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1.
bioRxiv ; 2024 Mar 17.
Article in English | MEDLINE | ID: mdl-38559085

ABSTRACT

Genome organization is intricately tied to regulating genes and associated cell fate decisions. In this study, we examine the positioning and functional significance of human genes, grouped by their evolutionary age, within the 3D organization of the genome. We reveal that genes of different evolutionary origin have distinct positioning relationships with both domains and loop anchors, and remarkably consistent relationships with boundaries across cell types. While the functional associations of each group of genes are primarily cell type-specific, such associations of conserved genes maintain greater stability across 3D genomic features and disease than recently evolved genes. Furthermore, the expression of these genes across various tissues follows an evolutionary progression, such that RNA levels increase from young genes to ancient genes. Thus, the distinct relationships of gene evolutionary age, function, and positioning within 3D genomic features contribute to tissue-specific gene regulation in development and disease.

2.
Front Cell Dev Biol ; 10: 968145, 2022.
Article in English | MEDLINE | ID: mdl-36036013

ABSTRACT

Genome organization includes contacts both within a single chromosome and between distinct chromosomes. Thus, regulatory organization in the nucleus may include interplay of these two types of chromosomal interactions with genome activity. Emerging advances in omics and single-cell imaging technologies have allowed new insights into chromosomal contacts, including those of homologs and sister chromatids, and their significance to genome function. In this review, we highlight recent studies in this field and discuss their impact on understanding the principles of chromosome organization and associated functional implications in diverse cellular processes. Specifically, we describe the contributions of intra-chromosomal, inter-homolog, and inter-sister chromatid contacts to genome organization and gene expression.

3.
Nat Commun ; 10(1): 4486, 2019 10 03.
Article in English | MEDLINE | ID: mdl-31582744

ABSTRACT

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.


Subject(s)
Chromosome Pairing , Chromosomes, Insect/genetics , Drosophila melanogaster/genetics , Genome, Insect , Animals , Cell Culture Techniques , Cell Line , Chromatin/metabolism , Computational Biology , Datasets as Topic , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Embryo, Mammalian , Embryo, Nonmammalian , Female , Genomics/methods , High-Throughput Nucleotide Sequencing , Male , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Small Interfering/metabolism , Sequence Homology, Nucleic Acid , Transcription, Genetic , Zygote
4.
Nat Commun ; 10(1): 4485, 2019 10 03.
Article in English | MEDLINE | ID: mdl-31582763

ABSTRACT

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.


Subject(s)
Chromosome Pairing , Chromosomes, Insect/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Genome, Insect , Animals , Cell Culture Techniques , Cell Line , Chromatin/metabolism , Female , High-Throughput Nucleotide Sequencing , Male , Sequence Homology, Nucleic Acid
5.
PLoS Biol ; 17(5): e3000241, 2019 05.
Article in English | MEDLINE | ID: mdl-31086362

ABSTRACT

Centromeres are essential chromosomal regions that mediate kinetochore assembly and spindle attachments during cell division. Despite their functional conservation, centromeres are among the most rapidly evolving genomic regions and can shape karyotype evolution and speciation across taxa. Although significant progress has been made in identifying centromere-associated proteins, the highly repetitive centromeres of metazoans have been refractory to DNA sequencing and assembly, leaving large gaps in our understanding of their functional organization and evolution. Here, we identify the sequence composition and organization of the centromeres of Drosophila melanogaster by combining long-read sequencing, chromatin immunoprecipitation for the centromeric histone CENP-A, and high-resolution chromatin fiber imaging. Contrary to previous models that heralded satellite repeats as the major functional components, we demonstrate that functional centromeres form on islands of complex DNA sequences enriched in retroelements that are flanked by large arrays of satellite repeats. Each centromere displays distinct size and arrangement of its DNA elements but is similar in composition overall. We discover that a specific retroelement, G2/Jockey-3, is the most highly enriched sequence in CENP-A chromatin and is the only element shared among all centromeres. G2/Jockey-3 is also associated with CENP-A in the sister species D. simulans, revealing an unexpected conservation despite the reported turnover of centromeric satellite DNA. Our work reveals the DNA sequence identity of the active centromeres of a premier model organism and implicates retroelements as conserved features of centromeric DNA.


Subject(s)
Centromere/genetics , Drosophila/genetics , Retroelements/genetics , Animals , Centromere Protein A/genetics , Chromatin/metabolism , DNA Transposable Elements/genetics , DNA, Satellite/genetics , Drosophila/embryology , Drosophila Proteins/genetics , Embryo, Nonmammalian/metabolism , Genome, Insect , Terminal Repeat Sequences/genetics
6.
PLoS Genet ; 14(12): e1007872, 2018 12.
Article in English | MEDLINE | ID: mdl-30586358

ABSTRACT

Chromosome organization is crucial for genome function. Here, we present a method for visualizing chromosomal DNA at super-resolution and then integrating Hi-C data to produce three-dimensional models of chromosome organization. Using the super-resolution microscopy methods of OligoSTORM and OligoDNA-PAINT, we trace 8 megabases of human chromosome 19, visualizing structures ranging in size from a few kilobases to over a megabase. Focusing on chromosomal regions that contribute to compartments, we discover distinct structures that, in spite of considerable variability, can predict whether such regions correspond to active (A-type) or inactive (B-type) compartments. Imaging through the depths of entire nuclei, we capture pairs of homologous regions in diploid cells, obtaining evidence that maternal and paternal homologous regions can be differentially organized. Finally, using restraint-based modeling to integrate imaging and Hi-C data, we implement a method-integrative modeling of genomic regions (IMGR)-to increase the genomic resolution of our traces to 10 kb.


Subject(s)
Chromosome Walking/methods , Chromosomes, Human, Pair 19/genetics , Chromosomes, Human, Pair 19/ultrastructure , Models, Genetic , Cells, Cultured , Chromosome Painting/methods , Chromosome Structures/chemistry , Chromosome Structures/genetics , Chromosome Structures/ultrastructure , Chromosomes, Human, Pair 19/chemistry , Female , Fluorescent Dyes , Humans , Imaging, Three-Dimensional , In Situ Hybridization, Fluorescence/methods , Male , Oligonucleotide Probes , Pedigree
7.
Cell Rep ; 24(2): 479-488, 2018 07 10.
Article in English | MEDLINE | ID: mdl-29996107

ABSTRACT

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.


Subject(s)
Conserved Sequence/genetics , Genome , Mammals/genetics , Animals , Chromosomes, Mammalian/genetics , DNA, Intergenic/genetics , Exons/genetics , Humans , Introns/genetics , Kidney/metabolism , Mice , RNA Processing, Post-Transcriptional/genetics , Transcription Initiation Site
8.
Genes Dev ; 31(6): 590-602, 2017 03 15.
Article in English | MEDLINE | ID: mdl-28381411

ABSTRACT

Developmental gene expression is tightly regulated through enhancer elements, which initiate dynamic spatio-temporal expression, and Polycomb response elements (PREs), which maintain stable gene silencing. These two cis-regulatory functions are thought to operate through distinct dedicated elements. By examining the occupancy of the Drosophila pleiohomeotic repressive complex (PhoRC) during embryogenesis, we revealed extensive co-occupancy at developmental enhancers. Using an established in vivo assay for PRE activity, we demonstrated that a subset of characterized developmental enhancers can function as PREs, silencing transcription in a Polycomb-dependent manner. Conversely, some classic Drosophila PREs can function as developmental enhancers in vivo, activating spatio-temporal expression. This study therefore uncovers elements with dual function: activating transcription in some cells (enhancers) while stably maintaining transcriptional silencing in others (PREs). Given that enhancers initiate spatio-temporal gene expression, reuse of the same elements by the Polycomb group (PcG) system may help fine-tune gene expression and ensure the timely maintenance of cell identities.


Subject(s)
Drosophila Proteins/metabolism , Enhancer Elements, Genetic , Gene Expression Regulation, Developmental , Polycomb-Group Proteins/metabolism , Response Elements , Animals , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Embryonic Development/genetics
9.
Curr Opin Genet Dev ; 37: 119-128, 2016 04.
Article in English | MEDLINE | ID: mdl-27065367

ABSTRACT

The presence of maternal and paternal homologs appears to be much more than just a doubling of genetic material. We know this because genomes have evolved elaborate mechanisms that permit homologous regions to sense and then respond to each other. One way in which homologs communicate is to come into contact and, in fact, Dipteran insects such as Drosophila excel at this task, aligning all pairs of maternal and paternal chromosomes, end-to-end, in essentially all somatic tissues throughout development. Here, we reexamine the widely held tenet that extensive somatic pairing of homologous sequences cannot occur in mammals and suggest, instead, that pairing may be a widespread and significant potential that has gone unnoticed in mammals because they expend considerable effort to prevent it. We then extend this discussion to interchromosomal interactions, in general, and speculate about the potential of nuclear organization and pairing to impact inheritance.


Subject(s)
Chromosome Pairing/genetics , Drosophila/genetics , Meiosis/genetics , Animals , Cell Nucleus/genetics , Diploidy , Genome , Mammals/genetics
10.
Nat Commun ; 6: 7147, 2015 May 12.
Article in English | MEDLINE | ID: mdl-25962338

ABSTRACT

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.


Subject(s)
Chromosome Painting/methods , Chromosomes/genetics , Haplotypes , In Situ Hybridization, Fluorescence/methods , Oligonucleotide Array Sequence Analysis/methods , Animals , Cell Line , Drosophila , Gene Library , Oligonucleotide Probes/metabolism , Staining and Labeling
11.
PLoS Genet ; 10(1): e1004060, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24391522

ABSTRACT

Deciphering the specific contribution of individual motifs within cis-regulatory modules (CRMs) is crucial to understanding how gene expression is regulated and how this process is affected by sequence variation. But despite vast improvements in the ability to identify where transcription factors (TFs) bind throughout the genome, we are limited in our ability to relate information on motif occupancy to function from sequence alone. Here, we engineered 63 synthetic CRMs to systematically assess the relationship between variation in the content and spacing of motifs within CRMs to CRM activity during development using Drosophila transgenic embryos. In over half the cases, very simple elements containing only one or two types of TF binding motifs were capable of driving specific spatio-temporal patterns during development. Different motif organizations provide different degrees of robustness to enhancer activity, ranging from binary on-off responses to more subtle effects including embryo-to-embryo and within-embryo variation. By quantifying the effects of subtle changes in motif organization, we were able to model biophysical rules that explain CRM behavior and may contribute to the spatial positioning of CRM activity in vivo. For the same enhancer, the effects of small differences in motif positions varied in developmentally related tissues, suggesting that gene expression may be more susceptible to sequence variation in one tissue compared to another. This result has important implications for human eQTL studies in which many associated mutations are found in cis-regulatory regions, though the mechanism for how they affect tissue-specific gene expression is often not understood.


Subject(s)
DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Factors/genetics , Animals , Animals, Genetically Modified , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Genome , Humans , Mutation , Nucleotide Motifs/genetics , Quantitative Trait Loci/genetics , Sequence Analysis, DNA
12.
Dev Dyn ; 237(8): 2195-208, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18627100

ABSTRACT

Activity of zebrafish hoxb4a in the developing brain was analyzed in comparison to hoxa4a and hoxd4a using unique enhancer detection transgenes. Cytoplasmic YFP revealed shape and axonal projections of neurons in animals with insertions near the Hox4 genes and provided a means for the identification of neuronal subtypes. Despite an early activity of the genes in neuroepithelial cells and later in immature postmitotic neurons, we found reporter expression in distinct neuronal subtypes in the r7-r8-derived hindbrain. Most strikingly, hoxb4a neuronal subtypes projected through the vagus and into the pectoral fin while others formed symmetrically located fiber tracts innervating the cerebellum and the tectum, features that are partially shared by the other two paralogs. Collectively, our expression analysis indicates that hoxb4a in combination with its paralogs may play a significant role in the development of precerebellar, vagal, and pectoral fin neuronal subtypes.


Subject(s)
Enhancer Elements, Genetic/genetics , Homeodomain Proteins/genetics , Neurons/classification , Neurons/physiology , Rhombencephalon/embryology , Zebrafish Proteins/genetics , Animals , Branchial Region/cytology , Branchial Region/embryology , Branchial Region/physiology , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Reticular Formation/cytology , Reticular Formation/embryology , Reticular Formation/physiology , Rhombencephalon/cytology , Rhombencephalon/physiology , Transgenes/genetics , Vagus Nerve/cytology , Vagus Nerve/embryology , Vagus Nerve/physiology , Zebrafish
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