Firre locus produces a trans-acting RNA molecule that functions in hematopoiesis

RNA has been classically known to play central roles in biology, including maintaining telomeres1, protein synthesis2, and in sex chromosome compensation in certain species3,4. At the center of these important biological systems are noncoding RNAs. While thousands of long noncoding RNAs (lncRNAs) have been identified in mammalian genomes5-8, attributing RNA-based roles to lncRNA loci requires an assessment of whether the observed effect could be due to DNA regulatory elements, the act of transcription, or the lncRNA transcript. Here, we use the syntenically conserved lncRNA locus, Functional intergenic repeating RNA element (Firre), that is located on the X chromosome as a model to discriminate between DNA- and RNA-mediated effects in vivo. To this end, we generated genetically defined loss-of-function, gain-of-function, and rescue mouse models for Firre and provide genetic evidence that the Firre locus produces a trans-acting RNA. We report that: (i) Firre mutant mice have cell-specific defects during hematopoiesis and changes in gene expression that can be rescued by induction of Firre RNA from a transgene in the Firre knockout background, (ii) mice overexpressing Firre from a transgene exhibit increased levels of pro-inflammatory cytokines and impaired survival upon exposure to lipopolysaccharide, and (iii) deletion of the Firre locus did not result in changes in local gene expression on the X chromosome in 9 different biological contexts, suggesting that Firre does not function by cis-acting RNA or DNA elements. Together, our results provide genetic evidence that the Firre locus produces a trans-acting lncRNA that has physiological roles in hematopoiesis and immune function.

immunology↗

bioRxiv2019

In vivo Firre and Dxz4 deletion elucidates roles for autosomal gene regulation

Recent evidence has determined that the conserved X chromosome \"mega-structures\" controlled by the Firre and Dxz4 alleles are not required for X chromosome inactivation (XCI) in cell lines. Here we determined the in vivo contribution of these alleles by generating mice carrying a single or double deletion of Firre and Dxz4. We found that these mutants are viable, fertile and show no defect in random or imprinted XCI. However, the lack of these elements results in many dysregulated genes on autosomes in an organ-specific manner. By comparing the dysregulated genes between the single and double deletion, we identified superloop, megadomain, and Firre locus dependent gene sets. The largest transcriptional effect was observed in all strains lacking the Firre locus, indicating that this locus is the main driver for these autosomal expression signatures. Collectively, these findings suggest that these X-linked loci are involved in autosomal gene regulation rather than XCI biology.

genetics↗

bioRxiv2019

The Tug1 Locus is Essential for Male Fertility

BackgroundSeveral long noncoding RNAs (lncRNAs) have been shown to function as central components of molecular machines that play fundamental roles in biology. While the number of annotated lncRNAs in mammalian genomes has greatly expanded, their functions remain largely uncharacterized. This is compounded by the fact that identifying lncRNA loci that have robust and reproducible phenotypes when mutated has been a challenge. ResultsWe previously generated a cohort of 20 lncRNA loci knockout mice. Here, we extend our initial study and provide a more detailed analysis of the highly conserved lncRNA locus, Taurine Upregulated Gene 1 (Tug1). We report that Tug1 knockout male mice are sterile with complete penetrance due to a low sperm count and abnormal sperm morphology. Having identified a lncRNA loci with a robust phenotype, we wanted to determine which, if any, potential elements contained in the Tug1 genomic region (DNA, RNA, protein, or the act of transcription) have activity. Using engineered mouse models and cell-based assays, we provide evidence that the Tug1 locus harbors three distinct regulatory activities - two noncoding and one coding: (i) a cis DNA repressor that regulates many neighboring genes, (ii) a lncRNA that can regulate genes by a trans-based function, and finally (iii) Tug1 encodes an evolutionary conserved peptide that when overexpressed impacts mitochondrial membrane potential. ConclusionsOur results reveal an essential role for the Tug1 locus in male fertility and uncover three distinct regulatory activities in the Tug1 locus, thus highlighting the complexity present at lncRNA loci.

genomics↗

bioRxiv2018

High-throughput functional analysis of lncRNA core promoters elucidates rules governing tissue-specificity

Transcription initiates at both coding and non-coding genomic elements, including mRNA and long non-coding RNA (lncRNA) core promoters and enhancer RNAs (eRNAs). However, each class has different expression profiles with lncRNAs and eRNAs being the most tissue-specific. How these complex differences in expression profiles and tissue-specificities are encoded in a single DNA sequence, however, remains unresolved. Here, we address this question using computational approaches and massively parallel reporter assays (MPRA) surveying hundreds of promoters and enhancers. We find that both divergent lncRNA and mRNA core promoters have higher capacities to drive transcription than non-divergent lncRNA and mRNA core promoters, respectively. Conversely, lincRNAs and eRNAs have lower capacities to drive transcription and are more tissue-specific than divergent genes. This higher tissue-specificity is strongly associated with having less complex TF motif profiles at the core promoter. We experimentally validated these findings by testing both engineered single-nucleotide deletions and human single-nucleotide polymorphisms (SNPs) in MPRA. In both cases, we observe that single nucleotides associated with many motifs are important drivers of promoter activity. Thus, we suggest that high TF motif density serves as a robust mechanism to increase promoter activity at the expense of tissue-specificity. Moreover, we find that 22% of common SNPs in core promoter regions have significant regulatory effects. Collectively, our findings show that high TF motif density provides redundancy and increases promoter activity at the expense of tissue specificity, suggesting that specificity of expression may be regulated by simplicity of motif usage.

genomics↗

bioRxiv2017

High-throughput identification of RNA nuclear enrichment sequences

One of the biggest surprises since the sequencing of the human genome has been the discovery of thousands of long noncoding RNAs (lncRNAs)1-6. Although lncRNAs and mRNAs are similar in many ways, they differ with lncRNAs being more nuclear-enriched and in several cases exclusively nuclear7,8. Yet, the RNA-based sequences that determine nuclear localization remain poorly understood9-11. Towards the goal of systematically dissecting the lncRNA sequences that impart nuclear localization, we developed a massively parallel reporter assay (MPRA). Unlike previous MPRAs12-15 that determine motifs important for transcriptional regulation, we have modified this approach to identify sequences sufficient for RNA nuclear enrichment for 38 human lncRNAs. Using this approach, we identified 109 unique, conserved nuclear enrichment regions, originating from 29 distinct lncRNAs. We also discovered two shorter motifs within our nuclear enrichment regions. We further validated the sufficiency of several regions to impart nuclear localization by single molecule RNA fluorescence in situ hybridization (smRNA-FISH). Taken together, these results provide a first systematic insight into the sequence elements responsible for the nuclear enrichment of lncRNA molecules.

genomics↗

bioRxiv2016

Chromatin environment, transcriptional regulation, and splicing distinguish lincRNAs and mRNAs

While long intergenic noncoding RNAs (lincRNAs) and mRNAs share similar biogenesis pathways, these transcript classes differ in many regards. LincRNAs are less evolutionarily conserved, less abundant, and more tissue-specific, suggesting that their pre- and post-transcriptional regulation is different from that of mRNAs. Here, we perform an in-depth characterization of the features that contribute to lincRNA regulation in multiple human cell lines. We find that lincRNA promoters are depleted of transcription factor (TF) binding sites, yet enriched for some specific factors such as GATA and FOS relative to mRNA promoters. Surprisingly, we find that H3K9me3--a histone modification typically associated with transcriptional repression--is more enriched at the promoters of active lincRNA loci than at those of active mRNAs. Moreover, H3K9me3-marked lincRNA genes are more tissue-specific. The most discriminant differences between lincRNAs and mRNAs involve splicing. LincRNAs are less efficiently spliced, which cannot be explained by differences in U1 binding or the density of exonic splicing enhancers, but may be partially attributed to lower U2AF65 binding and weaker splicing-related motifs. Conversely, the stability of lincRNAs and mRNAs is similar, differing only with regard to the location of stabilizing protein binding sites. Finally, we find that certain transcriptional properties are correlated with higher evolutionary conservation in both DNA and RNA motifs, and are enriched in lincRNAs that have been functionally characterized.

genomics↗