Unraveling the three-dimensional (3D) genome architecture in Neurodevelopmental Disorders (NDDs).

The human genome, comprising millions of pairs of bases, serves as the blueprint of life, encoding instructions for cellular processes. However, genomes are not merely linear sequences; rather, the complex of DNA and histones, known as chromatin, exhibits complex organization across various levels, which profoundly influence gene expression and cellular function. Central to understanding genome organization is the emerging field of three-dimensional (3D) genome studies. Utilizing advanced techniques such as Hi-C, researchers have unveiled non-random dispositions of genomic elements, highlighting their importance in transcriptional regulation and disease mechanisms. Topologically Associating Domains (TADs), that demarcate regions of chromatin with preferential internal interactions, play crucial roles in gene regulation and are increasingly implicated in various diseases such as cancer and schizophrenia. However, their role in Neurodevelopmental Disorders (NDDs) remains poorly understood. Here, we focus on TADs and 3D conservation across the evolution and between cell types in NDDs. The investigation into genome organization and its impact on disease has led to significant breakthroughs in understanding NDDs etiology such ASD (Autism Spectrum Disorder). By elucidating the wide spectrum of ASD manifestations, researchers aim to uncover the underlying genetic and epigenetic factors contributing to its heterogeneity. Moreover, studies linking TAD disruption to NDDs underscore the importance of spatial genome organization in maintaining proper brain development and function. In summary, this review highlights the intricate interplay between genome organization, transcriptional control, and disease pathology, shedding light on fundamental biological processes and offering insights into the mechanisms underlying NDDs like ASD.

Rice RS2-9, which is bound by transcription factor OSH1, blocks enhancer-promoter interactions in plants.

Insulators characterized in Drosophila and mammals have been shown to play a key role in the restriction of promiscuous enhancer-promoter interactions, as well as reshaping the topological landscape of chromosomes. Yet the role of insulators in plants remains poorly understood, in large part because of a lack of well-characterized insulators and binding factor(s). In this study, we isolated a 1.2-kb RS2-9 insulator from the Oryza sativa (rice) genome that can, when interposed between an enhancer and promoter, efficiently block the activation function of both constitutive and floral organ-specific enhancers in transgenic Arabidopsis and Nicotiana tabacum (tobacco). In the rice genome, the genes flanking RS2-9 exhibit an absence of mutual transcriptional interactions, as well as a lack of histone modification spread. We further determined that O. sativa Homeobox 1 (OSH1) bound two regions of RS2-9, as well as over 50 000 additional sites in the rice genome, the majority of which resided in intergenic regions. Mutation of one of the two OSH1-binding sites in RS2-9 impaired insulation activity by up to 60%, whereas the mutation of both binding sites virtually abolished insulator function. We also demonstrated that OSH1 binding sites were associated with 72% of the boundaries of topologically associated domains (TADs) identified in the rice genome, which is comparable to the 77% of TAD boundaries bound by the insulator CCCTC-binding factor (CTCF) in mammals. Taken together, our findings indicate that OSH1-RS2-9 acts as a true insulator in plants, and highlight a potential role for OSH1 in gene insulation and topological organization in plant genomes.

Breakpoint analysis for cytogenetically balanced translocation revealed unexpected complex structural abnormalities and suggested the position effect for MEF2C.

Many disease-causing genes have been identified by determining the breakpoints of balanced chromosomal translocations. Recent progress in genomic analysis has accelerated the analysis of chromosomal translocation-breakpoints at the nucleotide level. Using a long-read whole-genome sequence, we analyzed the breakpoints of the cytogenetically balanced chromosomal translocation t(5;15)(q21;26.3), which was confirmed to be of de novo origin, in a patient with a neurodevelopmental disorder. The results showed complex rearrangements with seven fragments consisting of five breakpoint-junctions (BJs). Four of the five BJs showed microhomologies of 1-3-bp, and only one BJ displayed a signature of blunt-end ligation, indicating chromothripsis as the underlying mechanism. Although the BJs did not disrupt any disease-causing gene, the clinical features of the patient were compatible with MEF2C haploinsufficiency syndrome. Complex rearrangements were located approximately 2.5-Mb downstream of MEF2C. Therefore, position effects were considered the mechanism of the occurrence of MEF2C haploinsufficiency syndrome.

To be or not be (in the LAD): emerging roles of lamin proteins in transcriptional regulation.

Lamins are components of the nuclear lamina, a protein meshwork that underlies the nuclear membrane. Lamins interact with chromatin in transcriptionally silent regions defined as lamina-associated-domains (LADs). However, recent studies have shown that lamins regulate active transcription inside LADs. In addition, ChIP-seq analysis has shown that lamins interact with lamin-dependent promoters and enhancers located in the interior of the nucleus. Moreover, functional studies suggest that lamins regulate transcription at associated-promoters and long-range chromatin interactions of key developmental gene programs. This review will discuss emerging, non-canonical functions of lamins in controlling non-silent genes located both inside and outside of LADs, focusing on transcriptional regulation and chromatin organization in Drosophila and mammals as metazoan model organisms.

Fine Breakpoint Mapping by Genome Sequencing Reveals the First Large X Inversion Disrupting the NHS Gene in a Patient with Syndromic Cataracts.

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.

Tidying-up the plant nuclear space: domains, functions, and dynamics.

Understanding how the packaging of chromatin in the nucleus is regulated and organized to guide complex cellular and developmental programmes, as well as responses to environmental cues is a major question in biology. Technological advances have allowed remarkable progress within this field over the last years. However, we still know very little about how the 3D genome organization within the cell nucleus contributes to the regulation of gene expression. The nuclear space is compartmentalized in several domains such as the nucleolus, chromocentres, telomeres, protein bodies, and the nuclear periphery without the presence of a membrane around these domains. The role of these domains and their possible impact on nuclear activities is currently under intense investigation. In this review, we discuss new data from research in plants that clarify functional links between the organization of different nuclear domains and plant genome function with an emphasis on the potential of this organization for gene regulation.

Identification of OAF and PVRL1 as candidate genes for an ocular anomaly characterized by Peters anomaly type 2 and ectopia lentis.

Keratolenticular dysgenesis (KLD) and ectopia lentis are congenital eye defects. The aim of this study is the identification of molecular genetic alterations responsible for those ocular anomalies with neurologic impairment in an individual with a de novo balanced chromosome translocation t(11;18)(q23.3;q11.2)dn. Disruption of OAF, the human orthologue of the Drosophila oaf, by the 11q23.3 breakpoint results in reduced expression of this transcriptional regulator. Furthermore, four most likely nonfunctional chimeric transcripts comprising up to OAF exon 3, derived from the der(11) allele, have also been identified. This locus has been implicated by publicly available genome-wide association data in corneal disease and corneal topography. The expression of the poliovirus receptor-related 1(PVRL1) or nectin cell adhesion molecule 1 (NECTIN1), a paralogue of nectin cell adhesion molecule 3 (PVRL3) associated with congenital ocular defects, situated 500 kb upstream from 11q23.3 breakpoint, is increased. The 18q11.2 breakpoint is localized between cutaneous T-cell lymphoma-associated antigen 1(CTAGE1) and retinoblastoma binding protein 8 (RBBP8) genes. Genomic imbalance that could contribute to the observed phenotype was excluded. Analysis of gene expression datasets throughout normal murine ocular lens embryogenesis suggests that OAF expression is significantly enriched in the lens from early stages of development through adulthood, whereas PVRL1 is lens-enriched until E12.5 and then down-regulated. This contrasts with the observation that the proposita's lymphoblastoid cell lines exhibit low OAF and high PVRL1 expression as compared to control, which offers further support that the alterations described above are most likely responsible for the clinical phenotype. Finally, gene interaction topology data for PVRL1 also agree with our proposal that disruption of OAF by the translocation breakpoint and misregulation of PVRL1 due to a position effect contribute to the observed ocular and neurological phenotype.

Transcriptomic study reveals lncRNA-mediated downregulation of innate immune and inflammatory response in the SARS-CoV-2 vaccination breakthrough infections.

Introduction: The emergence of multiple variants of concerns (VOCs) with higher number of Spike mutations have led to enhanced immune escape by the SARS-CoV-2. With the increasing number of vaccination breakthrough (VBT) infections, it is important to understand the possible reason/s of the breakthrough infections. Methods: We performed transcriptome sequencing of 57 VBT and unvaccinated COVID-19 patients, followed by differential expression and co-expression analysis of the lncRNAs and the mRNAs. The regulatory mechanism was highlighted by analysis towards repeat element distribution within the co-expressed lncRNAs, followed by repeats driven homologous interaction between the lncRNAs and the promoter regions of genes from the same topologically associated domains (TAD). Results: We identified 727 differentially expressed lncRNAs (153 upregulated and 574 downregulated) and 338 mRNAs (34 up- and 334 downregulated) in the VBT patients. This includes LUCAT1, MALAT1, ROR1-AS1, UGDH-AS1 and LINC00273 mediated modulation of immune response, whereas MALAT1, NEAT1 and GAS5 regulated inflammatory response in the VBT. LncRNA-mRNA co-expression analysis highlighted 34 lncRNAs interacting with 267 mRNAs. We also observed a higher abundance of Alu, LINE1 and LTRs within the interacting lncRNAs of the VBT patients. These interacting lncRNAs have higher interaction with the promoter region of the genes from the same TAD, compared to the non-interacting lncRNAs with the enrichment of Alu and LINE1 in the gene promoter. Discussion: Significant downregulation and GSEA of the TAD gene suggest Alu and LINE1 driven homologous interaction between the lncRNAs and the TAD genes as a possible mechanism of lncRNA-mediated suppression of innate immune/inflammatory responses and activation of adaptive immune response. The lncRNA-mediated suppression of innate immune/inflammatory responses and activation of adaptive immune response might explain the SARS-CoV-2 breakthrough infections with milder symptoms in the VBT. Besides, the study also highlights repeat element mediated regulation of genes in 3D as another possible way of lncRNA-mediated immune-regulation modulating vaccination breakthroughs milder disease phenotype and shorter hospital stay.

Prolonged FOS activity disrupts a global myogenic transcriptional program by altering 3D chromatin architecture in primary muscle progenitor cells.

BACKGROUND: The AP-1 transcription factor, FBJ osteosarcoma oncogene (FOS), is induced in adult muscle satellite cells (SCs) within hours following muscle damage and is required for effective stem cell activation and muscle repair. However, why FOS is rapidly downregulated before SCs enter cell cycle as progenitor cells (i.e., transiently expressed) remains unclear. Further, whether boosting FOS levels in the proliferating progeny of SCs can enhance their myogenic properties needs further evaluation. METHODS: We established an inducible, FOS expression system to evaluate the impact of persistent FOS activity in muscle progenitor cells ex vivo. We performed various assays to measure cellular proliferation and differentiation, as well as uncover changes in RNA levels and three-dimensional (3D) chromatin interactions. RESULTS: Persistent FOS activity in primary muscle progenitor cells severely antagonizes their ability to differentiate and form myotubes within the first 2 weeks in culture. RNA-seq analysis revealed that ectopic FOS activity in muscle progenitor cells suppressed a global pro-myogenic transcriptional program, while activating a stress-induced, mitogen-activated protein kinase (MAPK) transcriptional signature. Additionally, we observed various FOS-dependent, chromosomal re-organization events in A/B compartments, topologically associated domains (TADs), and genomic loops near FOS-regulated genes. CONCLUSIONS: Our results suggest that elevated FOS activity in recently activated muscle progenitor cells perturbs cellular differentiation by altering the 3D chromosome organization near critical pro-myogenic genes. This work highlights the crucial importance of tightly controlling FOS expression in the muscle lineage and suggests that in states of chronic stress or disease, persistent FOS activity in muscle precursor cells may disrupt the muscle-forming process.

Data mining of bulk and single-cell RNA sequencing introduces OBI1-AS1 as an astrocyte marker with possible role in glioma recurrence and progression.

Long non-coding RNAs (LncRNAs) are widely known for their various functions in cancer from tumor initiation to tumor progression and metastasis. Gliomas are the most prevalent primary forms of brain tumor, classified into grades I to IV according to their malignant histological features with grade IV, also known as glioblastoma multiforme (GBM), displaying the highest level of malignancy. Thus, the search for differentially expressed LncRNAs in GBM versus low-grade glioma to uncover new insights into the molecular mechanisms of glioma progression have intensified. Bulk RNA sequencing pinpointed decreased expression of OBI1-AS1 in GBM compared to low-grade glioma samples. Subsequent single nuclei RNA sequencing revealed OBI1-AS1 to be a super-exclusive astrocyte marker with AUC = 0.99 and the potential to fully differentiate astrocytes from other brain cell types. Additional supplementary bioinformatics analysis exhibited OBI1-AS1 role in synaptic signal transduction and glutamatergic signaling. In addition, ChIP-Seq data were analyzed to explore transcription factors that can regulate OBI1-AS1 expression in neural cells. Results of Hi-C, methylation and ChIP-Seq analysis strongly suggest methylation of the CTCF binding site serving a central role in regulation of OBI1-AS1 expression via managing chromatin interactions. Our study indicated that lncRNAs, like OBI1-AS1, could be extremely precise in identifying the astrocyte cluster in the single-cell transcriptome and demonstrating superiority to well-established astrocyte markers such as GFAP, S100B, ALDH1L1, and AQP4.

Towards understanding the Regulation of Histone H1 Somatic Subtypes with OMICs.

Histone H1 is involved in the regulation of chromatin higher-order structure and compaction. In humans, histone H1 is a multigene family with seven subtypes differentially expressed in somatic cells. Which are the regulatory mechanisms that determine the variability of the H1 complement is a long-standing biological question regarding histone H1. We have used a new approach based on the integration of OMICs data to address this issue. We have examined the 3D-chromatin structure, the binding of transcription factors (TFs), and the expression of somatic H1 genes in human cell lines, using data from public repositories, such as ENCODE. Analysis of Hi-C, ChIP-seq, and RNA-seq data, have revealed that transcriptional control has a greater impact on H1 regulation than previously thought. Somatic H1 genes located in topologically associated domains (TADs) show higher expression than in boundary regions. H1 genes are targeted by a variable number of transcription factors including cell cycle-related TFs, and tissue-specific TFs, suggesting a fine-tuned, subtype-specific transcriptional control. We describe, for the first time, that all H1 somatic subtypes are under transcriptional co-regulation. The replication-independent subtypes, which are encoded in different chromosomes isolated from other histone genes, are also co-regulated with the rest of the somatic H1 genes, indicating that transcriptional co-regulation extends beyond the histone cluster. Transcriptional control and transcriptional co-regulation explain, at least in part, the variability of H1 complement, the fluctuations of H1 subtypes during development, and also the compensatory effects observed, in model systems, after perturbation of one or more H1 subtypes.

Differentially accessible, single copy sequences form contiguous domains along metaphase chromosomes that are conserved among multiple tissues.

BACKGROUND: During mitosis, chromatin engages in a dynamic cycle of condensation and decondensation. Condensation into distinct units to ensure high fidelity segregation is followed by rapid and reproducible decondensation to produce functional daughter cells. Factors contributing to the reproducibility of chromatin structure between cell generations are not well understood. We investigated local metaphase chromosome condensation along mitotic chromosomes within genomic intervals showing differential accessibility (DA) between homologs. DA was originally identified using short sequence-defined single copy (sc) DNA probes of < 5 kb in length by fluorescence in situ hybridization (scFISH) in peripheral lymphocytes. These structural differences between metaphase homologs are non-random, stable, and heritable epigenetic marks which have led to the proposed function of DA as a marker of chromatin memory. Here, we characterize the organization of DA intervals into chromosomal domains by identifying multiple DA loci in close proximity to each other and examine the conservation of DA between tissues. RESULTS: We evaluated multiple adjacent scFISH probes at 6 different DA loci from chromosomal regions 2p23, 3p24, 12p12, 15q22, 15q24 and 20q13 within peripheral blood T-lymphocytes. DA was organized within domains that extend beyond the defined boundaries of individual scFISH probes. Based on hybridizations of 2 to 4 scFISH probes per domain, domains ranged in length from 16.0 kb to 129.6 kb. Transcriptionally inert chromosomal DA regions in T-lymphocytes also demonstrated conservation of DA in bone marrow and fibroblast cells. CONCLUSIONS: We identified novel chromosomal regions with allelic differences in metaphase chromosome accessibility and demonstrated that these accessibility differences appear to be aggregated into contiguous domains extending beyond individual scFISH probes. These domains are encompassed by previously established topologically associated domain (TAD) boundaries. DA appears to be a conserved feature of human metaphase chromosomes across different stages of lymphocyte differentiation and germ cell origin, consistent with its proposed role in maintenance of intergenerational cellular chromosome memory.

TADCompare: An R Package for Differential and Temporal Analysis of Topologically Associated Domains.

Recent research using chromatin conformation capture technologies, such as Hi-C, has demonstrated the importance of topologically associated domains (TADs) and smaller chromatin loops, collectively referred hereafter as "interacting domains." Many such domains change during development or disease, and exhibit cell- and condition-specific differences. Quantification of the dynamic behavior of interacting domains will help to better understand genome regulation. Methods for comparing interacting domains between cells and conditions are highly limited. We developed TADCompare, a method for differential analysis of boundaries of interacting domains between two or more Hi-C datasets. TADCompare is based on a spectral clustering-derived measure called the eigenvector gap, which enables a loci-by-loci comparison of boundary differences. Using this measure, we introduce methods for identifying differential and consensus boundaries of interacting domains and tracking boundary changes over time. We further propose a novel framework for the systematic classification of boundary changes. Colocalization- and gene enrichment analysis of different types of boundary changes demonstrated distinct biological functionality associated with them. TADCompare is available on https://github.com/dozmorovlab/TADCompare and Bioconductor (submitted).