Pericentromeric heterochromatin is hierarchically organized and spatially contacts H3K9me2 islands in euchromatin.

Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct territories. Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin

LMDA Review, volume 11, issue 2

Contents include: A Letter to the Membership from the LMDA Board Chair, Dixon to the Guthrie, Office Update, Technology Notes, News from CEAD Montreal, LMDA Affiliates with ATHE, ATHE 2001 Practice, Theory, Technology and the New Student, Report on Canadian Caucus, Note to the Editor, The Experiment Tradition, Community and the Development of Scripts for Stage and Screen, A Collaborative Workshop, An Intern on Internships, Spotlight on Early Career Dramaturgs, Exciting News from the West Coast, Coming this Way, and Playscripts.com.https://soundideas.pugetsound.edu/lmdareview/1021/thumbnail.jp

Oligopaints: Highly Programmable Oligonucleotide Probes for Visualizing Genomes in Situ

Fluorescence in situ hybridization (FISH) is a powerful assay that can visualize the position of DNA and RNA molecules in individual cells. Here, I describe the development of a method that utilizes complex oligonucleotide (oligo) libraries as a renewable source of FISH probes, which we term ‘Oligopaints’. Our novel FISH platform includes a reliable and robust protocol for the bulk production of fluorescently labeled, strand-specific, single-stranded DNA (ssDNA) probe sets and a bioinformatic pipeline able to identify optimal target sequences for in situ hybridization on a genome-wide scale. A key advantage of Oligopaints is that it permits the researcher to precisely define the genomic sequence contained within each probe molecule, specify the placement of fluorophores, and engineer ssDNA overhangs to which activities can be targeted. We harness this control to make two significant technological advances in FISH- based imaging. In one, Oligopaint probes are programmed to carry 5’ ssDNA overhangs that enable stochastic super-resolution microscopy via two methodologies, STORM and DNA- PAINT. We have used these probes to produce <25 nm resolution images of developmentally regulated chromatin in Drosophila and mouse, which are to our knowledge the first images at this resolution of single-copy chromosomal regions produced by FISH. In the second, we utilize single nucleotide polymorphism (SNP) data to generate FISH probes that can for the first time visually distinguish single-copy regions of the maternal and paternal homologous chromosomes, thus allowing the examination of parent-of-origin dependent effects on chromosome positioning and gene expression in individual cells

Socio-Economic Practices of Resilience in Ireland

This working paper is a qualitative analysis of socio-economic practices of resilience in Irish households. It was produced as the Irish national report for Work Package 4 (D4.19) within the FP7 Project, ‘RESCuE: Patterns of resilience during socio-economic crisis among households in Europe.’ The paper includes an overview of the socio-economic characteristics of participating households in Ireland. It discusses the narratives of hardship provided by Irish participants and considers the varying practices adopted within households to cope with hardship. It concludes with an analysis of the degrees of hardship and forms of resilience experienced and displayed across different household types, and discusses some of the limitations of the concept of resilience

Homolog separation is maintained in primordial germ cells throughout embryogenesis.

<p>A, Drosophila life-cycle. The Drosophila embryo develops through a series of synchronized, rapid divisions for the first 2.5 hours (h) after egg lay (AEL). Approximately 8–10 nuclei separate from the somatic divisions, migrate to the posterior pole of the embryo, and, following up to two further divisions, give rise to ∼15–30 primordial germ cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004013#pgen.1004013-Williamson1" target="_blank">[38]</a>. These cells will eventually produce the adult GSCs, from which haploid gametes are derived. B, Using DAPI (blue) and an antibody to the germline-specific protein VASA (red), primordial germ cells (PGCs) are identified at the posterior pole of embryos 2.5 h AEL and within the embryonic gonad 14 h AEL. Right-most column are magnified images of PGCs and somatic cells at the respective embryonic stages with FISH targeting AACAC (green). White arrowheads denote PGC loci and orange arrowheads denote somatic loci. Scale bars represent 10 µm. C–D, Percentage of pairing in embryos 2.5 h (C) and 14 h (D) AEL within somatic and PGC nuclei (n.s. not significant, *p<0.05, **p<0.0001). The chromatin state (Het, heterochromatin, or Eu, euchromatin), and chromosome are noted below each FISH target. For each data point, 46–98 nuclei were scored from a total of 6–7 embryos (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004013#s3" target="_blank">Materials and Methods</a>).</p

Germline progenitors contain large nuclear volumes with chromosomes juxtaposed to the nuclear envelope.

<p>A, PGC nuclei (VASA, red) are larger than surrounding somatic nuclei in embryonic gonads 14 h AEL. DAPI, blue. Dashed circles denote nuclear periphery. Scale bar represents 10 µm. Right: Average nuclear volume of PGCs and surrounding somatic cells ± SEM. B, Average nuclear volume of germline and somatic follicle cells of the adult ovary ± SEM (**p<0.0001). C, Wild-type germarium stained for DNA (grey) and SXL (green). Shown on right are cross-sections of representative GSC and somatic nuclei with 3D and 2D (insets) surface plots displaying increased peripheral intensity in the nucleus of the GSC and more uniform intensity in the nucleus of the somatic cell. Scale bar represents 10 µm in the image of the germarium and 5 µm in images of single nuclei. D, Left: GSC nucleus with FISH targeting 24D (red) and lamin staining (nuclear envelope, green). Scale bar represents 5 µm. Right: Average distance between FISH signals and the nuclear envelope (NE) ± SEM, normalized to the nuclear radius, in germline and somatic follicle cells of the adult ovary. Asterisks denote significant differences in the normalized distances between somatic and GSCs (*p<0.05, **p<0.0001). For each data point, a minimum number of 30 nuclei were scored (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004013#s3" target="_blank">Materials and Methods</a>).</p

Homologous chromosomes enter the germline unpaired.

<p>A, Left: Schematic of a germarium showing pre-meiotic mitotic cell divisions as well as maturation of the meiotic cysts. The GSCs (purple nuclei) are positioned adjacent to the somatic niche (brown) and express high levels of SXL (green cytoplasm). Each GSC divides asymmetrically to produce a renewed stem cell and a differentiating cystoblast (CB, blue nucleus surrounded by green cytoplasm), which is positioned distal to the niche. The CB will undergo four more rounds of mitotic divisions to form a 16-cell cyst. Following these pre-meiotic stages, the 16-cell cyst will enter meiotic prophase, as defined by the initiation (zygotene) and complete formation (pachytene) of the synaptonemal complex (SC, red) between the paired homologs in two of the sixteen cells. Only a single cell will complete meiosis within each 16-cell cyst to form a mature egg (not shown) Arrow, direction of maturation. Right: Wild-type germarium stained for DNA (blue) and SXL (green). A GSC and CB are indicated by arrows and identified by SXL staining and relative position to somatic niche. Approximately 1–2 GSCs and 1–2 CBs are present in each germarium. Scale bar represents 10 µm. B, Drosophila chromosomes and targets of FISH probes (red). Heterochromatin is denoted in grey and rDNA cluster on the X-chromosome is in purple. C, Image of a GSC nucleus (dashed circle) at the tip of a germarium identified by DAPI (blue) surrounded by cytoplasmic SXL (green) staining and combined with FISH targeting AACAC (red) and dodeca (grey). Two signals for each FISH target represent separated homologous loci. Scale bar represents 5 µm. D, Percentage of nuclei exhibiting paired and unpaired loci in GSCs (left panel) and CBs (right panel). 15–30 ovaries were scored for each stage with a combined total of 242 GSC nuclei and 262 CB nuclei (approximately 30 nuclei for each locus at each stage). E, CB nuclei identified with SXL staining in combination with two-color FISH targeting AACAC (grey) and 24D (red) on Chromosome 2. Cartoon depicts hypothetical arrangement of homologous chromosomes as either unpaired or partially paired. Scale bars represents 5 µm.</p

Homolog pairing is established during the mitotic cell cycles prior to meiosis.

<p>A, Left: Schematic of a germarium identifying the pre-meiotic stages with BAM (red) and Spectrin (white) and meiotic stages with C(3)G (green). Right: Wild-type germarium in which GSCs are identified by the position near the niche, absence of BAM staining, and presence of a spectrosome (white). Developing cysts are identified by the presence of BAM staining and a branched fusome (white). DAPI, blue. Approximately 1–2 germline cysts are present in each germarium, with equal occurrence of the 2-, 4-, 8-, and 16-cell stages. Scale bar represents 10 µm. B, FISH targeting dodeca (grey). 2-cell and 4-cell cysts (the 4^th cell is out of focus) identified with BAM∶GFP (pseudo-colored red). Scale bars represent 10 µm. C, FISH targeting 24D (red) and AACAC (grey) in a germarium identifying pachytene nuclei in meiosis with an antibody against the SC protein C(3)G (green). Scale bar represents 10 µm in upper panel and 5 µm in lower panel. D, Percentage of nuclei exhibiting paired and unpaired loci in pre-meiotic stages as well as in meiotic pachytene with FISH targeting AACAC, dodeca, 5A, 24D, 69C, and 100B. Pre-meiotic cysts were identified using BAM∶GFP and Spectrin. Pachytene nuclei were identified in a separate experiment using an antibody against C(3)G. 15–30 ovaries were scored for each stage with a minimum of 20 nuclei counted for 2- and 4-cell stages, 40 nuclei for the 8-cell stage, and 80 nuclei for the 16-cell stage (*<i>P</i><0.01, **<i>P</i><0.0001).</p