Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the ...in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis.
A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome ...positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide- and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.
Trans-homolog interactions have been studied extensively in Drosophila, where homologs are paired in somatic cells and transvection is prevalent. Nevertheless, the detailed structure of pairing and ...its functional impact have not been thoroughly investigated. Accordingly, we generated a diploid cell line from divergent parents and applied haplotype-resolved Hi-C, showing that homologs pair with varying precision genome-wide, in addition to establishing trans-homolog domains and compartments. We also elucidate the structure of pairing with unprecedented detail, observing significant variation across the genome and revealing at least two forms of pairing: tight pairing, spanning contiguous small domains, and loose pairing, consisting of single larger domains. Strikingly, active genomic regions (A-type compartments, active chromatin, expressed genes) correlated with tight pairing, suggesting that pairing has a functional implication genome-wide. Finally, using RNAi and haplotype-resolved Hi-C, we show that disruption of pairing-promoting factors results in global changes in pairing, including the disruption of some interaction peaks.
Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focus on trans interactions in Drosophila, where homologous ...chromosomes are paired in somatic cells from embryogenesis through adulthood. We first address long-standing questions regarding the structure of embryonic homolog pairing and, to this end, develop a haplotype-resolved Hi-C approach to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which we call Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. We also find a significant genome-wide correlation between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, we demonstrate the versatility of our haplotype-resolved approach by applying it to mammalian embryos.
This study explores the relationship between three-dimensional genome organization and ultraconserved elements (UCEs), an enigmatic set of DNA elements that are perfectly conserved between the ...reference genomes of distantly related species. Examining both human and mouse genomes, we interrogate the relationship of UCEs to three features of chromosome organization derived from Hi-C studies. We find that UCEs are enriched within contact domains and, further, that the subset of UCEs within domains shared across diverse cell types are linked to kidney-related and neuronal processes. In boundaries, UCEs are generally depleted, with those that do overlap boundaries being overrepresented in exonic UCEs. Regarding loop anchors, UCEs are neither overrepresented nor underrepresented, but those present in loop anchors are enriched for splice sites. Finally, as the relationships between UCEs and human Hi-C features are conserved in mouse, our findings suggest that UCEs contribute to interspecies conservation of genome organization and, thus, genome stability.
Display omitted
•UCEs are enriched in domains, depleted from boundaries, and neither at loop anchors•UCEs in domains shared across cell types are linked to kidney and neuronal processes•UCEs that do occur in boundaries and loop anchors are predominantly exonic•UCEs that are present in loop anchors are enriched in splice sites
McCole et al. demonstrate the non-random relationship between the positions of perfectly conserved genomic regions, termed the ultraconserved elements (UCEs), and three-dimensional genome organization within mammalian nucleus as defined by Hi-C studies. They postulate that these connections aid in orchestrating genome packaging and preserving genome function and integrity.
Ultraconserved elements (UCEs) are strongly depleted from segmental duplications and copy number variations (CNVs) in the human genome, suggesting that deletion or duplication of a UCE can be ...deleterious to the mammalian cell. Here we address the process by which CNVs become depleted of UCEs. We begin by showing that depletion for UCEs characterizes the most recent large-scale human CNV datasets and then find that even newly formed de novo CNVs, which have passed through meiosis at most once, are significantly depleted for UCEs. In striking contrast, CNVs arising specifically in cancer cells are, as a rule, not depleted for UCEs and can even become significantly enriched. This observation raises the possibility that CNVs that arise somatically and are relatively newly formed are less likely to have established a CNV profile that is depleted for UCEs. Alternatively, lack of depletion for UCEs from cancer CNVs may reflect the diseased state. In support of this latter explanation, somatic CNVs that are not associated with disease are depleted for UCEs. Finally, we show that it is possible to observe the CNVs of induced pluripotent stem (iPS) cells become depleted of UCEs over time, suggesting that depletion may be established through selection against UCE-disrupting CNVs without the requirement for meiotic divisions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
An unexpectedly large number of human autosomal genes are subject to monoallelic expression (MAE). Our analysis of 4,227 such genes uncovers surprisingly high genetic variation across human ...populations. This increased diversity is unlikely to reflect relaxed purifying selection. Remarkably, MAE genes exhibit an elevated recombination rate and an increased density of hypermutable sequence contexts. However, these factors do not fully account for the increased diversity. We find that the elevated nucleotide diversity of MAE genes is also associated with greater allelic age: variants in these genes tend to be older and are enriched in polymorphisms shared by Neanderthals and chimpanzees. Both synonymous and nonsynonymous alleles of MAE genes have elevated average population frequencies. We also observed strong enrichment of the MAE signature among genes reported to evolve under balancing selection. We propose that an important biological function of widespread MAE might be the generation of cell-to-cell heterogeneity; the increased genetic variation contributes to this heterogeneity.
DNA binding factors are essential for regulating gene expression. CTCF and cohesin are DNA binding factors with central roles in chromatin organization and gene expression. We determined the sites of ...CTCF and cohesin binding to DNA in mouse brain, genome wide and in an allele-specific manner with high read-depth ChIP-seq. By comparing our results with existing data for mouse liver and embryonic stem (ES) cells, we investigated the tissue specificity of CTCF binding sites. ES cells have fewer unique CTCF binding sites occupied than liver and brain, consistent with a ground-state pattern of CTCF binding that is elaborated during differentiation. CTCF binding sites without the canonical consensus motif were highly tissue specific. In brain, a third of CTCF and cohesin binding sites coincide, consistent with the potential for many interactions between cohesin and CTCF but also many instances of independent action. In the context of genomic imprinting, CTCF and/or cohesin bind to a majority but not all differentially methylated regions, with preferential binding to the unmethylated parental allele. Whether the parental allele-specific methylation was established in the parental germlines or post-fertilization in the embryo is not a determinant in CTCF or cohesin binding. These findings link CTCF and cohesin with the control regions of a subset of imprinted genes, supporting the notion that imprinting control is mechanistically diverse.
Genomic imprinting is a form of gene dosage regulation in which a gene is expressed from only one of the alleles, in a manner dependent on the parent of origin. The mechanisms governing imprinted ...gene expression have been investigated in detail and have greatly contributed to our understanding of genome regulation in general. Both DNA sequence features, such as CpG islands, and epigenetic features, such as DNA methylation and non-coding RNAs, play important roles in achieving imprinted expression. However, the relative importance of these factors varies depending on the locus in question. Defining the minimal features that are absolutely required for imprinting would help us to understand how imprinting has evolved mechanistically. Imprinted retrogenes are a subset of imprinted loci that are relatively simple in their genomic organisation, being distinct from large imprinting clusters, and have the potential to be used as tools to address this question. Here, we compare the repeat element content of imprinted retrogene loci with non-imprinted controls that have a similar locus organisation. We observe no significant differences that are conserved between mouse and human, suggesting that the paucity of SINEs and relative abundance of LINEs at imprinted loci reported by others is not a sequence feature universally required for imprinting.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Bladder Cancer-Associated Protein gene (BLCAP; previously BC10) is a tumour suppressor that limits cell proliferation and stimulates apoptosis. BLCAP protein or message are downregulated or ...absent in a variety of human cancers. In mouse and human, the first intron of Blcap/BLCAP contains the distinct Neuronatin (Nnat/NNAT) gene. Nnat is an imprinted gene that is exclusively expressed from the paternally inherited allele. Previous studies found no evidence for imprinting of Blcap in mouse or human. Here we show that Blcap is imprinted in mouse and human brain, but not in other mouse tissues. Moreover, Blcap produces multiple distinct transcripts that exhibit reciprocal allele-specific expression in both mouse and human. We propose that the tissue-specific imprinting of Blcap is due to the particularly high transcriptional activity of Nnat in brain, as has been suggested previously for the similarly organized and imprinted murine Commd1/U2af1-rs1 locus. For Commd1/U2af1-rs1, we show that it too produces distinct transcript variants with reciprocal allele-specific expression. The imprinted expression of BLCAP and its interplay with NNAT at the transcriptional level may be relevant to human carcinogenesis.
PDF
