DNA FISH Research Articles

Comparative performance of methylation DNA markers, brushing cytology, and FISH in diagnosing malignant biliary strictures.

Comparative performance of methylation DNA markers, brushing cytology, and FISH in diagnosing malignant biliary strictures.

High-throughput image processing software for the study of nuclear architecture and gene expression

High-throughput imaging (HTI) generates complex imaging datasets from a large number of experimental perturbations. Commercial HTI software programs for image analysis workflows typically do not allow full customization and adoption of new image processing algorithms in the analysis modules. While open-source HTI analysis platforms provide individual modules in the workflow, like nuclei segmentation, spot detection, or cell tracking, they are often limited in integrating novel analysis modules or algorithms. Here, we introduce the High-Throughput Image Processing Software (HiTIPS) to expand the range and customization of existing HTI analysis capabilities. HiTIPS incorporates advanced image processing and machine learning algorithms for automated cell and nuclei segmentation, spot signal detection, nucleus tracking, nucleus registration, spot tracking, and quantification of spot signal intensity. Furthermore, HiTIPS features a graphical user interface that is open to integration of new analysis modules for existing analysis pipelines and to adding new analysis modules. To demonstrate the utility of HiTIPS, we present three examples of image analysis workflows for high-throughput DNA FISH, immunofluorescence (IF), and live-cell imaging of transcription in single cells. Altogether, we demonstrate that HiTIPS is a user-friendly, flexible, and open-source HTI software platform for a variety of cell biology applications.

SnapFISH-IMPUTE: an imputation method for multiplexed DNA FISH data

Chromatin spatial organization plays a crucial role in gene regulation. Recently developed and prospering multiplexed DNA FISH technologies enable direct visualization of chromatin conformation in the nucleus. However, incomplete data caused by limited detection efficiency can substantially complicate and impair downstream analysis. Here, we present SnapFISH-IMPUTE that imputes missing values in multiplexed DNA FISH data. Analysis on multiple published datasets shows that the proposed method preserves the distribution of pairwise distances between imaging loci, and the imputed chromatin conformations are indistinguishable from the observed conformations. Additionally, imputation greatly improves downstream analyses such as identifying enhancer-promoter loops and clustering cells into distinct cell types. SnapFISH-IMPUTE is freely available at https://github.com/hyuyu104/SnapFISH-IMPUTE.

Chromosome segregation during spermatogenesis occurs through a unique center-kinetic mechanism in holocentric moth species.

Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the center of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments co-orienting these separated kinetochores. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.

3D genomic analysis reveals novel enhancer-hijacking caused by complex structural alterations that drive oncogene overexpression.

Cancer genomes are composed of many complex structural alterations on chromosomes and extrachromosomal DNA (ecDNA), making it difficult to identify non-coding enhancer regions that are hijacked to activate oncogene expression. Here, we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking. HAPI analysis of HiChIP data from 34 cancer cell lines identified enhancer hijacking events that activate both known and potentially novel oncogenes such as MYC, CCND1 , ETV1 , CRKL , and ID4 . Furthermore, we found enhancer hijacking among multiple oncogenes from different chromosomes, often including MYC , on the same complex amplicons such as ecDNA. We characterized a MYC - ERBB2 chimeric ecDNA, in which ERBB2 heavily hijacks MYC 's enhancers. Notably, CRISPRi of the MYC promoter led to increased interaction of ERBB2 with MYC enhancers and elevated ERBB2 expression. Our HAPI analysis tool provides a robust strategy to detect enhancer hijacking and reveals novel insights into oncogene activation.

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.

Survival implications and radiographic predictors of prognosis in patients with IDH-mutant astrocytoma with CDKN2A/b homozygous deletion: An international, multi-centered study.

e14035 Background: Isocitrate Dehydrogenase (IDH) 1 or 2 mutations are associated with improved prognosis in glioma patients. Despite their less aggressive clinical course when compared to IDH-wild type gliomas, molecularly-defined IDH-mutant glioma subtypes with poor prognosis have been identified. Among these, CDKN2A/B homozygous deletion represents the strongest negative genetic predictor of prognosis yet identified in IDH-mutant astrocytoma. With the WHO 2021 classification recognizing CDKN2A/B homozygous deletion as sufficient for the designation of astrocytoma, IDH-mutant, WHO Grade 4, the translational implications and potential for therapies targeting CDKN2A/B homozygous deletion are an area of active investigation. We sought to further characterize overall (OS) and progression-free survival (PFS) of CDKN2A/B deleted patients within a large, multi-centered, international cohort and identify clinical characteristics that predict survival for patients with CDKN2A/B deletion. Methods: Patients with IDH-mutant glioma with CDKN2A/B homozygous deletion were retrospectively identified across four institutions (Massachusetts General Hospital, USA; University Hospital Heidelberg, Germany; University Hospital Dresden, Germany, and Foudan-Shanghai, China). IDH-mutation status was determined by immunohistochemistry or next-generation sequencing. CDKN2A/B status was determined by hybridized DNA capture or FISH. Results: We identified 75 patients with IDH-mutant astrocytoma with CDKN2A/B homozygous deletion. The cohort had a median follow-up time of 5.0 years, was 56% male, and had an average age of 38.8 years. Patients were treated with chemotherapy and radiation following initial surgery in 77% of cases. Preoperative contrast enhancement and T2 FLAIR mismatch were identified in 56 and 44% of cases, respectively. Compared to a cohort of 51 patients with CDKN2A/B intact, grade 4, IDH-mutant astrocytoma, those with CDKN2A/B-deleted tumors had significantly shorter median OS and PFS (OS: 4 vs 13.5 years, HR = 6.8, HR 95% CI = 3.1, 14.6, p < 0.0001; PFS: 2.8 vs 5.9 years, HR = 3.0, HR 95% CI = 1.7, 5.2, p = 0.0001). Within the CDKN2A/B deleted cohort, preoperative contrast enhancement predicted worse OS, but not PFS (OS: HR = 2.7, 95% CI = 1.1, 7.4, p = 0.04; PFS: HR = 1.17, 95% CI = 0.7, 3.7, p = 0.24). Conclusions: These data reinforce the strong negative prognostic impact of CDKN2A/B homozygous deletion on OS and PFS and identify a novel association between preoperative contrast enhancement and shorter OS in patients with CDKN2A/Bdeleted, IDH-mutant astrocytoma.

Abstract 3501: Concurrent amplification of TBXT and highly activated enhancers in extrachromosomal DNA (ecDNA) drives chordoma tumorigenesis

Abstract Chordoma, a rare bone cancer characterized by limited actionable clinical targets, exhibits a pivotal hallmark in the activation of the developmental transcription factor gene TBXT (Brachyury) through its association with highly active enhancers. TBXT amplification emerges as a major causative factor in familial chordomas, yet the amplification mechanism and its consequential impact on genomic instability remain inadequately elucidated. In this study, we conducted a comprehensive analysis of multiple genomic and epigenomic sequencing datasets, including WGS-seq, H3K27ac ChIP-seq, and ATAC-seq in six chordoma cell lines, and HiC data in two of them, to unravel the mechanisms of structural variations and their biological implications. Notably, our focus extends to JHC7 cells, which exhibit extrachromosomal amplification of a 2.4M region encompassing TBXT and the associated enhancers. The existence of such extrachromosomal DNA (ecDNA) in JHC7 was further validated through DNA FISH and Sanger sequencing. Comparing JHC7 with CH22, a chordoma cell line lacking TBXT amplification, revealed heightened chromatin relaxation and increased DNA rearrangements in JHC7. Interestingly, FISH results showed significant genomic ploidy heterogeneity within JHC7 cells, a finding demonstrated by nucleus FACS sorting. Single-cell multiomic analysis, combining snRNA-seq and snATAC-seq, demonstrated heterogeneous gene expression and distinct copy number variations among JHC7 cells. However, the amplification of the TBXT locus was observed in nearly all cells examined, indicating the rapid genomic evolution in the cultured JHC7 cells. These results collectively show that TBXT is extensively amplified through the formation of ecDNA, providing a fitness advantage to chordoma cells and concurrently exacerbating genomic instability. The findings in this study provide an insight for understanding the pathogenesis of chordoma and may inform the development of targeted therapeutic strategies. Citation Format: Bo Zhao, Martin Barrio Ines, Lingqun Ye, Xizi Wu, Alice Soragni, Andrew Futreal, Kadir Akdemir. Concurrent amplification of TBXT and highly activated enhancers in extrachromosomal DNA (ecDNA) drives chordoma tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3501.

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

Abstract 6599: 3D genomic analysis reveals novel enhancer hijacking mechanisms caused by complex structural alterations that drive oncogene overexpression

Abstract Enhancer hijacking, caused by structural alterations on chromosomes as well as extrachromosomal DNA (ecDNA), is a common cancer driver event. The complexity and ubiquity of structural alterations in cancer genomes make it difficult to identify enhancer hijacking with genome sequencing alone. Here we describe a 3D genomics-based analysis called HAPI (Highly Active Promoter Interactions) to characterize enhancer hijacking caused by various types of structural alterations. Applying HAPI analysis to HiChIP data from 34 cancer cell lines, we identified novel enhancers hijacked through chromosomal rearrangements to activate both known and potentially novel oncogenes such as MYC, CCND1, ETV1, CRKL, and ID4, which we validated using CRISPRi assays and RNA-seq analysis. Furthermore, we found that ecDNAs often contain multiple oncogenes from different chromosomes, which cause nested enhancer hijacking among them. For instance, we found that MYC ecDNAs relocate additional oncogenes from other chromosomes such as CDX2, ERBB2, or CD44 near the MYC locus, co-opting MYC’s enhancers for their overexpression, which we validated using dual-color DNA FISH and CRISPRi assays. This multiple oncogenes-involved enhancer hijacking mechanism may suggest novel therapeutic strategies such as targeting either the co-opting oncogenes or the hijacked enhancers for ecDNAs. Our study provides a robust strategy to detect enhancer hijacking events using our publicly available HAPI analysis tool and reveals novel mechanisms underlying oncogene activation caused by chromosomal and extrachromosomal structural alterations. Citation Format: Katelyn L. Mortenson, Courtney Dawes, Emily R. Wilson, Nathan E. Patchen, Hailey Johnson, Jason Gertz, Swneke D. Bailey, Yan Liu, Katherine E. Varley, Xiaoyang Zhang. 3D genomic analysis reveals novel enhancer hijacking mechanisms caused by complex structural alterations that drive oncogene overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6599.

Environmental DNA (eDNA) metabarcoding and fish visual census reveals the first record of Doboatherina magnidentata in the Philippines

Abstract. Bautista JA, Manubag JJ, Sumaya NH, Martinez JG, Tabugo SR. 2023. Environmental DNA (eDNA) metabarcoding and fish visual census reveals the first record of Doboatherina magnidentatain the Philippines. Biodiversitas 24: 3063-3072. Biodiversity monitoring is the cornerstone for conserving marine fish species. However, classical methods, like the Fish Visual Census (FVC), are often limited due to sampling difficulties, the occurrence of rare and cryptic organisms, and reliance on a taxonomic expert for species identification. Recently, environmental DNA (eDNA) metabarcoding has been suggested as a non-invasive, powerful tool for biomonitoring. This study evaluates the eDNA approach as complementary tool for the FVC data in species detection and identification of important marine fishes from the marine sanctuary of Dalipuga, Iligan City, Philippines. The findings obtained through the eDNA approach provide insights into identifying significant fish species. Notably, the presence of the Hippocampus kuda Bleeker, 1852 (yellow seahorse), categorized as a vulnerable and threatened species, was detected. Additionally, the study identified Herklotsichthys quadrimaculatus Rüppell, 1837 (bluestripe herring), a native species to the Philippines that may pose potential risks to humans and the ecological balance. Furthermore, two demersal fish species, namely Large-scale whiting (Sillaginops macrolepis Bleeker, 1858) and Large-scale mullet (Planiliza macrolepis Smith, 1846), were also detected. The eDNA approach also delineated the morphologically cryptic fishes from Scaridae (parrotfishes) and Mugilidae (mullet fish) taxa to the species level. The highlight of this study was the detectionof the new Indo-pacific atherinomorine fish species Doboatherina magnidentata, which to the best of our knowledge, was the first record in the Philippine marine waters. Despite the efficiency of the eDNA metabarcoding in fish species detection and identification, the viability of eDNA in the marine environment and biases of the primer limit this method. Thus, the classical method must complement the molecular approach for better taxonomic resolution and community analysis. Future studies were also recommended to use a multigene eDNA approach to improve taxonomic sensitivity and reduce primer biases.

UNUSUAL COLOR OF URINE AND SERUM, SHOULD ONE INVESTIGATE THE CAUSE OR NOT?

Laboratory diagnosis has become the cornerstone for all clinical diagnosis and treatment, with the advent of genetic workups like DNA sequencing and FISH prognosis of diseases also coming under the umbrella of lab tests. However, the time-tested methods for the analysis of samples before processing (which in the era of accreditation comes under the pre-analytical phase) are still very important in suspecting if not diagnosing disorders apart from preventing pre-analytical errors because of hemolysis, lipemia, QNS, mislabeling, etc. A Yellow serum sample in a rack of tubes is quite common in the laboratory which suggests hyperbilirubinemia. We here like to report a case of 12 years old female child who presented for routine blood and urine testing as she was suffering from low grazed fever, abdominal pain, and loose stool. One look per se the picture favors an infective etiology. But on a collection of specimens the serum turned out to be greenish and the urine too was dark green in color (Fig 1&2) So the questions that we tried to answer by this article are what caused this change in plasma color? what other conditions could cause blood discoloration? And are there concerns or possible interference with laboratory tests? On reviewing the literature, we came across various causes of green discoloration of urine, and interferences encountered routinely which we have discussed in our article. On follow up the child was all right after treatment and has no further episodes of such discoloration

The nematode Oscheius tipulae as a genetic model for programmed DNA elimination

Programmed DNA elimination (PDE) is a notable exception to the paradigm of genome integrity. In metazoa, PDE often occurs coincident with germline to somatic cell differentiation. During PDE, portions of genomic DNA are lost, resulting in reduced somatic genomes. Prior studies have described the sequences lost, as well as chromosome behavior, during metazoan PDE. However, a system for studying the mechanisms and consequences of PDE in metazoa is lacking. Here, we present a functional and genetic model for PDE in the free-living Rhabditidae nematode Oscheius tipulae, a family that also includes Caenorhabditis elegans. O.tipulae was recently suggested to eliminate DNA. Using staged embryos and DNA FISH, we showed that O.tipulae PDE occurs during embryogenesis at the 8-16 cell stages. We identified a conserved motif, named Sequence For Elimination (SFE), for all 12 break sites on the six chromosomes at the junctions of retained and eliminated DNA. SFE mutants exhibited a "fail-to-eliminate" phenotype only at the modified sites. END-seq revealed that breaks can occur at multiple positions within the SFE, with extensive end resection followed by telomere addition to both retained and eliminated ends. We identified many functional SFEs at the chromosome ends through END-seq in the wild-type embryos, genome sequencing of SFE mutants, and comparative genomics of 23 wild isolates. We suggest that these alternative SFEs provide flexibility in the sequences eliminated and a fail-safe mechanism for PDE. These studies establish O.tipulae as a new, attractive model for studying the mechanisms and consequences of PDE in a metazoan.

ALL-268 Genetic Classification of B-Cell Precursor Adult Acute Lymphoblastic Leukemia Patients Enrolled in LAL19 Trial from the Pethema Group: Response to Treatment and Survival

B-cell precursor acute lymphoblastic leukemia (BCP ALL) is a genetically heterogeneous neoplasm with >20 biologic subtypes. Each subtype shows specific genetic traits that determine relapse risk and patients' survival. To establish the genetic subtype (primary alteration) of adult BCP ALL patients enrolled in the PETHEMA LAL19 trial (NCT04179929) and to correlate them with measurable residual disease (MRD) level and survival. In the LAL19 trial (NCT04179929), Ph-negative patients (18-65 y) with MRD≥0.01% at day+35 or high-risk genetics receive alloHSCT and MRD<0.01% patients with standard-risk genetics receive maintenance chemotherapy. The genetic analyses are centralized: FISH and NGS DNA panel (Hospital de Salamanca), RNAseq panel (Hospital 12 de Octubre), FISH panel (Hospital La Fe), and SNP array (Josep Carreras Institute/ICO-Hospital Germans Trias i Pujol). MRD determinations are centrally done by next-generation flow cytometry in the Cytometry Service, NUCLEUS, University of Salamanca. The genetic subtype was identified in 54% (82/152) of patients. The most recurrent subtypes were KMT2Ar (11%), Ph-like (mostly CRLF2::IGH, 11%), low-hypodiploid (7%), PAX5 P80R (7%), high-hyperdiploid (6%), and t(1;19)/TCF3::PBX1 (6%). In addition, t(12;21)/ETV6::RUNX1, ZNF384r, and iAMP21 subtypes (1.5% each) and MEF2Dr, MYCr, IDH1 R132 subtypes (<1% each) were found. Regarding secondary alterations, NRAS (15%), TP53 (13%), PAX5 (13%), and KRAS (10%) mutations were the most frequently observed. Twelve patients were refractory (mainly low-hypodiploid, Ph-like, MYCr, and B-other/unclassified patients). Statistically significant differences were observed for day+35 MRD levels between genetic subtypes. Ph-like, low-hypodiploid, and KMT2Ar showed lower frequencies of MRD<0.01% (17%, 33%, and 57%, respectively) than patients with PAX5P80R (100%), t(1;19)/TCF3::PBX1 (83%), and high-hyperdiploid (75%) (P=0.006). Despite the short median follow-up (11 months), differences in response to treatment were reflected in patients' survival. Significant differences in survival were observed between poor-response subtypes (Ph-like, KMT2Ar, and low-hypodiploid) and good-response subtypes (PAX5 P80R, t(1;19)/TCF3::PBX1, and high-hyperdiploid). Knowing the genetic subtype of each ALL is crucial to better predict relapse risk and offer the best (personalized) treatment for each patient.

NODeJ: an ImageJ plugin for 3D segmentation of nuclear objects

BackgroundThe three-dimensional nuclear arrangement of chromatin impacts many cellular processes operating at the DNA level in animal and plant systems. Chromatin organization is a dynamic process that can be affected by biotic and abiotic stresses. Three-dimensional imaging technology allows to follow these dynamic changes, but only a few semi-automated processing methods currently exist for quantitative analysis of the 3D chromatin organization.ResultsWe present an automated method, Nuclear Object DetectionJ (NODeJ), developed as an imageJ plugin. This program segments and analyzes high intensity domains in nuclei from 3D images. NODeJ performs a Laplacian convolution on the mask of a nucleus to enhance the contrast of intra-nuclear objects and allow their detection. We reanalyzed public datasets and determined that NODeJ is able to accurately identify heterochromatin domains from a diverse set of Arabidopsis thaliana nuclei stained with DAPI or Hoechst. NODeJ is also able to detect signals in nuclei from DNA FISH experiments, allowing for the analysis of specific targets of interest.Conclusion and availabilityNODeJ allows for efficient automated analysis of subnuclear structures by avoiding the semi-automated steps, resulting in reduced processing time and analytical bias. NODeJ is written in Java and provided as an ImageJ plugin with a command line option to perform more high-throughput analyses. NODeJ can be downloaded from https://gitlab.com/axpoulet/image2danalysis/-/releases with source code, documentation and further information avaliable at https://gitlab.com/axpoulet/image2danalysis. The images used in this study are publicly available at https://www.brookes.ac.uk/indepth/images/ and https://doi.org/10.15454/1HSOIE.

Environmental DNA persistence and fish detection in captive sponges

Environmental DNA persistence and fish detection in captive sponges

Environmental DNA persistence and fish detection in captive sponges

Environmental DNA persistence and fish detection in captive sponges

Enhancer of trithorax/polycomb, Corto, regulates timing of hunchback gene relocation and competence in Drosophila neuroblasts

BackgroundNeural progenitors produce diverse cells in a stereotyped birth order, but can specify each cell type for only a limited duration. In the Drosophila embryo, neuroblasts (neural progenitors) specify multiple, distinct neurons by sequentially expressing a series of temporal identity transcription factors with each division. Hunchback (Hb), the first of the series, specifies early-born neuronal identity. Neuroblast competence to generate early-born neurons is terminated when the hb gene relocates to the neuroblast nuclear lamina, rendering it refractory to activation in descendent neurons. Mechanisms and trans-acting factors underlying this process are poorly understood. Here we identify Corto, an enhancer of Trithorax/Polycomb (ETP) protein, as a new regulator of neuroblast competence.MethodsWe used the GAL4/UAS system to drive persistent misexpression of Hb in neuroblast 7–1 (NB7-1), a model lineage for which the early competence window has been well characterized, to examine the role of Corto in neuroblast competence. We used immuno-DNA Fluorescence in situ hybridization (DNA FISH) in whole embryos to track the position of the hb gene locus specifically in neuroblasts across developmental time, comparing corto mutants to control embryos. Finally, we used immunostaining in whole embryos to examine Corto’s role in repression of Hb and a known target gene, Abdominal B (Abd-B).ResultsWe found that in corto mutants, the hb gene relocation to the neuroblast nuclear lamina is delayed and the early competence window is extended. The delay in gene relocation occurs after hb transcription is already terminated in the neuroblast and is not due to prolonged transcriptional activity. Further, we find that Corto genetically interacts with Posterior Sex Combs (Psc), a core subunit of polycomb group complex 1 (PRC1), to terminate early competence. Loss of Corto does not result in derepression of Hb or its Hox target, Abd-B, specifically in neuroblasts.ConclusionsThese results show that in neuroblasts, Corto genetically interacts with PRC1 to regulate timing of nuclear architecture reorganization and support the model that distinct mechanisms of silencing are implemented in a step-wise fashion during development to regulate cell fate gene expression in neuronal progeny.

High-Throughput DNA FISH (hiFISH).

High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence.

Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPβ signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from 'permissive' H3K9me3 regions in senescent cells.