Cohesin-dependence of neuronal gene expression relates to chromatin loop length.

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 mouse primary cortical neurons. Immediate early genes (IEGs) remained fully inducible by KCl and BDNF, and short-range enhancer-promoter contacts at the IEGs Fos formed robustly in the absence of cohesin. In contrast, cohesin was required for full expression of a subset of secondary response genes characterized 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 on 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.

Neuronal Yin Yang1 in the prefrontal cortex regulates transcriptional and behavioral responses to chronic stress in mice.

Although the synaptic alterations associated with the stress-related mood disorder major depression has been well-documented, the underlying transcriptional mechanisms remain poorly understood. Here, we perform complementary bulk nuclei- and single-nucleus transcriptome profiling and map locus-specific chromatin interactions in mouse neocortex to identify the cell type-specific transcriptional changes associated with stress-induced behavioral maladaptation. We find that cortical excitatory neurons, layer 2/3 neurons in particular, are vulnerable to chronic stress and acquire signatures of gene transcription and chromatin structure associated with reduced neuronal activity and expression of Yin Yang 1 (YY1). Selective ablation of YY1 in cortical excitatory neurons enhances stress sensitivity in both male and female mice and alters the expression of stress-associated genes following an abbreviated stress exposure. These findings demonstrate how chronic stress impacts transcription in cortical excitatory neurons and identify YY1 as a regulator of stress-induced maladaptive behavior in mice.

Publisher Correction: Three-dimensional genome restructuring across timescales of activity-induced neuronal gene expression.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Three-dimensional genome restructuring across timescales of activity-induced neuronal gene expression.

Neuronal activation induces rapid transcription of immediate early genes (IEGs) and longer-term chromatin remodeling around secondary response genes (SRGs). Here, we use high-resolution chromosome-conformation-capture carbon-copy sequencing (5C-seq) to elucidate the extent to which long-range chromatin loops are altered during short- and long-term changes in neural activity. We find that more than 10% of loops surrounding select IEGs, SRGs, and synaptic genes are induced de novo during cortical neuron activation. IEGs Fos and Arc connect to activity-dependent enhancers via singular short-range loops that form within 20 min after stimulation, prior to peak messenger RNA levels. By contrast, the SRG Bdnf engages in both pre-existing and activity-inducible loops that form within 1-6 h. We also show that common single-nucleotide variants that are associated with autism and schizophrenia are colocalized with distinct classes of activity-dependent, looped enhancers. Our data link architectural complexity to transcriptional kinetics and reveal the rapid timescale by which higher-order chromatin architecture reconfigures during neuronal stimulation.

On the existence and functionality of topologically associating domains.

Genomes across a wide range of eukaryotic organisms fold into higher-order chromatin domains. Topologically associating domains (TADs) were originally discovered empirically in low-resolution Hi-C heat maps representing ensemble average interaction frequencies from millions of cells. Here, we discuss recent advances in high-resolution Hi-C, single-cell imaging experiments, and functional genetic studies, which provide an increasingly complex view of the genome's hierarchical structure-function relationship. On the basis of these new findings, we update the definitions of distinct classes of chromatin domains according to emerging knowledge of their structural, mechanistic and functional properties.

LADL: light-activated dynamic looping for endogenous gene expression control.

Mammalian genomes are folded into tens of thousands of long-range looping interactions. The cause-and-effect relationship between looping and genome function is poorly understood, and the extent to which loops are dynamic on short time scales remains an unanswered question. Here, we engineer a new class of synthetic architectural proteins for directed rearrangement of the three-dimensional genome using blue light. We target our light-activated-dynamic-looping (LADL) system to two genomic anchors with CRISPR guide RNAs and induce their spatial colocalization via light-induced heterodimerization of cryptochrome 2 and a dCas9-CIBN fusion protein. We apply LADL to redirect a stretch enhancer (SE) away from its endogenous Klf4 target gene and to the Zfp462 promoter. Using single-molecule RNA-FISH, we demonstrate that de novo formation of the Zfp462-SE loop correlates with a modest increase in Zfp462 expression. LADL facilitates colocalization of genomic loci without exogenous chemical cofactors and will enable future efforts to engineer reversible and oscillatory loops on short time scales.

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.

5C-ID: Increased resolution Chromosome-Conformation-Capture-Carbon-Copy with in situ 3C and double alternating primer design.

Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs, and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C can generate genome-wide maps of looping interactions but is intractable for high-throughput comparison of loops across multiple conditions due to the enormous number of reads (>6 Billion) required per library. Here, we describe 5C-ID, a new version of Chromosome-Conformation-Capture-Carbon-Copy (5C) with restriction digest and ligation performed in the nucleus (in situ Chromosome-Conformation-Capture (3C)) and ligation-mediated amplification performed with a double alternating primer design. We demonstrate that 5C-ID produces higher-resolution 3D genome folding maps with reduced spatial noise using markedly lower cell numbers than canonical 5C. 5C-ID enables the creation of high-resolution, high-coverage maps of chromatin loops in up to a 30 Megabase subset of the genome at a fraction of the cost of Hi-C.

YY1 and CTCF orchestrate a 3D chromatin looping switch during early neural lineage commitment.

CTCF is an architectural protein with a critical role in connecting higher-order chromatin folding in pluripotent stem cells. Recent reports have suggested that CTCF binding is more dynamic during development than previously appreciated. Here, we set out to understand the extent to which shifts in genome-wide CTCF occupancy contribute to the 3D reconfiguration of fine-scale chromatin folding during early neural lineage commitment. Unexpectedly, we observe a sharp decrease in CTCF occupancy during the transition from naïve/primed pluripotency to multipotent primary neural progenitor cells (NPCs). Many pluripotency gene-enhancer interactions are anchored by CTCF, and its occupancy is lost in parallel with loop decommissioning during differentiation. Conversely, CTCF binding sites in NPCs are largely preexisting in pluripotent stem cells. Only a small number of CTCF sites arise de novo in NPCs. We identify another zinc finger protein, Yin Yang 1 (YY1), at the base of looping interactions between NPC-specific genes and enhancers. Putative NPC-specific enhancers exhibit strong YY1 signal when engaged in 3D contacts and negligible YY1 signal when not in loops. Moreover, siRNA knockdown of Yy1 specifically disrupts interactions between key NPC enhancers and their target genes. YY1-mediated interactions between NPC regulatory elements are often nested within constitutive loops anchored by CTCF. Together, our results support a model in which YY1 acts as an architectural protein to connect developmentally regulated looping interactions; the location of YY1-mediated interactions may be demarcated in development by a preexisting topological framework created by constitutive CTCF-mediated interactions.

CRISPR/Cas9 genome editing throws descriptive 3-D genome folding studies for a loop.

CRISPR/Cas9 genome editing studies have recently shed new light into the causal link between the linear DNA sequence and 3-D chromatin architecture. Here we describe current models for the folding of genomes into a nested hierarchy of higher-order structures and discuss new insights into the organizing principles governing genome folding at each length scale. WIREs Syst Biol Med 2016, 8:286-299. doi: 10.1002/wsbm.1338 For further resources related to this article, please visit the WIREs website.

Local Genome Topology Can Exhibit an Incompletely Rewired 3D-Folding State during Somatic Cell Reprogramming.

Pluripotent genomes are folded in a topological hierarchy that reorganizes during differentiation. The extent to which chromatin architecture is reconfigured during somatic cell reprogramming is poorly understood. Here we integrate fine-resolution architecture maps with epigenetic marks and gene expression in embryonic stem cells (ESCs), neural progenitor cells (NPCs), and NPC-derived induced pluripotent stem cells (iPSCs). We find that most pluripotency genes reconnect to target enhancers during reprogramming. Unexpectedly, some NPC interactions around pluripotency genes persist in our iPSC clone. Pluripotency genes engaged in both "fully-reprogrammed" and "persistent-NPC" interactions exhibit over/undershooting of target expression levels in iPSCs. Additionally, we identify a subset of "poorly reprogrammed" interactions that do not reconnect in iPSCs and display only partially recovered, ESC-specific CTCF occupancy. 2i/LIF can abrogate persistent-NPC interactions, recover poorly reprogrammed interactions, reinstate CTCF occupancy, and restore expression levels. Our results demonstrate that iPSC genomes can exhibit imperfectly rewired 3D-folding linked to inaccurately reprogrammed gene expression.

ALS-associated peripherin spliced transcripts form distinct protein inclusions that are neuroprotective against oxidative stress.

Intracellular proteinaceous inclusions are well-documented hallmarks of the fatal motor neuron disorder amyotrophic lateral sclerosis (ALS). The pathological significance of these inclusions remains unknown. Peripherin, a type III intermediate filament protein, is upregulated in ALS and identified as a component within different types of ALS inclusions. The formation of these inclusions may be associated with abnormal peripherin splicing, whereby an increase in mRNA retaining introns 3 and 4 (Per-3,4) leads to the generation of an aggregation-prone isoform, Per-28. During the course of evaluating peripherin filament assembly in SW-13 cells, we identified that expression of both Per-3,4 and Per-28 transcripts formed inclusions with categorically distinct morphology: Per-3,4 was associated with cytoplasmic condensed/bundled filaments, small inclusions (<10µM), or large inclusions (≥10µM); while Per-28 was associated with punctate inclusions in the nucleus and/or cytoplasm. We found temporal and spatial changes in inclusion morphology between 12 and 48h post-transfected cells, which were accompanied by unique immunofluorescent and biochemical changes of other ALS-relevant proteins, including TDP-43 and ubiquitin. Despite mild cytotoxicity associated with peripherin transfection, Per-3,4 and Per-28 expression increased cell viability during H2O2-mediated oxidative stress in BE(2)-M17 neuroblastoma cells. Taken together, this study shows that ALS-associated peripherin isoforms form dynamic cytoplasmic and intranuclear inclusions, effect changes in local endogenous protein expression, and afford cytoprotection against oxidative stress. These findings may have important relevance to understanding the pathophysiological role of inclusions in ALS.

Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection.

Adeno-associated viral (AAV) gene transfer holds great promise for treating a wide-range of neurodegenerative disorders. The AAV9 serotype crosses the blood-brain barrier and shows enhanced transduction efficiency compared to other serotypes, thus offering advantageous targeting when global transgene expression is required. Neonatal intravenous or intracerebroventricular (i.c.v.) delivery of recombinant AAV9 (rAAV9) have recently proven effective for modeling and treating several rodent models of neurodegenerative disease, however, the technique is associated with variable cellular tropism, making tailored gene transfer a challenge. In the current study, we employ the human synapsin 1 (hSYN1) gene promoter to drive neuron-specific expression of green fluorescent protein (GFP) after neonatal i.c.v. injection of rAAV9 in mice. We observed widespread GFP expression in neurons throughout the brain, spinal cord, and peripheral nerves and ganglia at 6 weeks-of-age. Region-specific quantification of GFP expression showed high neuronal transduction rates in substantia nigra pars reticulata (43.9±5.4%), motor cortex (43.5±3.3%), hippocampus (43.1±2.7%), cerebellum (29.6±2.3%), cervical spinal cord (24.9±3.9%), and ventromedial striatum (16.9±4.3%), among others. We found that 14.6±2.2% of neuromuscular junctions innervating the gastrocnemius muscle displayed GFP immunoreactivity. GFP expression was identified in several neuronal sub-types, including nigral tyrosine hydroxylase (TH)-positive dopaminergic cells, striatal dopamine- and cAMP-regulated neuronal phosphoprotein (DARPP-32)-positive neurons, and choline acetyltransferase (ChAT)-positive motor neurons. These results build on contemporary gene transfer techniques, demonstrating that the hSYN1 promoter can be used with rAAV9 to drive robust neuron-specific transgene expression throughout the nervous system.