Spatially coordinated heterochromatinization of long synaptic genes in fragile X syndrome.

Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.

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.

Cohesin-mediated loop anchors confine the locations of human replication origins.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

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.

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.

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 Alzheimer's 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.

New insights in addressing cerebral small vessel disease: Associated with extracellular fluid in white matter.

Objective: To explore the role of extracellular fluid, assessed by diffusion tensor imaging (DTI) metrics of free water (FW), in the white matter of patients with cerebral small vessel disease (CSVD). Materials and methods: The baseline clinical and imaging data of 129 patients with CSVD were collected and reviewed. CSVD MR markers, including periventricular white matter hyperintensity (PWMH), deep white matter hyperintensity (DWMH), cerebral microbleed (CMB), enlarged perivascular space (PVS), and lacunar infarction (LI), were identified, and CSVD burden was calculated. According to total CSVD MR marker score, cases were classified as mild, moderate, or severe. The mean FW and fractional anisotropy (FA) values were calculated using DTI images. Results: The mean white matter FW was associated with the CSVD MR markers, including PWMH, DWMH, LI and PVS (P < 0.05). Moreover, age, hypertension, diabetes mellitus, and FW value were associated with total CSVD MR marker score (P < 0.05). Ordinal logistic regression analysis revealed that FW and age were independently associated with CSVD burden (P < 0.05). Finally, FW in white matter was associated with FA (r = -0.334, P < 0.001). Conclusion: Extracellular fluid changes, assessed by DTI metrics of FW in white matter, were associated with CSVD markers and burden. An increased extracellular fluid volume in the white matter was associated with lower FA.

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.

Hypertonic saline resuscitation of hemorrhagic shock does not decrease in vivo neutrophil interactions with endothelium in the blood-brain microcirculation.

BACKGROUND: Resuscitation of hemorrhagic shock with isotonic crystalloids has been shown to activate polymorphonuclear neutrophils (PMNs). Although hypertonic saline (HTS) can reduce PMN activation and interactions with endothelial cells (EC) in systemic microvascular beds, no data exist demonstrating that the same occurs in the unique blood-brain barrier microcirculation. We hypothesized that resuscitation of hemorrhagic shock with HTS would blunt brain in vivo PMN-EC interactions. METHODS: Wistar rats (250-350 g) underwent craniotomy and placement of a window for live intravital viewing of pial vessels. Twenty animals were bled to a mean arterial pressure of 30 mm Hg to 35 mm Hg for 1 hour and resuscitated with shed blood and either 5% HTS (6 mL/kg) or Ringer's lactate (RL) (2× shed blood volume). Circulating rhodamine-6G-labeled PMN in pial venules were captured by videomicroscopy at baseline (preshock), end of the shock period, after resuscitation, and every 15 minutes to 30 minutes for 2 hours. Hemodynamics and arterial gases were monitored. Off-line footage analysis allowed comparisons of PMN-EC interactions between groups. RESULTS: Animals in both groups developed significant metabolic acidosis (p < 0.01) after hemorrhage, but postresuscitation blood pressures were similar at all time points. Crystalloid resuscitation volumes were 10× greater in RL than HTS animals (p < 0.001). For all time points, we did not observe the expected reduction in PMN rolling and adhesion in HTS animals, instead noted trends of consistently lower interactions in RL counterparts. CONCLUSIONS: In contradistinction to studies evaluating the systemic microcirculation, HTS may activate PMN-EC crosstalk in the blood-brain microcirculation. Further studies are needed to analyze whether this effect is due to the unique nature of the blood-brain interface.

Comparing the diagnostic value of 18F-FDG-PET/CT versus CT for differentiating benign and malignant solitary pulmonary nodules: a meta-analysis.

BACKGROUND: This quantitative meta-analysis was conducted to provide an indirect comparison of the diagnostic value of computed tomography (CT) with positron emission tomography (PET)/CT for differentiating benign and malignant solitary pulmonary nodules (SPNs). METHODS: PubMed, Embase, and the Cochrane Library were searched to identify eligible studies throughout November 2018, which differentiated benign and malignant SPNs using CT or PET/CT. The summary sensitivity, specificity, positive and negative likelihood ratio (PLR and NLR), diagnostic odds ratio (DOR), and area under the receiver operating characteristic curve (AUC) were calculated using bivariate generalized linear mixed model and random-effects model. The diagnostic value of CT with PET/CT was indirectly evaluated using the ratio for diagnostic parameters. RESULTS: The sensitivity, specificity, PLR, NLR, DOR, and AUC for CT were 0.94 [95% confidence interval (CI): 0.87-0.97], 0.73 (95% CI: 0.64-0.80), 3.45 (95% CI: 2.60-4.58), 0.09 (95% CI: 0.04-0.17), 32.01 (95% CI: 15.10-67.86), and 0.89 (95% CI: 0.86-0.91), respectively. The pooled sensitivity, specificity, PLR, NLR, DOR, and AUC for PET/CT were 0.89 (95% CI: 0.85-0.92), 0.78 (95% CI: 0.66-0.86), 3.97 (95% CI: 2.57-6.13), 0.15 (95% CI: 0.10-0.20), 24.04 (95% CI: 12.71-45.48), and 0.91 (95% CI: 0.89-0.94), respectively. No significant differences were observed between CT and PET/CT for sensitivity, specificity, PLR, NLR, DOR, and AUC. CONCLUSIONS: This study used both CT and PET/CT with a moderate-to-high diagnostic value for differentiating benign and malignant SPNs and showed no significant differences in diagnostic parameters between CT and PET/CT.

Neuroprotective effects of progesterone in traumatic brain injury: blunted in vivo neutrophil activation at the blood-brain barrier.

BACKGROUND: Progesterone (PRO) may confer a survival advantage in traumatic brain injury (TBI) by reducing cerebral edema. We hypothesized that PRO reduces edema by blocking polymorphonuclear (PMN) interactions with endothelium (EC) in the blood-brain barrier (BBB). METHODS: CD1 mice received repeated PRO (16 mg/kg intraperitoneally) or vehicle (cyclodextrin) for 36 hours after TBI. Sham animals underwent craniotomy without TBI. The modified Neurological Severity Score graded neurologic recovery. A second craniotomy allowed in vivo observation of pial EC/PMN interactions and vascular macromolecule leakage. Wet/dry ratios assessed cerebral edema. RESULTS: Compared with the vehicle, PRO reduced subjective cerebral swelling (2.9 ± .1 vs 1.2 ± .1, P < .001), PMN rolling (95 ± 1.8 vs 57 ± 2.0 cells/100 µm/min, P < .001), total EC/PMN adhesion (2.0 ± .4 vs .8 ± .1 PMN/100 µm, P < .01), and vascular permeability (51.8% ± 4.9% vs 27.1% ± 4.6%, P < .01). TBI groups had similar a Neurological Severity Score and cerebral wet/dry ratios (P > .05). CONCLUSIONS: PRO reduces live pericontusional EC/PMN and BBB macromolecular leakage after TBI. Direct PRO effects on the microcirculation warrant further investigation.

Similar effects of hypertonic saline and mannitol on the inflammation of the blood-brain barrier microcirculation after brain injury in a mouse model.

BACKGROUND: There has been substantial debate regarding the efficacy of hypertonic saline (HTS) versus mannitol (MTL) in treating moderate and severe traumatic brain injury (TBI). HTS blunts polymorphonuclear neutrophil (PMN) and endothelial cell (EC) activation and reduces tissue edema after resuscitated shock in systemic microvascular beds. MTL also modulates PMN activation markers. It remains unknown if either of these osmotherapies exert similar anti-inflammatory effects along the blood-brain barrier (BBB). We hypothesized that HTS, as compared with MTL, would more greatly reduce PMN-EC interactions, thereby reducing BBB permeability and tissue edema after simulated TBI. METHODS: CD1 male mice (25-30 g) underwent craniotomy and window placement for observation of in vivo PMN-EC interactions in pial venules using intravital video microscopy. TBI was simulated through local suffusion of the brain surface with interleukin 1ß (100 ng/0.1 mL). Animals were randomized to receive a single, equiosmolar, intravenous dose of 20% MTL or 5% HTS after injury. Live microcirculatory footage was obtained every 15 minutes for 2 hours, after which fluorescent-labeled albumin was administered to assess microvascular permeability. PMN rolling and adhesion and macromolecular leakage were analyzed offline by a blinded observer and postmortem brain and lung edema assessed by wet-to-dry ratios. Student's t test and Mann-Whitney U test determined significance (p ≤ 0.05). RESULTS: Neither osmotherapy resulted in significant differences in PMN rolling or adhesion; however, both trended higher in HTS. Similarly, vessel permeability did not differ between groups but also trended higher with HTS. In contrast, brain and lung edema was greater in MTL than HTS as compared with controls (p = 0.05). CONCLUSION: MTL and HTS have indistinguishable effects on PMN-EC interactions in the brain after simulated TBI. Additional studies are needed to determine if either osmotherapy has more subtle effects on BBB PMN-EC interactions after injury exerting a potential clinical advantage.

LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling.

The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.