Biology subjects

Phillips-Cremins, J. E.

Publications and source records attributed to Phillips-Cremins, J. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Integrative spatial profiling of 3D genome organization and gene expression in tissue

The interplay between 3D genome architecture and transcriptional activity is fundamental to gene regulation. However, existing methodologies cannot simultaneously measure these modalities within intact tissues, limiting our understanding of how genome organization coordinates transcriptional programs across diverse cell types and spatial microenvironments. Here, we introduce Spatial Hi-C-RNA, a spatial multi-omics technology that enables the genome-wide co-mapping of chromatin conformation and transcriptome directly from the same tissue section at near- single-cell resolution. Applied to the mouse embryo and adult brains, Spatial Hi-C-RNA generated high-resolution tissue maps revealing that chromatin organization and gene expression jointly define spatially coherent domains aligned with histological structures. While concordant features were observed across modalities, distinct domain patterns also emerged, indicating that chromatin structure and transcription each contribute complementary layers of spatial regulation. We further demonstrated the robustness and biological insight of Spatial Hi-C-RNA in human melanoma, where both modalities delineated tumor boundaries and microenvironmental niches. Notably, chromatin maps revealed fine-scale tumor subdomains undetectable by transcriptomic profiling alone, highlighting the added resolution provided by spatial chromatin architecture. Integrated analysis revealed that multiscale 3D genome features, from A/B compartments and topologically associating domains to chromatin loops, are closely coupled with domain- and cell-type-specific transcriptional programs. In addition, Spatial Hi-C-RNA resolves spatiotemporal dynamics underlying embryonic lineage specification and tumor progression. Together, these capabilities extend the spatial omics landscape beyond transcriptome and epigenome profiling to the level of chromatin organization, establishing an integrative framework for understanding tissue biology across development and disease.

genomics↗

bioRxiv2025

Persistent chromatin loops shape gene expression plasticity upon stimulation and restimulation of human neurons

Persistent molecular correlates of long-term memory storage remain an open question. Here, we stimulate and re-stimulate human neurons and use multi-modal single-nucleus technologies to query DNA methylation, higher-order chromatin folding, and gene expression. We find enduring traces of activity-gained and activity-lost chromatin loops. Genes anchoring persistent activity-gained loops exhibit activity-upregulated expression, whereas persistent activity-lost loops anchor activity-downregulated genes that remain repressed five days post-stimulation. CTCF-bound looped enhancers and promoters are refractory to activity-dynamic DNA methylation. Looped enhancers bound by CTCF can exhibit memory of activity-induced histone modifications and persistent expression of activity-upregulated genes. Upon second stimulation, activity-upregulated genes are robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. Activity-independent gene expression can be downregulated when unlooped but protected from homeostatic downscaling when anchored in persistent loops. Our data reveal long-term genome folding persistence linked to plasticity of activity-dependent gene expression during recall in human neurons. Structured AbstractO_ST_ABSIntroductionC_ST_ABSA long-standing question in neuroscience is how memories of previous experiences are stored in the mammalian brain to facilitate recall over the lifetime of an individual. Classic models of memory posit that both synapse-specific events and cell-wide transcriptional programs are required for encoding, consolidation, and long-term storage of memory (1, 2). Learning involves synapse strengthening, and synapse weakening has been linked to memory loss (3, 4). Nascent transcription and protein synthesis also occur in response to neural activation in vitro and in vivo (5-7). A leading hypothesis, the synaptic tagging and capture model, asserts that specific synapses are biochemically marked during encoding and newly made cell-wide RNA/proteins act specifically on tagged synapses to maintain long-term memory (8). Multiple historic studies have pursued the identification of proteins linked to potentiated synapses (9-13), but few explore the possibility of persistent, activity-dependent patterns of DNA, chromatin, higher-order chromatin folding, or RNA in human models of long-term memory. Over the last decade, the molecular technique of Chromatin-Conformation-Capture has been employed to discover that the mammalian genome folds into thousands loops of (5, 14-28). Loops bring distal non-coding cis regulatory elements into contact with their target genes to influence gene expression (29). They form via the processive motion of the cohesin ring along chromatin until it stalls at the architectural protein CTCF, thus extruding out the intervening DNA (22, 30-32). Loops can markedly reconfigure during lineage commitment in development and in response to genetic perturbations, and their formation is critical for spatiotemporal regulation of expression (22, 30, 33-41). RationaleMultiple recent works suggest that loops connecting activity-dependent enhancers to distal genes and can be induced during neural stimulation in vitro and behavior paradigms in vivo (5, 27, 42, 43). Genetic elimination of the architectural proteins CTCF and cohesin prior to learning substantially impairs memory encoding across multiple behavior tasks in vivo (42, 44, 45). Cohesin-mediated loops are necessary for the establishment of new gene expression programs in post-mitotic neurons, including the upregulation of genes encoding axon guidance, dendritic spine morphology, and synaptic plasticity during neuron maturation in vivo and activity-dependent gene expression during neural stimulation in vitro (27). Fear conditioning recruits epigenetically plastic neurons to form the memory engram (46) and induces chromatin accessibility changes at enhancers that might persist at least five days after fear conditioning (43). Together, these data provide the rationale for our hypothesis that key structural features of higher-order chromatin folding could be persistent long after the exposure and removal of a pharmacological, environmental, or behavioral stimulus that causes neurons to fire action potentials. ResultsHere, we apply cutting-edge single-nucleus multi-modal technologies toward the goal of ascertaining the extent to which there are enduring traces of higher-order chromatin folding, DNA methylation, histone modifications at activity-dependent enhancers, and/or mRNA levels in single human neurons upon exposure to a repetitive stimulation and re-stimulation paradigm. We find that pharmacological stimulation of human induced pluripotent stem cell (iPSC)-derived neurons induces chromatin loop plasticity, including persistent activity-gained (PG) and persistent activity-lost (PL) loops that are dependent on CREB phosphorylation. We also query activity-dependent patterns of DNA methylation genome-wide and demonstrate that the majority of looped enhancers and promoters are negligibly differentially methylated in human neurons in response to pharmacological stimulation. CTCF binding to looped enhancers and promoters further protects such regulatory elements from activity-stimulated changes in DNA methylation. What are the potential functional effects of persistent chromatin loops? We find that activity-lost persistent loops anchor activity-downregulated genes that remain repressed five days post-stimulation. By contrast, activity-gained persistent loops are enriched for activity-induced gene expression at 2-5 hours post-stimulation, but mRNA levels resume baseline levels by five days after removal of the stimulus. A subset of promoter-enhancer loops bound by CTCF can exhibit enduring traces of the histone modification H3K27ac. Promoters persistently looped to CTCF-bound persistent activity-induced enhancers can exhibit memory of activity-upregulated gene expression for at least five days after stimulation. Finally, we re-stimulated our human neurons five days after the original stimulation event and assessed gene expression with single-nucleus RNA-seq. Upon second stimulation, we unexpectedly find that activity-upregulated genes can be robustly re-induced when unlooped but remain nonresponsive at persistent loops akin to habituation. We also unexpectedly find that activity-independent, invariant gene expression can be downregulated at second stimulation when unlooped but protected from homeostatic downscaling when anchored in persistent loops. ConclusionTaken together, our data reveal enduring traces of higher-order chromatin loops in human neurons after activity-simulation and their link to gene expression plasticity and habituation during re-stimulation and re-stimulation of human neurons. Our work sheds light on the role for chromatin and gene expression in an important unsolved paradox in neuroscience: How is information encoded in neural circuits on the timescale of years despite the rapid turnover of synaptic proteins/RNAs in hours to weeks? Persistent chromatin loops shape plasticity in cell-wide RNA levels, thus laying the foundation for future studies linking genes identified here to their subcellular localization and possible functional role at the synapse.

neuroscience↗

bioRxiv2025

Learning induces persistent chromatin loops underlying robust gene expression during memory recall

Long-term memories are stored in neuronal ensembles called engrams, but the existence of persistent molecular traces in nuclei of engram neurons remain unknown. Using activity-dependent nuclear tagging in vivo, we profiled higher-order chromatin folding and DNA methylation in thousands of single hippocampal neurons up to a month after contextual fear conditioning (CFC). We find CFC-induced chromatin loop plasticity genome-wide, including persistently gained and lost loops with enduring traces in vivo. DNA methylation showed minimal CFC-induced persistence at promoters and enhancers. Persistently gained and lost loops connect distinct enhancers and promoters in excitatory and inhibitory subtypes and correlate with robust CFC-upregulated and -downregulated gene expression, respectively, upon recall. Synaptic genes associated with post-traumatic stress disorder and autism anchor neuronal subtype-specific persistent loops, suggesting relevance to neuropsychiatric dysfunction. We harness the power and sensitivity of multi-modal single-cell measurements to find enduring chromatin traces linked to robust gene expression during fear memory recall. Structured AbstractO_ST_ABSIntroductionC_ST_ABSLong-term memory is thought to be stored in specific neural circuits called engrams (1). Mechanisms centered at the synapse have been proposed, but what is altered within each engram cell that persists for the duration of the memory is unknown. Neural circuits fire action potentials in response to experiences. Such electrochemical signals are converted to molecular signaling pathways which travel from the synapse to the nucleus to activate new gene expression programs. However, experience-dependent RNA and protein molecules as well as synaptic plasticity phenomena are short-lived, lasting for only a few hours to several days (2). Thus, the extent to which chromatin and gene expression changes induced by behavior persist and functionally contribute to memory encoding, consolidation, and retrieval remains an important answered question. DNA is folded in the mammalian nucleus into higher-order long-range chromatin looping interactions (3). Loops form mechanistically through extrusion in which cohesin subunits form a ring that shuttles along chromatin and extrudes out the intervening DNA until it stalls at boundaries occupied by architectural proteins such as CTCF (4, 5). CTCF-independent looping mechanisms have also been uncovered (6-10). A subset of loops regulates gene expression by bringing distant enhancers into spatial proximity with their target promoters. Loops connect activity-dependent enhancers to their distal target genes to govern gene expression during in vitro neural stimulation and in vivo behavior paradigms (11-14). However, the extent to which chromatin changes persist on time scales to support long-term memory storage in vivo remains unclear. RationaleMultiple studies support the idea that higher-order chromatin architecture is plastic and can undergo activity- and experience-dependent remodeling linked to gene expression. Electron microscopy measurements provide direct evidence of chromatin architecture plasticity within minutes, with some changes persisting for up to an hour after potassium chloride stimulation of primary hippocampal neurons (15). Perturbing loops by selectively eliminating either CTCF or cohesin subunits impairs memory encoding in multiple behavior models (11, 14, 16-18), disrupts long-term potentiation (17, 18), and alters dendritic morphology (13, 19, 20). Cohesin-mediated loops are necessary for the establishment of new gene expression programs in post-mitotic neurons, including the upregulation of genes encoding axon guidance, dendritic spine morphology, and synaptic plasticity during neuron maturation in vivo as well as activity-dependent regulation of secondary response genes during neural stimulation in vitro (12, 13). The state of histone modifications before learning influences which neurons are recruited into memory traces, suggesting chromatin carries long-lasting yet adaptable information during memory encoding and long-term storage (21). Moreover, a subset of enhancers retains chromatin accessibility after learning in vivo (11). Together, these observations suggest that chromatin might provide a durable regulatory scaffold that persists long after the initial experience to govern gene expression programs required for the aspects of long-term memory storage. ResultsHere, we employ activity-dependent nuclear labeling in TRAP2 (targeted recombination in active populations) mice to isolate nuclei from neurons stimulated during contextual fear conditioning (CFC) (22, 23). Using single-nucleus methyl 3C-sequencing (snm3C-seq3), we simultaneously profiled DNA methylation and 3D genome folding in the same single tagged neurons from the hippocampus in a time course up to 28 days after CFC and during fear memory recall. Our multi-modal inquiry offered ability to group neurons by their neuronal subtype-specific DNA methylation profile, and thereby computationally generate pseudobulk Chromosome Conformation Capture heatmaps per each sorted hippocampal neuron subtype. We find CFC-induced chromatin loop plasticity genome-wide, including persistently gained and persistently lost loops with enduring structural traces up to 28 days after training. Persistent loops connect distinct distal enhancers to target genes encoding synaptic plasticity and neurotransmitter signaling pathways unique to each excitatory and inhibitory neuron cell type. By contrast to loops, we observe negligible persistence of CFC-induced changes in DNA methylation at enhancers and promoters. Persistently gained and lost loops show upregulation and downregulation of gene expression upon recall, respectively, with the functional effect focused on genes with mRNA levels influenced during training. We uncovered a strong enrichment for genes associated with post-traumatic stress disorder and autism spectrum disorder anchoring the base of persistent loops, suggesting the relevance of enduring chromatin architecture traces to dysregulation of fear, anxiety, and stress in neuropsychiatric disorders. ConclusionOur findings reveal that learning induces neuronal subtype-specific changes in chromatin loops, a subset of which remain durable for at least a month in vivo. Persistent chromatin loops are linked to expression of disease-associated synaptic genes during fear memory recall, thus highlighting relevance to neuropsychiatric and neurodevelopmental disorders. Our work opens up new avenues for investigating enduring chromatin traces for their role in governing cell-wide mRNA levels of genes that impact synaptic plasticity during fear learning and long-term memory storage.

neuroscience↗

bioRxiv2025

Persistent chromatin alterations and gene expression reprogramming follow widespread DNA damage in glioblastoma

DNA damage from routine cellular processes or exogenous insults can have a lasting impact on gene regulation beyond genetic mutations. The prevailing paradigm for the consequences of DNA damage repair revolves around restoration of the original genetic sequence, but long-term changes in chromatin configuration, gene expression and DNA modifications have not been analyzed. We introduced numerous, simultaneous Cas9-mediated DNA double strand breaks (DSBs) at defined locations in human glioblastoma cells and tracked both non-genetic and genetic alterations over time. Megabase-scale genomic alterations that endured two weeks after the initial damage were detected, involving a shift from transiently increased intra-TAD interactions to persistent long range cis and trans contacts, alterations in gene-expression and associated large structural variations. These findings reveal that widespread DNA damage, such as chemotherapy or radiotherapy, can trigger long-term genetic and non-genetic modifications which alter cellular function and may impact tumor outcome and the emergence of resistant cells.

genomics↗

bioRxiv2025

Autism spectrum disorder risk genes have convergent effects on transcription and neuronal firing patterns in primary neurons

Autism spectrum disorder (ASD) is a highly heterogenous neurodevelopmental disorder with numerous genetic risk factors. Notably, a disproportionate number of risk genes encode transcription regulators including transcription factors and proteins that regulate chromatin. Here, we teste the function of nine such ASD-linked transcription regulators by depleting them in primary cultured neurons. We then define the resulting gene expression disruptions using RNA-sequencing and test effects on neuronal firing using multielectrode array recordings. We identify shared gene expression signatures across many ASD risk genes that converge on disruption of critical synaptic genes. Fitting with this, we detect robust disruptions to neuronal firing throughout neuronal maturation. Together, these findings provide evidence that loss of multiple ASD-linked transcriptional regulators disrupts transcription of synaptic genes and has convergent effects on neuronal firing that may contribute to enhanced ASD risk.

neuroscience↗

bioRxiv2025

Mosaic H3K9me3 at BREACHes predicts synaptic gene expression associated with fragile X syndrome cognitive severity

Diseases vary in clinical presentation across individuals despite the same molecular diagnosis. In fragile X syndrome (FXS), mutation-length expansion of a CGG short tandem repeat (STR) in FMR1 causes reduced gene expression and FMRP loss. Nevertheless, FMR1 and FMRP are limited predictors of adaptive functioning and cognition in FXS patients, suggesting that molecular correlates of clinical measures would add diagnostic value. We recently uncovered Megabase-scale domains of heterochromatin (BREACHes) in FXS patient-derived iPSCs. Here, we identify BREACHes in FXS brain tissue (N=4) and absent from sex/age-matched neurotypical controls (N=4). BREACHes span >250 genes and exhibit patient-specific H3K9me3 variation. Using N=4 FXS iPSC lines and N=7 single-cell isogenic FXS iPSC subclones, we observe a strong correlation between inter-sample H3K9me3 variation and heterogeneous BREACH gene repression. We demonstrate improved prediction of cognitive metrics in FXS patients with an additive model of blood FMRP and mRNA levels of H3K9me3-mosaic, but not H3K9me3-invariant, BREACH genes. Our results highlight the utility of H3K9me3 variation at BREACHes for identifying genes associated with FXS clinical metrics.

genomics↗

bioRxiv2024

FISHnet: Detecting chromatin domains in single-cell sequential Oligopaints imaging data

Sequential Oligopaints DNA FISH is an imaging technique that measures higher-order genome folding at single-allele resolution via multiplexed, probe-based tracing. Currently there is a paucity of algorithms to identify 3D genome features in sequential Oligopaints data. Here, we present FISHnet, a graph theory method based on optimization of network modularity to detect chromatin domains and boundaries in pairwise distance matrices. FISHnet uncovers cell type-specific domain-like folding patterns on single alleles, thus enabling future studies aiming to elucidate the role for single-cell folding variation on genome function.

molecular biology↗

bioRxiv2024

MASTR-seq: Multiplexed Analysis of Short Tandem Repeats with sequencing

More than 60 human disorders have been linked to unstable expansion of short tandem repeat (STR) tracts. STR length and the extent of DNA methylation is linked to disease pathology and can be mosaic in a cell type-specific manner in several repeat expansion disorders. Mosaic phenomenon have been difficult to study to date due to technical bias intrinsic to repeat sequences and the need for multi-modal measurements at single-allele resolution. Nanopore long-read sequencing accurately measures STR length and DNA methylation in the same single molecule but is cost prohibitive for studies assessing a target locus across multiple experimental conditions or patient samples. Here, we describe MASTR-seq, Multiplexed Analysis of Short Tandem Repeats, for cost-effective, high-throughput, accurate, multi-modal measurements of DNA methylation and STR genotype at single-allele resolution. MASTR-seq couples long-read sequencing, Cas9-mediated target enrichment, and PCR-free multiplexed barcoding to achieve a >ten-fold increase in on-target read mapping for 8-12 pooled samples in a single MinION flow cell. We provide a detailed experimental protocol and computational tools and present evidence that MASTR-seq quantifies tract length and DNA methylation status for CGG and CAG STR loci in normal-length and mutation-length human cell lines. The MASTR-seq protocol takes approximately eight days for experiments and one additional day for data processing and analyses. Key pointsO_LIWe provide a protocol for MASTR-seq: Multiplexed Analysis of Short Tandem Repeats using Cas9-mediated target enrichment and PCR-free, multiplexed nanopore sequencing. C_LIO_LIMASTR-seq achieves a >10-fold increase in on-target read proportion for highly repetitive, technically inaccessible regions of the genome relevant for human health and disease. C_LIO_LIMASTR-seq allows for high-throughput, efficient, accurate, and cost-effective measurement of STR length and DNA methylation in the same single allele for up to 8-12 samples in parallel in one Nanopore MinION flow cell. C_LI

genomics↗

bioRxiv2024

The chromatin conformation landscape of Alzheimer's disease

We have been investigating epigenetic alterations in the brain during human aging and Alzheimers disease (AD), and have evidence for histone acetylation both protecting the aging epigenome and driving AD. Here we extend our studies to chromatin architecture via looping studies, and with binding studies of key proteins required for looping: CTCF and RAD21. We detected changes in CTCF and RAD21 levels and localization, finding major changes in CTCF in AD compared to fewer changes in healthy aging. In our study of 3D genome conformation changes, we identified stable topological associating domains (TADs) in Old and AD; in contrast, in AD, there is loss of interaction at genomic sites/loops within TADs, likely reflecting the loss of CTCF. We identified genes and potential transcription factor binding at the loops that are lost in AD. in addition, we found enrichment of CTCF peak losses for AD eQTLs, suggesting that architectural dysfunction has a role in Alzheimers. Functional experiments lowering the homologues of several key genes in a Drosophila model of A{beta}42 toxicity exacerbate neurodegeneration. Taken together, these data indicate both functional protections and losses occur in the Alzheimers brain genome compared to normal aging.

neuroscience↗

bioRxiv2024

A multi-looping chromatin signature predicts dysregulated gene expression in neurons with familial Alzheimer's disease mutations

Mammalian genomes fold into tens of thousands of long-range loops, but their functional role and physiologic relevance remain poorly understood. Here, using human post-mitotic neurons with rare familial Alzheimers disease (FAD) mutations, we identify hundreds of reproducibly dysregulated genes and thousands of miswired loops prior to amyloid accumulation and tau phosphorylation. Single loops do not predict expression changes; however, the severity and direction of change in mRNA levels and single-cell burst frequency strongly correlate with the number of FAD-gained or -lost promoter-enhancer loops. Classic architectural proteins CTCF and cohesin do not change occupancy in FAD-mutant neurons. Instead, we unexpectedly find TAATTA motifs amenable to binding by DLX homeodomain transcription factors and changing noncoding RNAPolII signal at FAD-dynamic promoter-enhancer loops. DLX1/5/6 mRNA levels are strongly upregulated in FAD-mutant neurons coincident with a shift in excitatory-to-inhibitory gene expression and miswiring of multi-loops connecting enhancers to neural subtype genes. DLX1 overexpression is sufficient for loop miswiring in wildtype neurons, including lost and gained loops at enhancers with tandem TAATTA arrays and singular TAATTA motifs, respectively. Our data uncover a genome structure-function relationship between multi-loop miswiring and dysregulated excitatory and inhibitory transcriptional programs during lineage commitment of human neurons homozygously-engineered with rare FAD mutations.

molecular biology↗

bioRxiv2023

Cell type-specific loops linked to RNA polymerase II elongation in human neural differentiation

DNA is folded into higher-order structures that shape and are shaped by genome function. The role for long-range loops in the establishment of new gene expression patterns during cell fate transitions remains poorly understood. Here, we investigate the link between cell-specific loops and RNA polymerase II (RNAPolII) during neural lineage commitment. We find thousands of loops decommissioned or gained de novo upon differentiation of human induced pluripotent stem cells (hiPSCs) to neural progenitors (NPCs) and post-mitotic neurons. During hiPSC-to-NPC and NPC-to-neuron transitions, genes changing from RNAPolII initiation to elongation are >4-fold more likely to anchor cell-specific loops than repressed genes. Elongated genes exhibit significant mRNA upregulation when connected in cell-specific promoter-enhancer loops but not invariant promoter-enhancer loops, promoter-promoter loops, or unlooped. Genes transitioning from repression to RNAPolII initiation exhibit slight mRNA increase independent of loop status. Our data link cell-specific loops and robust RNAPolII-mediated elongation during neural cell fate transitions. HighlightsO_LIThousands of loops are decommissioned and gained upon human iPSC differentiation to NPCs/neurons C_LIO_LIGenes transitioning from initiated-to-elongated exhibit robust mRNA upregulation when connected in cell type-specific promoter-enhancer loops C_LIO_LIGenes transitioning from repressed-to-initiated exhibit slight increases in mRNA levels independent of loop status C_LIO_LIUpon short-term RNAPolII degradation, loops formed by elongated genes are more severely disrupted than those anchoring initiated genes C_LI

genomics↗

bioRxiv2022

Remodeling and compaction of the inactive X is regulated by Xist during female B cell activation

X Chromosome Inactivation (XCI) equalizes X-linked gene expression between sexes. B cells exhibit unusually dynamic XCI, as Xist RNA/heterochromatic marks on the inactive X (Xi) are absent in naive B cells, but return following mitogenic stimulation. Xi gene expression analysis supports dosage compensation, but reveals high levels of XCI escape genes in both naive and activated B cells. Allele-specific OligoPaints indicate similar Xi and Xa territories in B cells that is less compact than in fibroblasts. Allele-specific Hi-C maps reveal a lack of TAD-like structures on the Xi of naive B cells, and alterations in TADs and stronger TAD boundaries at Xi escape genes after mitogenic stimulation. Notably, Xist deletion in B cells reduces Xi compaction and changes TAD boundaries, independent of its localization to the Xi. Our findings provide the first evidence that Xi compaction/small scale organization in lymphocytes impact XCI maintenance and female biased X-linked gene expression.

genetics↗

bioRxiv2022

High-throughput Oligopaint screen identifies druggable regulators of genome folding

Although the molecular rules governing genome organization are being quickly elucidated, relatively few proteins regulating this process have been identified. To address this gap, we developed a fully automated imaging pipeline, called HiDRO (high-throughput DNA or RNA labeling with optimized Oligopaints), that permits quantitative measurement of chromatin interactions across a large number of samples. Using HiDRO, we screened the human druggable genome and identified >300 factors that regulate chromatin folding during interphase, including 43 validated hits that either increase or decrease interactions between topological associating domains (TADs). We discovered that genetic or chemical inhibition of the ubiquitous kinase GSK3A enhances long-range interactions by dysregulating cohesin-mediated chromatin looping. Collectively, these results highlight a noncanonical role for GSK3A signaling in nuclear architecture and underscore the broader utility of HiDRO-based screening to identify novel mechanisms that drive the spatial organization of the genome.

genetics↗

bioRxiv2021

Acetylated Chromatin Domains Link Chromosomal Organization to Cell- and Circuit-level Dysfunction in Schizophrenia and Bipolar Disorder

To explore modular organization of chromosomes in schizophrenia (SCZ) and bipolar disorder (BD), we applied population-scale correlational structuring of 739 histone H3-lysine 27 acetylation and H3-lysine 4 trimethylation profiles, generated from the prefrontal cortex (PFC) of 568 cases and controls. Neuronal histone acetylomes and methylomes assembled as thousands of cis-regulatory domains (CRDs), revealing fine-grained, kilo-to megabase scale chromatin organization at higher resolution but firmly integrated into Hi-C chromosomal conformations. Large clusters of domains that were hyperacetylated in disease shared spatial positioning within the nucleus, predominantly regulating PFC projection neuron function and excitatory neurotransmission. Hypoacetylated domains were linked to inhibitory interneuron- and myelination-relevant genes. Chromosomal modular architecture is affected in SCZ and BD, with hyperacetylated domains showing unexpectedly strong convergences defined by cell type, nuclear topography, genetic risk, and active chromatin state across a wide developmental window.

genomics↗

bioRxiv2021

Spatially coordinated heterochromatinization of distal short tandem repeats in fragile X syndrome

Short tandem repeat (STR) instability is causally linked to pathologic transcriptional silencing in a subset of repeat expansion disorders. In fragile X syndrome (FXS), instability of a single CGG STR tract is thought to repress FMR1 via local DNA methylation. Here, we report the acquisition of more than ten Megabase-sized H3K9me3 domains in FXS, including a 5-8 Megabase block around FMR1. Distal H3K9me3 domains encompass synaptic genes with STR instability, and spatially co-localize in trans concurrently with FMR1 CGG expansion and the dissolution of TADs. CRISPR engineering of mutation-length FMR1 CGG to normal-length preserves heterochromatin, whereas cut-out to pre-mutation-length attenuates a subset of H3K9me3 domains. Overexpression of a pre-mutation-length CGG de-represses both FMR1 and distal heterochromatinized genes, indicating that long-range H3K9me3-mediated silencing is exquisitely sensitive to STR length. Together, our data uncover a genome-wide surveillance mechanism by which STR tracts spatially communicate over vast distances to heterochromatinize the pathologically unstable genome in FXS. One-Sentence SummaryHeterochromatinization of distal synaptic genes with repeat instability in fragile X is reversible by overexpression of a pre-mutation length CGG tract.

genomics↗

bioRxiv2021

Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

Cohesin and CTCF are major drivers of 3D genome organization, but their role in neurons is still emerging. Here we show a prominent role for cohesin in the expression of genes that facilitate neuronal maturation and homeostasis. Unexpectedly, we observed two major classes of activity-regulated genes with distinct reliance on cohesin in primary cortical neurons. Immediate early genes remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the Immediate early gene Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterised by long-range chromatin contacts. Cohesin-dependence of constitutive neuronal genes with key functions in synaptic transmission and neurotransmitter signaling also scaled with chromatin loop length. Our data demonstrate that key genes required for the maturation and activation of primary cortical neurons depend cohesin for their full expression, and that the degree to which these genes rely on cohesin scales with the genomic distance traversed by their chromatin contacts.

genomics↗

bioRxiv2021

Cohesin-mediated loop anchors confine the location of human replication origins

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability across dividing cells in metazoans. It is currently unknown how the location of replication origins and the timing of their activation is determined in the human genome. Here, we dissect the role for G1 phase topologically associating domains (TADs), subTADs, and loops in the activation of replication initiation zones (IZs). We identify twelve subtypes of self-interacting chromatin domains distinguished by their degree of nesting, the presence of corner dot structures indicative of loops, and their co-localization with A/B compartments. Early replicating IZs localize to boundaries of nested corner-dot TAD/subTADs anchored by high density arrays of co-occupied CTCF+cohesin binding sites with divergently oriented motifs. By contrast, late replicating IZs localize to weak TADs/subTAD boundaries devoid of corner dots and most often anchored by singlet CTCF+cohesin sites. Upon global knock-down of cohesin-mediated loops in G1, early wave focal IZs replicate later in S phase and convert to diffuse placement along the genome. Moreover, IZs in mid-late S phase are delayed to the final minutes before entry into G2 when cohesin-mediated dot-less boundaries are ablated. We also delete a specific loop anchor and observe a sharp local delay of an early wave IZ to replication in late S phase. Our data demonstrate that cohesin-mediated loops at genetically-encoded TAD/subTAD boundaries in G1 phase are an essential determinant of the precise genomic placement of human replication origins in S phase.

genomics↗

bioRxiv2020

Stress-induced epigenetic regulation of transcription in neocortical excitatory neurons drives depression-like behavior

Prolonged stress exposure is a major risk factor for the development of depression and comorbid anxiety. Efforts to understand how recurrent stress induces behavioral maladaptation have largely concentrated on the neuronal synapse with limited understanding of the underlying epigenetic mechanisms. Here, we performed complementary bulk nuclei- and single-nucleus transcriptome profiling and mapped fine-scale chromatin architecture in mice subjected to chronic unpredictable stress (CUS) to identify the cell type-specific epigenetic alterations that drive complex behavior. We find that neocortical excitatory neurons are particularly vulnerable to chronic stress and acquire signatures of transcription and chromatin configuration that denote reduced neuronal activity and expression of Yin Yang 1 (YY1). Selective ablation of YY1 in excitatory neurons of the prefrontal cortex (PFC) enhances stress sensitivity in mice, inducing depressive- and anxiety-related behaviors and deregulating the expression of stress-associated genes following an abbreviated stress exposure. Notably, we find that loss of YY1 in PFC excitatory neurons provokes more maladaptive behaviors in stressed females than males. These findings demonstrate how chronic stress provokes maladaptive behavior by epigenetically shaping excitatory neurons in the PFC, identifying a novel molecular target for therapeutic treatment of stress-related mood and anxiety disorders.

neuroscience↗