LHX2- and LDB1-mediated trans interactions regulate olfactory receptor choice.

The genome is partitioned into topologically associated domains and genomic compartments with shared chromatin valence. This architecture is constrained by the DNA polymer, which precludes interactions between genes on different chromosomes. Here we report a marked divergence from this pattern of nuclear organization that occurs in mouse olfactory sensory neurons. Chromatin conformation capture using in situ Hi-C on fluorescence-activated cell-sorted olfactory sensory neurons and their progenitors shows that olfactory receptor gene clusters from 18 chromosomes make specific and robust interchromosomal contacts that increase with differentiation of the cells. These contacts are orchestrated by intergenic olfactory receptor enhancers, the 'Greek islands', which first contribute to the formation of olfactory receptor compartments and then form a multi-chromosomal super-enhancer that associates with the single active olfactory receptor gene. The Greek-island-bound transcription factor LHX2 and adaptor protein LDB1 regulate the assembly and maintenance of olfactory receptor compartments, Greek island hubs and olfactory receptor transcription, providing mechanistic insights into and functional support for the role of trans interactions in gene expression.

Excess crossovers impede faithful meiotic chromosome segregation in C. elegans.

During meiosis, diploid organisms reduce their chromosome number by half to generate haploid gametes. This process depends on the repair of double strand DNA breaks as crossover recombination events between homologous chromosomes, which hold homologs together to ensure their proper segregation to opposite spindle poles during the first meiotic division. Although most organisms are limited in the number of crossovers between homologs by a phenomenon called crossover interference, the consequences of excess interfering crossovers on meiotic chromosome segregation are not well known. Here we show that extra interfering crossovers lead to a range of meiotic defects and we uncover mechanisms that counteract these errors. Using chromosomes that exhibit a high frequency of supernumerary crossovers in Caenorhabditis elegans, we find that essential chromosomal structures are mispatterned in the presence of multiple crossovers, subjecting chromosomes to improper spindle forces and leading to defects in metaphase alignment. Additionally, the chromosomes with extra interfering crossovers often exhibited segregation defects in anaphase I, with a high incidence of chromatin bridges that sometimes created a tether between the chromosome and the first polar body. However, these anaphase I bridges were often able to resolve in a LEM-3 nuclease dependent manner, and chromosome tethers that persisted were frequently resolved during Meiosis II by a second mechanism that preferentially segregates the tethered sister chromatid into the polar body. Altogether these findings demonstrate that excess interfering crossovers can severely impact chromosome patterning and segregation, highlighting the importance of limiting the number of recombination events between homologous chromosomes for the proper execution of meiosis.

Nuclear architecture dictates HIV-1 integration site selection.

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

Causes and consequences of nuclear gene positioning.

The eukaryotic genome is organized in a manner that allows folding of the genetic material in the confined space of the cell nucleus, while at the same time enabling its physiological function. A major principle of spatial genome organization is the non-random position of genomic loci relative to other loci and to nuclear bodies. The mechanisms that determine the spatial position of a locus, and how position affects function, are just beginning to be characterized. Initial results suggest that there are multiple, gene-specific mechanisms and the involvement of a wide range of cellular machineries. In this Commentary, we review recent findings from candidate approaches and unbiased screening methods that provide initial insight into the cellular mechanisms of positioning and their functional consequences. We highlight several specific mechanisms, including tethering of genome regions to the nuclear periphery, passage through S-phase and histone modifications, that contribute to gene positioning in yeast, plants and mammals.

Sgo1 regulates both condensin and Ipl1/Aurora B to promote chromosome biorientation.

Correct chromosome segregation is essential in order to prevent aneuploidy. To segregate sister chromatids equally to daughter cells, the sisters must attach to microtubules emanating from opposite spindle poles. This so-called biorientation manifests itself by increased tension and conformational changes across kinetochores and pericentric chromatin. Tensionless attachments are dissolved by the activity of the conserved mitotic kinase Aurora B/Ipl1, thereby promoting the formation of correctly attached chromosomes. Recruitment of the conserved centromeric protein shugoshin is essential for biorientation, but its exact role has been enigmatic. Here, we identify a novel function of shugoshin (Sgo1 in budding yeast) that together with the protein phosphatase PP2A-Rts1 ensures localization of condensin to the centromeric chromatin in yeast Saccharomyces cerevisiae. Failure to recruit condensin results in an abnormal conformation of the pericentric region and impairs the correction of tensionless chromosome attachments. Moreover, we found that shugoshin is required for maintaining Aurora B/Ipl1 localization on kinetochores during metaphase. Thus, shugoshin has a dual function in promoting biorientation in budding yeast: first, by its ability to facilitate condensin recruitment it modulates the conformation of the pericentric chromatin. Second, shugoshin contributes to the maintenance of Aurora B/Ipl1 at the kinetochore during gradual establishment of bipolarity in budding yeast mitosis. Our findings identify shugoshin as a versatile molecular adaptor that governs chromosome biorientation.

Global nucleosome positioning regulates salicylic acid mediated transcription in Arabidopsis thaliana.

BACKGROUND: The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level. RESULTS: In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5' regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes. CONCLUSIONS: This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.

The role of nucleosome positioning in the evolution of gene regulation.

Chromatin organization plays a major role in gene regulation and can affect the function and evolution of new transcriptional programs. However, it can be difficult to decipher the basis of changes in chromatin organization and their functional effect on gene expression. Here, we present a large-scale comparative genomic analysis of the relationship between chromatin organization and gene expression, by measuring mRNA abundance and nucleosome positions genome-wide in 12 Hemiascomycota yeast species. We found substantial conservation of global and functional chromatin organization in all species, including prominent nucleosome-free regions (NFRs) at gene promoters, and distinct chromatin architecture in growth and stress genes. Chromatin organization has also substantially diverged in both global quantitative features, such as spacing between adjacent nucleosomes, and in functional groups of genes. Expression levels, intrinsic anti-nucleosomal sequences, and trans-acting chromatin modifiers all play important, complementary, and evolvable roles in determining NFRs. We identify five mechanisms that couple chromatin organization to evolution of gene regulation and have contributed to the evolution of respiro-fermentation and other key systems, including (1) compensatory evolution of alternative modifiers associated with conserved chromatin organization, (2) a gradual transition from constitutive to trans-regulated NFRs, (3) a loss of intrinsic anti-nucleosomal sequences accompanying changes in chromatin organization and gene expression, (4) re-positioning of motifs from NFRs to nucleosome-occluded regions, and (5) the expanded use of NFRs by paralogous activator-repressor pairs. Our study sheds light on the molecular basis of chromatin organization, and on the role of chromatin organization in the evolution of gene regulation.

The nuclear lamina as a gene-silencing hub.

There is accumulating evidence that the nuclear periphery is a transcriptionally repressive compartment. A surprisingly large fraction of the genome is either in transient or permanent contact with nuclear envelope, where the majority of genes are maintained in a silent state, waiting to be awakened during cell differentiation. The integrity of the nuclear lamina and the histone deacetylase activity appear to be essential for gene repression at the nuclear periphery. However, the molecular mechanisms of silencing, as well as the events that lead to the activation of lamina-tethered genes, require further elucidation. This review summarizes recent advances in understanding of the mechanisms that link nuclear architecture, local chromatin structure, and gene regulation.

CD8 locus nuclear dynamics during thymocyte development.

Nuclear architecture and chromatin reorganization have recently been shown to orchestrate gene expression and act as key players in developmental pathways. To investigate how regulatory elements in the mouse CD8 gene locus are arranged in space and in relation to each other, three-dimensional fluorescence in situ hybridization and chromosome conformation capture techniques were employed to monitor the repositioning of the locus in relation to its subchromosomal territory and to identify long-range interactions between the different elements during development. Our data demonstrate that CD8 gene expression in murine lymphocytes is accompanied by the relocation of the locus outside its subchromosomal territory. Similar observations in the CD4 locus point to a rather general phenomenon during T cell development. Furthermore, we show that this relocation of the CD8 gene locus is associated with a clustering of regulatory elements forming a tight active chromatin hub in CD8-expressing cells. In contrast, in nonexpressing cells, the gene remains close to the main body of its chromosomal domain and the regulatory elements appear not to interact with each other.

Changes in chromatin structure and mobility in living cells at sites of DNA double-strand breaks.

The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.

Interchromosomal associations between alternatively expressed loci.

The T-helper-cell 1 and 2 (T(H)1 and T(H)2) pathways, defined by cytokines interferon-gamma (IFN-gamma) and interleukin-4 (IL-4), respectively, comprise two alternative CD4+ T-cell fates, with functional consequences for the host immune system. These cytokine genes are encoded on different chromosomes. The recently described T(H)2 locus control region (LCR) coordinately regulates the T(H)2 cytokine genes by participating in a complex between the LCR and promoters of the cytokine genes Il4, Il5 and Il13. Although they are spread over 120 kilobases, these elements are closely juxtaposed in the nucleus in a poised chromatin conformation. In addition to these intrachromosomal interactions, we now describe interchromosomal interactions between the promoter region of the IFN-gamma gene on chromosome 10 and the regulatory regions of the T(H)2 cytokine locus on chromosome 11. DNase I hypersensitive sites that comprise the T(H)2 LCR developmentally regulate these interchromosomal interactions. Furthermore, there seems to be a cell-type-specific dynamic interaction between interacting chromatin partners whereby interchromosomal interactions are apparently lost in favour of intrachromosomal ones upon gene activation. Thus, we provide an example of eukaryotic genes located on separate chromosomes associating physically in the nucleus via interactions that may have a function in coordinating gene expression.

Three-Dimensional Confocal Analysis of Chromosome Positioning Coupled with Immunofluorescence in Mouse Sperm Nuclei.

Male infertility is associated with several causes affecting the paternal nucleus such as DNA lesions (breaks, deletions, mutations, ...) or numerical chromosome anomalies. More recently, male infertility has also been associated with changes in the sperm epigenome, including modification in the topology of chromatin (Olszewska et al., Chromosome Research 16:875-890, 2008; Alladin et al., Syst Biol Reprod Med 59: 146-152, 2013) ref with number 1, 2. Indeed, the positioning of chromosomes in the sperm nucleus is nonrandom and defines chromosome territories (Champroux et al., Genes (Basel) 9:501, 2018) ref with number 3 whose optimal organization determines the success of embryonic development. In this context, the study of the spatial distribution of chromosomes in sperm cells could be relevant for clinical diagnosis. We describe here a in situ fluorescence hybridization (FISH) strategy coupled with a fluorescent immunocytochemistry approach followed by confocal analysis and reconstruction (2D/3D) as a powerful tool to analyze the location of chromosomes in the sperm nucleus using the mouse sperm as a model. Already, the two-dimensional (2D) analysis of FISH and immunofluorescence data reveal the location of chromosomes as well as the different markings on the spermatic nucleus. In addition, a good 3D rendering after Imaris software processing was obtained when Z-stacks of images were acquired over a defined volume (10 µm × 13 µm × 15 µm) with a sequential scanning mode to minimize bleed-through effects and avoid overlapping wavelengths.

Promiscuity of translocation partners in multiple myeloma.

Multiple myeloma (MM) is characterized by karyotypic instability, including chromosomal translocations involving the IGH locus. MM cells display a promiscuity of translocation partners, only some of which are recurrent. We propose that several factors, including temporal and spatial nuclear positioning of potential partner loci, "off-target" IGH diversification mechanisms, and aberrant repair pathways contribute to the promiscuity of translocation partners in MM. We speculate that in MM, IGH diversification processes [V(D)J recombination, somatic hypermutation, and class switch recombination] in B cells may not be restricted to specific stages of B-cell development or within specific immune tissues, but may occur in different temporal "windows." Before or during MM evolution, off-target activities of the enzymes involved in IGH modification processes may contribute to the generation of double-strand breaks (DSB) in translocation partner loci. In the parent B cells from which MM originates, spatial proximity within the nucleus of IGH and potential translocation partners contributes to the selection of a translocation partner and the clinical frequency at which a specific translocation occurs. The spatial proximity of IGH and specific translocation partners may be temporal and contribute not only to partner selection but also to the promiscuity of partners seen in MM. Lastly, aberrant repair mechanisms in MM progenitors (including the possibility that a Ku 86 variant allows for positional instability at DSBs) may also contribute to the promiscuity of chromosome translocation partners in MM.

Coordinate gene regulation during hematopoiesis is related to genomic organization.

Gene loci are found in nuclear subcompartments that are related to their expression status. For instance, silent genes are often localized to heterochromatin and the nuclear periphery, whereas active genes tend to be found in the nuclear center. Evidence also suggests that chromosomes may be specifically positioned within the nucleus; however, the nature of this organization and how it is achieved are not yet fully understood. To examine whether gene regulation is related to a discernible pattern of genomic organization, we analyzed the linear arrangement of co-regulated genes along chromosomes and determined the organization of chromosomes during the differentiation of a hematopoietic progenitor to erythroid and neutrophil cell types. Our analysis reveals that there is a significant tendency for co-regulated genes to be proximal, which is related to the association of homologous chromosomes and the spatial juxtaposition of lineage-specific gene domains. We suggest that proximity in the form of chromosomal gene distribution and homolog association may be the basis for organizing the genome for coordinate gene regulation during cellular differentiation.

Nuclear filaments: role in chromosomal positioning and gene expression.

Nuclear lamins form an elastic meshwork underlying the inner nuclear membrane and provide mechanical rigidity to the nucleus and maintain shape. Lamins also maintain chromosome positioning and play important roles in several nuclear processes like replication, DNA damage repair, transcription, and epigenetic modifications. LMNA mutations affect cardiac tissue, muscle tissues, adipose tissues to precipitate several diseases collectively termed as laminopathies. However, the rationale behind LMNA mutations and laminopathies continues to elude scientists. During interphase, several chromosomes form inter/intrachromosomal contacts inside nucleoplasm and several chromosomal loops also stretch out to make a 'loop-cluster' which are key players to regulate gene expressions. In this perspective, we have proposed that the lamin network in tandem with nuclear actin and myosin provide mechanical rigidity to the chromosomal contacts and facilitate loop-clusters movements. LMNA mutations thus might perturb the landscape of chromosomal contacts or loop-clusters positioning which can impair gene expression profile.

Positioning of human chromosomes in murine cell hybrids according to synteny.

Chromosomes occupy non-random spatial positions in interphase nuclei. It remains unclear what orchestrates this high level of organisation. To determine how the nuclear environment influences the spatial positioning of chromosomes, we utilised a panel of stable mouse hybrid cell lines carrying a single, intact human chromosome. Eleven of 22 human chromosomes revealed an alternative location in hybrid nuclei compared to that of human fibroblasts, with the majority becoming more internally localised. Human chromosomes in mouse nuclei position according to neither their gene density nor size, but rather the position of human chromosomes in hybrid nuclei appears to mimic that of syntenic mouse chromosomes. These results suggest that chromosomes adopt the behaviour of their host species chromosomes and that the nuclear environment is an important determinant of the interphase positioning of chromosomes.

Lamin A/C and Emerin depletion impacts chromatin organization and dynamics in the interphase nucleus.

BACKGROUND: Nuclear lamins are type V intermediate filament proteins that maintain nuclear structure and function. Furthermore, Emerin - an interactor of Lamin A/C, facilitates crosstalk between the cytoskeleton and the nucleus as it also interacts with actin and Nuclear Myosin 1 (NM1). RESULTS: Here we show that the depletion of Lamin A/C or Emerin, alters the localization of the nuclear motor protein - Nuclear Myosin 1 (NM1) that manifests as an increase in NM1 foci in the nucleus and are rescued to basal levels upon the combined knockdown of Lamin A/C and Emerin. Furthermore, Lamin A/C-Emerin co-depletion destabilizes cytoskeletal organization as it increases actin stress fibers. This further impinges on nuclear organization, as it enhances chromatin mobility more toward the nuclear interior in Lamin A/C-Emerin co-depleted cells. This enhanced chromatin mobility was restored to basal levels either upon inhibition of Nuclear Myosin 1 (NM1) activity or actin depolymerization. In addition, the combined loss of Lamin A/C and Emerin alters the otherwise highly conserved spatial positions of chromosome territories. Furthermore, knockdown of Lamin A/C or Lamin A/C-Emerin combined, deregulates expression levels of a candidate subset of genes. Amongst these genes, both KLK10 (Chr.19, Lamina Associated Domain (LAD+)) and MADH2 (Chr.18, LAD-) were significantly repressed, while BCL2L12 (Chr.19, LAD-) is de-repressed. These genes differentially reposition with respect to the nuclear envelope. CONCLUSIONS: Taken together, these studies underscore a remarkable interplay between Lamin A/C and Emerin in modulating cytoskeletal organization of actin and NM1 that impinges on chromatin dynamics and function in the interphase nucleus.

5-Bromouracil disrupts nucleosome positioning by inducing A-form-like DNA conformation in yeast cells.

5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the alpha2 repressor complex bound to its alpha2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA.

Genetic positioning of centromeres using half-tetrad analysis in a 4x-2x cross population of potato.

From biological and genetic standpoints, centromeres play an important role in the delivery of the chromosome complement to the daughter cells at cell division. The positions of the centromeres of potato were determined by half-tetrad analysis in a 4x-2x population where the male parent produced 2n pollen by first-division restitution (FDR). The genetic linkage groups and locations of 95 male parent-derived amplified fragment length polymorphism markers could be determined by comparing their position on a 2x-2x highly saturated linkage map of potato. Ten centromere positions were identified by 100% heterozygosity transmitted from the 2n heterozygous gametes of the paternal parent into the tetraploid offspring. The position of these centromeric marker loci was in accordance with those predicted by the saturated 2x-2x map using the level of marker clustering as a criterion. Two remaining centromere positions could be determined by extrapolation. The frequent observation of transmission of 100% heterozygosity proves that the meiotic restitution mechanism is exclusively based on FDR. Additional investigations on the position of recombination events of three chromosomes with sufficient numbers of markers showed that only one crossover occurred per chromosome arm, proving strong interference of recombination between centromere and telomere.

MLL/WDR5 Complex Regulates Kif2A Localization to Ensure Chromosome Congression and Proper Spindle Assembly during Mitosis.

Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer.