DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.

With the exception of lamina-associated domains, the radial organization of chromatin in mammalian cells remains largely unexplored. Here, we describe genomic loci positioning by sequencing (GPSeq), a genome-wide method for inferring distances to the nuclear lamina all along the nuclear radius that works by gradual enzymatic restriction of chromatin from the nuclear lamina towards the nucleus center, followed by sequencing of the generated cut sites. Using GPSeq, we mapped the radial organization of the human genome at 100 kb resolution, which revealed radial patterns of genomic and epigenomic features, gene expression, as well as A/B subcompartments. By combining radial information with chromosome contact frequencies measured by Hi-C, we substantially improved the accuracy of wholegenome structure modeling. Finally, we charted the radial topography of DNA double-strand breaks, germline variants and cancer mutations, and found that they have distinctive radial arrangements in A/B subcompartments. We conclude that GPSeq can reveal fundamental aspects of genome architecture.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-β signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed “wound and keratinocyte migration-associated lncRNA 1” (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

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Efferocytosis is critical for tissue homeostasis, as its deregulation is associated with several autoimmune pathologies. While engulfing apoptotic cells, phagocytes activate transcription factors, such as peroxisome proliferator-activated receptors (PPAR) or liver X receptors (LXR) that orchestrate metabolic, phagocytic, and inflammatory responses towards the ingested material. Coordination of these transcription factors in efferocytotic human macrophages is not fully understood. In this study, we evaluated the transcriptional profile of macrophages following the uptake of apoptotic Jurkat T cells using RNA-seq analysis. Results indicated upregulation of PPAR and LXR pathways but downregulation of sterol regulatory element-binding proteins (SREBP) target genes. Pharmacological inhibition and RNA interference pointed to LXR and PPARδ as relevant transcriptional regulators, while PPARγ did not substantially contribute to gene regulation. Mechanistically, lysosomal digestion and lysosomal acid lipase (LIPA) were required for PPAR and LXR activation, while PPARδ activation also demanded an active lysosomal phospholipase A2 (PLA2G15). Pharmacological interference with LXR signaling attenuated ABCA1-dependent cholesterol efflux from efferocytotic macrophages, but suppression of inflammatory responses following efferocytosis occurred independently of LXR and PPARδ. These data provide mechanistic details on LXR and PPARδ activation in efferocytotic human macrophages.

Background Ballet dancers are a risk group for body image (BI) distortion, dissatisfaction and eating disorders (ED), but few studies have investigated these aspects in amateur adult practitioners. This study aimed to evaluate if amateur female adult classical ballet dancers presented different BI and behaviors for ED than gym users and sedentary women. Methods This is a cross-sectional study where classical ballet dancers (n = 19) were compared to gym users (n = 19) and sedentary women (n = 19). Body mass index (BMI) was assessed, and a figure rating scale was applied to assess BI distortion/dissatisfaction. The body shape questionnaire (BSQ) was used to measure BI concern. The eating attitudes test (EAT-26) and the bulimic investigatory test, Edinburgh (BITE) were used for behaviors toward anorexia and bulimia. Results BMI was significantly lower in ballet dancers than gym users and sedentary women (F, p = .04). BI distortion did not differ among the studied groups. BI dissatisfaction was lower (X2, p = .041) in ballet dancers (75.0%) and gym users (70.6%) compared to sedentary women (100%). Correspondence analysis showed ballet dancers were mostly not concerned with BI, which was not observed among the other groups. The EAT-26 did not differ between the studied groups. The BITE score was lower (Tukey’s post hoc test, p = .005) in the ballet dancers [mean 5.3 (5.6)] compared to the sedentary women [mean 10.9 (4.8)]. Conclusions Data suggest that amateur classical ballet practicing is associated to better BI and fewer behaviors for ED in the studied population. The lower BMI in ballet dancers might explain these findings, and further studies should explore these associations.

Chromatin compaction is a key biophysical property that influences multiple DNA transactions. Lack of chromatin accessibility is frequently used as proxy for chromatin compaction. However, we currently lack tools for directly probing chromatin compaction at individual genomic loci. To fill this gap, here we present FRET-FISH, a method combining fluorescence resonance energy transfer (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at select loci in single cells. We first validate FRET-FISH by comparing it with ATAC-seq, demonstrating that local compaction and accessibility are strongly correlated. FRET-FISH also detects expected differences in compaction upon treatment with drugs perturbing global chromatin condensation. We then leverage FRET-FISH to study local chromatin compaction on the active and inactive X chromosome, along the nuclear radius, in different cell cycle phases, and during increasing passage number. FRET-FISH is a robust tool for probing local chromatin compaction in single cells.

DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools, which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface (http://ifish4u.org) that can be used to design DNA FISH probes. We individually validated 153 probes in our repository, and harnessed our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics. heterologous chromosomes. Reagents SYBRTM Select Master Mix for CFX (Thermo Fisher Scientific, cat. no. 4472942) Forward primer (Integrated DNA Technologies) Reverse primer (Integrated DNA Technologies) Customized oligopool (CustomArray) Nuclease-free H2O (Thermo Fisher Scientific, cat. no. AM9932) DNA Wash Buffer (Zymo Research, cat. no. D4003-2-48) Oligo Binding Buffer (Zymo Research, cat. no. D4060-1-40) Ethanol Absolute (VMR, cat. no. 20816.367) AMPure XP (Beckman Coulter, cat. no. A63881) RNAclean XP (Beckman Coulter, cat. no. A63987) RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher Scientific, cat. no. 10777019) DNase I, RNase-free (NEB, cat. no. M0303S) HiScribeTM T7 Quick high yield RNA synthesis kit (NEB, cat. no. E2040S) dNTP mix (Thermo Fisher Scientific, cat. no. R0191) Maxima H reverse transcriptase (Thermo Fisher Scientific, cat. no. EP0751) 0.5M EDTA (Thermo Fisher Scientific, cat. no. AM9260G) 1M NaOH (Sigma, cat. no. 1.06467) Novex TBE Running buffer (Thermo Fisher Scientific, cat. no. LC6675) Qubit® dsDNA HS Assay Kit (Thermo Fisher Scientific, cat. no. Q32851) Qubit® RNA BR Assay Kit (Thermo Fisher Scientific, cat. no. Q10210) Qubit® ssDNA Assay Kit (Thermo Fisher Scientific, cat. no. Q10212) TBE-Urea 15% gel (Thermo Fisher Scientific, cat. no. EC6885BOX Equipment PCR cycler DynaMag™-2 Magnet (Thermo Fisher Scientific, cat. no. 12321D) Agencourt SPRIPlate 96 Super Magnet Plate (Beckman Coulter, cat. no. A32782) Multichannel pipette (e.g., Eppendorf Xplorer Plus, 8-channel pipette: 0.5-10 µL, cat. no. 4861000767; 5-100 µL, cat. no. 4861000783)) Tabletop centrifuge (e.g., Eppendorf 5424, cat. no. 5424000410) Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, cat. no. Q32866) Gel imager (e.g., Amersham Imager 600) Microcentrifuge tubes 0.5 ml (e.g., Eppendorf RNA/DNA LoBind) Microcentrifuge tubes 1.5 ml (e.g., Eppendorf RNA/DNA LoBind) Zymo columns IC (Zymo Research, cat. no. C1004) Collecting tube (Zymo Research, cat. no. C1001) 96-well plate (Thermo Fisher Scientific, cat. no. 4316813