Biology subjects

Eggan, K.

Publications and source records attributed to Eggan, K..

25 records · Page 2Linked to original sources

bioRxiv2020

The Genetic Architecture of DNA Replication Timing in Human Pluripotent Stem Cells

DNA replication follows a strict spatiotemporal program that intersects with chromatin structure and gene regulation. However, the genetic basis of the mammalian DNA replication timing program is poorly understood1-3. To systematically identify genetic regulators of DNA replication timing, we exploited inter-individual variation in 457 human pluripotent stem cell lines from 349 individuals. We show that the human genomes replication program is broadly encoded in DNA and identify 1,617 cis-acting replication timing quantitative trait loci (rtQTLs4) - base-pair-resolution sequence determinants of replication initiation. rtQTLs function individually, or in combinations of proximal and distal regulators, to affect replication timing. Analysis of rtQTL locations reveals a histone code for replication initiation, composed of bivalent histone H3 trimethylation marks on a background of histone hyperacetylation. The H3 trimethylation marks are individually repressive yet synergize to promote early replication. We further identify novel positive and negative regulators of DNA replication timing, the former comprised of pluripotency-related transcription factors while the latter involve boundary elements. Human replication timing is controlled by a multi-layered mechanism that operates on target DNA sequences, is composed of dozens of effectors working combinatorially, and follows principles analogous to transcription regulation: a histone code, activators and repressors, and a promoter-enhancer logic.

genomics↗

bioRxiv2020

Connexin 43 hemichannels mediate spatial and temporal disease spread in ALS

Connexin 43 (Cx43) gap junctions and hemichannels mediate astrocyte intercellular communication in the central nervous system under normal conditions and may contribute to astrocyte-mediated neurotoxicity in amyotrophic lateral sclerosis (ALS). Here we show that astrocyte-specific knockout of Cx43 in a mouse model of ALS slows disease progression both spatially and temporally, provides motor neuron (MN) protection, and improves survival. In human ALS tissues and biofluids, we observe that higher levels of Cx43 correlate with accelerated disease progression. Using human iPSC-derived astrocytes (hiPSC-A) from both familial and sporadic ALS, we show that Cx43 is upregulated and that Cx43-hemichannels are enriched at the astrocyte membrane. We then demonstrate that the pharmacological blockade of Cx43-hemichannels in ALS astrocytes, during a specific temporal window, provides neuroprotection of hiPSC-MN and reduces ALS astrocyte-mediated neuronal hyperexcitability. Our data identify Cx43 hemichannels as novel conduits of astrocyte-mediated disease progression and a pharmacological target for disease-modifying ALS therapies.

neuroscience↗

bioRxiv2019

Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional genomic studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design new fluorescently labeled piggyBac (PB) vectors to deliver robust and stable expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene and conduct a rigorous assessment of expression levels of the dCas9 effectors, gRNAs and targeted gene. Collectively, these data provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system would facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

molecular biology↗

bioRxiv2018

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

Neural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe human Stem cell-derived NGN2-accelerated Progenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum gene PTEN.

neuroscience↗

bioRxiv2018

Enrichment of rare protein truncating variants in amyotrophic lateral sclerosis patients

To discover novel genetic risk factors underlying amyotrophic lateral sclerosis (ALS), we aggregated exomes from 3,864 cases and 7,839 ancestry matched controls. We observed a significant excess of ultra-rare and rare protein-truncating variants (PTV) among ALS cases, which was primarily concentrated in constrained genes; however, a significant enrichment in PTVs does persist in the remaining exome. Through gene level analyses, known ALS genes, SOD1, NEK1, and FUS, were the most strongly associated with disease status. We also observed suggestive statistical evidence for multiple novel genes including DNAJC7, which is a highly constrained gene and a member of the heat shock protein family (HSP40). HSP40 proteins, along with HSP70 proteins, facilitate protein homeostasis, such as folding of newly synthesized polypeptides, and clearance of degraded proteins. When these processes are not regulated, misfolding and accumulation of degraded proteins can occur leading to aberrant protein aggregation, one of the pathological hallmarks of neurodegeneration.

genomics↗

bioRxiv2018

All-optical electrophysiology for high-throughput functional characterization of human iPSC-derived motor neuron model of ALS

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch clamp electrophysiology. Here we describe an optimized all-optical electrophysiology, \"Optopatch\", pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron model of ALS. In a comparison of neurons with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus, and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results to numerical simulations of simple conductance-based neuronal models and to literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

neuroscience↗

bioRxiv2017

Whole Genome Sequencing in Psychiatric Disorders: the WGSPD Consortium

As technology advances, whole genome sequencing (WGS) is likely to supersede other genotyping technologies. The rate of this change depends on its relative cost and utility. Variants identified uniquely through WGS may reveal novel biological pathways underlying complex disorders and provide high-resolution insight into when, where, and in which cell type these pathways are affected. Alternatively, cheaper and less computationally intensive approaches may yield equivalent insights. Understanding the role of rare variants in the noncoding gene-regulating genome, through pilot WGS projects, will be critical to determine which of these two extremes best represents reality. With large cohorts, well-defined risk loci, and a compelling need to understand the underlying biology, psychiatric disorders have a role to play in this preliminary WGS assessment. The WGSPD consortium will integrate data for 18,000 individuals with psychiatric disorders, beginning with autism spectrum disorder, schizophrenia, bipolar disorder, and major depressive disorder, along with over 150,000 controls.

genomics↗