Organization Of Eukaryotic Genome Research Articles

Joint locus of a/b-globin genes in Danio rerio is segregated into structural subdomains active at different stages of development

In the domain model of eukaryotic genome organization, the functional unit of the genome, along with the relevant regulatory elements, is considered to be a gene or a gene family. In hot-blooded vertebrate animals, the domains of a- and b-globin genes are positioned at different chromosomes and are organized and regulated in different fashion. In cold-blooded animals, in particular in tropical fish Danio rerio, a- and b globin genes are located in a common gene cluster. However, the joint a/b-globin gene cluster is subdivided into two development stage-specific subdomains, the adult one and the embryonic-larval one. In an attempt to find out whether this functional segregation correlates with structural segregation of the domain we compared the DNase I sensitivity and profiles of histone modifications of adult and embryonic-larval segments of the domain in cultured D. rerio fibroblasts. We have demonstrated that, in these nonerythroid cells, adult and embryonic- larval subdomains possess different DNase I sensitivities and different profiles of H3K27me3, a histone modification introduced by PRC2 complex. These observations suggest that joint a/b globin gene domain of Danio rerio is segregated into two structural subdomain harboring adult and embryonic-larval globin genes.

Investigation of the spatial genome organization of mouse sperm and fibroblasts by the Hi-C method

The spatial organization of eukaryotic genome plays an important role in the control of gene expression. The new Hi-C method allows investigation of chromosome contact in the three-dimensional architecture of the whole genome; however, it has not yet been applied to study the spatial configuration of the germ cells genome. Here we describe a protocol for production and quality control of Hi-C DNA libraries from mouse sperm cells and fibroblasts. Our results demonstrate that the Hi-C method can be used for studying the spatial organization of the densely packed sperm genome.

Condensins are Required for Maintenance of Nuclear Architecture.

The 3-dimensional spatial organization of eukaryotic genomes is important for regulation of gene expression as well as DNA damage repair. It has been proposed that one basic biophysical property of all nuclei is that interphase chromatin must be kept in a condensed prestressed state in order to prevent entropic pressure of the DNA polymer from expanding and disrupting the nuclear envelope. Although many factors can contribute to specific organizational states to compact chromatin, the mechanisms through which such interphase chromatin compaction is maintained are not clearly understood. Condensin proteins are known to exert compaction forces on chromosomes in anticipation of mitosis, but it is not known whether condensins also function to maintain interphase prestressed chromatin states. Here we show that RNAi depletion of the N-CAP-H2, N-CAP-D3 and SMC2 subunits of human condensin II leads to dramatic disruption of nuclear architecture and nuclear size. This is consistent with the idea that condensin mediated chromatin compaction contributes significantly to the prestressed condensed state of the interphase nucleus, and when such compaction forces are disrupted nuclear size and shape change due to chromatin expansion.

Condensins and 3D Organization of the Interphase Nucleus

Condensins are conserved multi-subunit protein complexes that participate in eukaryotic genome organization. Well known for their role in mitotic chromosome condensation, condensins have recently emerged as integral components of diverse interphase processes. Recent evidence shows that condensins are involved in chromatin organization, gene expression, and DNA repair and indicates similarities between the interphase and mitotic functions of condensin. Recent work has enhanced our knowledge of how chromatin architecture is dynamically regulated by condensin to impact essential cellular processes.

Investigation of Latent Periodicity Phenomenon in the Genomes of Eukaryotic Organisms

Analysis of the data from the first release of HeteroGenome database, collecting the revealed regions of latent periodicity in the genomes of a number of eukaryotic organisms, is presented. Tandem repeats with different conservation of a pattern copies, including the highly diverged repeats, were identified in the genomes of S. cerevisiae, A. thaliana, C. elegans and D. melanogaster. The data were obtained with the help of original spectral-statistical approach to searching for the reliable regions of latent periodicity in DNA sequences. Introduction of the two-level structure for the data presentation (At the first, nonredundant level the regions of latent periodicity is generally viewed, at the second level only the fragments of conservative periodic structure are considered.) allowed to estimate a share of genome coverage by the regions of latent periodicity which counts ~10% of a whole genome length. The estimate is deduced according to the data of the first level. An analysis of quantitative and qualitative content (corresponding to the divergence levels) of the latent periodicity regions over all the chromosomes of the considered organisms revealed the characteristic types of periodicity in a genome of each organism. The histograms showing density distribution of the latent periodicity regions along every chromosome in the analyzed genomes were built. A repertoire of period lengths were revealed in the genomes. Moreover, HeteroGenome base offers some additional possibilities for its’ data analysis and is freely available at URL: http://www.jcbi.ru/lp_baze/.

Bioinformatic analysis reveals an evolutional selection for DNA:RNA hybrid G-quadruplex structures as putative transcription regulatory elements in warm-blooded animals

Recently, we reported the co-transcriptional formation of DNA:RNA hybrid G-quadruplex (HQ) structure by the non-template DNA strand and nascent RNA transcript, which in turn modulates transcription under both in vitro and in vivo conditions. Here we present bioinformatic analysis on putative HQ-forming sequences (PHQS) in the genomes of eukaryotic organisms. Starting from amphibian, PHQS motifs are concentrated in the immediate 1000-nt region downstream of transcription start sites, implying their potential role in transcription regulation. Moreover, their occurrence shows a strong bias toward the non-template versus the template strand. PHQS has become constitutional in genes in warm-blooded animals, and the magnitude of the strand bias correlates with the ability of PHQS to form HQ, suggesting a selection based on HQ formation. This strand bias is reversed in lower species, implying that the selection of PHQS/HQ depended on the living temperature of the organisms. In comparison with the putative intramolecular G-quadruplex-forming sequences (PQS), PHQS motifs are far more prevalent and abundant in the transcribed regions, making them the dominant candidates in the formation of G-quadruplexes in transcription. Collectively, these results suggest that the HQ structures are evolutionally selected to function in transcription and other transcription-mediated processes that involve guanine-rich non-template strand.

Elucidating Internucleosome Interactions and the Roles of Histone Tails

The nucleosome is the first level of genome organization and regulation in eukaryotes where negatively charged DNA is wrapped around largely positively charged histone proteins. Interaction between nucleosomes is dominated by electrostatics at long range and guided by specific contacts at short range, particularly involving their flexible histone tails. We have thus quantified how internucleosome interactions are modulated by salts (KCl, MgCl2) and histone tail deletions (H3, H4 N-terminal), using small-angle x-ray scattering and theoretical modeling. We found that measured effective charges at low salts are ∼1/5th of the theoretically predicted renormalized charges and that H4 tail deletion suppresses the attraction at high salts to a larger extent than H3 tail deletion.

The variant histone H2A.V of Drosophila—three roles, two guises

Histone variants play important roles in eukaryotic genome organization, the control of gene expression, cell division and DNA repair. Unlike other organisms that employ several H2A variants for different functions, the parsimonious fruit fly Drosophila melanogaster gets along with just a single H2A variant, H2A.V. Remarkably, H2A.V unites within one molecule features and functions of two different mammalian H2A variants, H2A.Z and H2A.X. Accordingly, H2A.V is involved in diverse functions, as an element of a class of active promoter structure, as a foundation for heterochromatin assembly and as a DNA damage sensor. Here, we comprehensively review the current knowledge of this fascinating histone variant.

Balancing of Histone H3K4 Methylation States by the Kdm5c/SMCX Histone Demethylase Modulates Promoter and Enhancer Function

The functional organization of eukaryotic genomes correlates with specific patterns of histone methylations. Regulatory regions in genomes such as enhancers and promoters differ in their extent of methylation of histone H3 at lysine-4 (H3K4), but it is largely unknown how the different methylation states are specified and controlled. Here, we show that the Kdm5c/Jarid1c/SMCX member of the Kdm5 family of H3K4 demethylases can be recruited to both enhancer and promoter elements in mouse embryonic stem cells and in neuronal progenitor cells. Knockdown of Kdm5c deregulates transcription via local increases in H3K4me3. Our data indicate that by restricting H3K4me3 modification at core promoters, Kdm5c dampens transcription, but at enhancers Kdm5c stimulates their activity. Remarkably, an impaired enhancer function activates the intrinsic promoter activity of Kdm5c-bound distal elements. Our results demonstrate that the Kdm5c demethylase plays a crucial and dynamic role in the functional discrimination between enhancers and core promoters.

Topological constraints and chromosome organization in eukaryotes: a physical point of view

New experimental tools capable of probing the three-dimensional organization of eukaryotic genomes with an unprecedented level of detail have been developed in the last few years. In the quest for a quantitative understanding of experimental results, several polymer models for chromatin organization were introduced and critically evaluated. In the present article, I give a brief introduction to the physical basis of chromosome organization, and recall the experimental evidence in favour of the importance of topological constraints for the description of chromosome conformations in eukaryotes.

Quantitative analysis of chaperone network throughput in budding yeast.

The network of molecular chaperones mediates the folding and translocation of the many proteins encoded in the genome of eukaryotic organisms, as well as a response to stress. It has been particularly well characterised in the budding yeast, Saccharomyces cerevisiae, where 63 known chaperones have been annotated and recent affinity purification and MS/MS experiments have helped characterise the attendant network of chaperone targets to a high degree. In this study, we apply our QconCAT methodology to directly quantify the set of yeast chaperones in absolute terms (copies per cell) via SRM MS. Firstly, we compare these to existing quantitative estimates of these yeast proteins, highlighting differences between approaches. Secondly, we cast the results into the context of the chaperone target network and show a distinct relationship between abundance of individual chaperones and their targets. This allows us to characterise the ‘throughput’ of protein molecules passing through individual chaperones and their groups on a proteome-wide scale in an unstressed model eukaryote for the first time. The results demonstrate specialisations of the chaperone classes, which display different overall workloads, efficiencies and preference for the sub-cellular localisation of their targets. The novel integration of the interactome data with quantification supports re-estimates of the level of protein throughout going through molecular chaperones. Additionally, although chaperones target fewer than 40% of annotated proteins we show that they mediate the folding of the majority of protein molecules (∼62% of the total protein flux in the cell), highlighting their importance.

The 3D Organization of the Yeast Genome Correlates with Co-Expression and Reflects Functional Relations between Genes

The spatial organization of eukaryotic genomes is thought to play an important role in regulating gene expression. The recent advances in experimental methods including chromatin capture techniques, as well as the large amounts of accumulated gene expression data allow studying the relationship between spatial organization of the genome and co-expression of protein-coding genes. To analyse this genome-wide relationship at a single gene resolution, we combined the interchromosomal DNA contacts in the yeast genome measured by Duan et al. with a comprehensive collection of 1,496 gene expression datasets. We find significant enhancement of co-expression among genes with contact links. The co-expression is most prominent when two gene loci fall within 1,000 base pairs from the observed contact. We also demonstrate an enrichment of inter-chromosomal links between functionally related genes, which suggests that the non random nature of the genome organization serves to facilitate coordinated transcription in groups of genes.

Sequence Analysis of KpnI Repeat Sequences to Revisit the Phylogeny of the Genus Carthamus L.

Repetitive DNA sequences constitute a significant proportion of eukaryotic genomes. Knowledge about the distribution of repetitive DNA sequences is necessary in order to gain insights into the organization, evolution and behavior of eukaryotic genomes. Therefore, we used two repetitive DNA sequences pCtKpnI-I and pCtKpnI-II, earlier reported in Carthamus tinctorius L. to study the phylogeny and to revise the taxonomic status of the taxa belonging to the genus. The study unraveled two major lines within the genus Carthamus; one line included all the diploid taxa (2n = 24) and the other line comprised the taxa with 2n = 20 and the polyploid taxa (2n = 44 and 64). The results of the present study will prove useful in molecular breeding for improving some targeted agronomic traits in genus Carthamus.

Intergenic region between TATA-box binding protein and proteasome subunit C3 genes of Medaka function as the bidirectional promoter in vitro and in vivo

In the genome of eukaryotic organisms, each protein-coding gene has the unique promoter in the 5′-flanking region, and the direction of the promoter is usually controlled unidirectional. In this study, we revealed that the intergenic region between TATA-box binding protein (tbp) and proteasome subunit C3 (psmc3) genes in Medaka functions as bidirectional promoter in vitro and in vivo. The tbp and psmc3 genes were allocated as a head-to-head configuration with a 719bp intergenic region. A comparative analysis of gene arrangement surrounding loci of tbp in vertebrates also illustrated that it was unique in Acanthopterygii lineage. The transcription activities were about 1.2 times for tbp direction and 0.7 times for psmc3 direction against that of SV40 promoter in Medaka fibroblasts, respectively. A dual fluorescent reporter assay directly showed that the bidirectional promoter could express two divergent genes concurrently without disruption of RNA polymerase II elongation. In addition, an analysis of sequential deletion of this promoter suggested that the ETS binding site was necessary for maximum expression of downstream gene, and only the ETS binding site was shared from fish to mammals. In mammals, high correlation with CpG islands was observed in such bidirectional promoters, no association was found in the tbp/psmc3 bidirectional promoter in Medaka. These results suggest that molecular machineries of fish bidirectional promoter may be somehow different from those of mammals but the cis-acting element for binding ETS transcription factors is essential for divergent gene expression.

Chicken β-globin genes: A model system to study the transcriptional regulation at the level of genome domains

In the last quarter of the 20th century, studies with a number of model systems produced a hypothesis that the higher eukaryotic genome consists of functionally isolated areas termed genomic domains. Each domain includes one or more genes and a regulatory system that is normally active only for the given domain and allows it to achieve a regulatory autonomy of the neighboring chromosome regions. A genome domain is characterized by spectra of covalent histone modifications, which determine the domain boundaries and the extent of chromatin condensation within the domain, thus determining whether transcriptional activation is possible for the domain genes. The domain hypothesis of genome organization is due, to a large extent, to studies of the mechanisms regulating transcription of the globin genes in vertebrates. The chicken β-globin gene domain is one of the most popular models in the field. The model was used to investigate the basic principles of the complex function of higher eukaryotic enhancers, the properties of insulators and functional units of eukaryotic enhancers and promoters, the influence of covalent histone modifications on the extent of chromatin condensation, and the role of such modifications in regulating transcription within the domain. The review summarizes the data on the chicken β-globin gene domain and considers the domain hypothesis of eukaryotic genome organization.

The genome in space and time: Does form always follow function?

AbstractRecent systematic studies using newly developed genomic approaches have revealed common mechanisms and principles that underpin the spatial organization of eukaryotic genomes and allow them to respond and adapt to diverse functional demands. Genomes harbor, interpret, and propagate genetic and epigenetic information, and the three‐dimensional (3D) organization of genomes in the nucleus should be intrinsically linked to their biological functions. However, our understanding of the mechanisms underlying both the topological organization of genomes and the various nuclear processes is still largely incomplete. In this essay, we focus on the functional relevance as well as the biophysical properties of common organizational themes in genomes (e.g. looping, clustering, compartmentalization, and dynamics), and examine the interconnection between genome structure and function from this angle. Present evidence supports the idea that, in general, genome architecture reflects and influences genome function, and is relatively stable. However, the answer as to whether genome architecture is a hallmark of cell identity remains elusive.

Feux croisés sur le nucléosome

In mammals, the post-meiotic phases of spermatogenesis direct a drastic compaction of the genome characterized by the replacement of the majority of histones by small basic non-histone proteins, transition proteins followed by protamines. Although the mechanisms controlling this process are poorly understood, available data suggest that the nucleosome, which represents the universal unit of genome organization in eukaryotes, is the target of specific events cooperating to globally transform DNA organization at a genomic scale. Indeed, after meiosis, nearly all nucleosomes associated with the male genome undergo successive modifications increasing their instability, finally leading to their dissociation and re-organization. The characterization of the underlying mechanisms is a real challenge and appears as essential for the understanding of male gametogenesis and associated pathologies.

Did the modulation of expression noise shape the evolution of three dimensional genome organizations in eukaryotes?

The pervasive role of distant chromatin interactions in transcriptional regulation is increasingly becoming evident. There is a possibility that the greater diversity in chromatin interactions of a genomic locus could contribute to stochastic variation in its gene expression. However, this issue has not been addressed. Here, I present a few lines of evidence, which suggest that the variation in trans chromatin interactions might occur at the cost of expression noise. Genomic regions with nucleosome depletion, abundant and rapid transcription and with essential gene clusters exhibit relatively fewer trans chromatin interactions in the nucleus. Moreover, loci with greater number of interactions tend to show higher expression noise. Based on these observations, I hypothesize that the three dimensional organization of eukaryotic genomes might have evolved under a selective pressure to minimize the expression noise of essential gene clusters in the nucleus.

Active nucleosome positioning beyond intrinsic biophysics is revealed by in vitro reconstitution

Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions ('genomic code for nucleosome positioning'). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called 'statistical positioning' may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5' ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.

Functional aspects of cytidine-guanosine dinucleotides and their locations in genes.

Originally, the finding of a particular distribution of cytidine-guanosine dinucleotides (CpGs) in genomic DNA was considered to be an interesting structural feature of eukaryotic genome organization. Despite a global depletion of CpGs, genes are frequently associated with CpG clusters called CpG islands (CGIs). CGIs are prevalently unmethylated but often found methylated in pathologic situations. On the other hand, CpGs outside of CGIs are generally methylated and are found mainly in the heterochromatic fraction of the genome. Hypomethylation of those CpGs is associated with genomic instability in malignancy. Additionally, CpG-rich and CpG-poor regions, as well as CpG-shores, are defined. Usually, the methylation status inversely correlates with gene expression. Methylation of CpGs, as well as demethylation and generation of hydroxmethyl-cytosines, is strictly regulated during development and differentiation. This review deals with the relevance of the organizational features of CpGs and their relation to each other.