Saturday, 22 December 2012

Nuclear organisation of gene expression

Dynamics and interplay of nuclear architecture, genome organisation, and gene expression by Schneider and Grosschedl
DNA transactions depend on genomic position of genes, subnuclear localisation of DNA sequences.

Chromatin domains
Traditionally euchromatin is associated with transcribed chromatin in an open chromatin conformation. Heterochromatin is associated with condensed chromatin which ins enriched in inactive and silenced chromatin region.

However, recently, Bickmore analysed chromatin structure. They fractionated chromatin into open, bulk and closed chromatin fibres and assayed distributed of gene density and activity in these fractions.

Open fibres are asssociated with highest gene density but not expression levels. Compact fibres have low density but can also have active genes. Ability of genes to be activated may not be lost when packed into compact fibres.

Inactive genes close to active genes in open chromatin can stay inactive. Location of gene in heterochromatin and euchromatin does not necessarily regulate gene activity. Covalent modifications of histones or DNA.

Transcription factories
Sites of active transcription are transcription factories. Traditionally it was thought that active genes recruit transcirption machinery. Machinery relocates to active genes.

3D-FISH, Immunofluorescence, and chromosome conformation capture (3C) analysis challenged this. Fraser et al showed that widely separated genes can colocalise to sites of active transcription  Genes are recruited to these sites, and most genes can move in and out of sites, causing activation or abatement of transcription.

Gene that can express at high levels seem to be constantly associated with transcription factories in cells that express these genes. Temporarily quiescent alleles are located away from the factories. Transcriptional activation of highly expressed genes eg immediate early genes involves relocalisation to preassembled transcription sites.

Organisation of chromosomes in interphase nuclei
Using probes for in situ hybridisation, it was observed that each chromosome occupies a specific territory in nucleus. The territories do not overlap. Most gene-rich territories concentrate in nuclear interior. Gene poor chromosomes tend to localise towards nuclear periphery.

On cell cycle exit a gene poor human chromosome can move from nuclear periphery to more internal site. After reentry into cell cycle, chromosome moves back to periphery.

Localisation in chromosome territories
Inactive genes tend to be in interior regions of CTs. Active genes concentrate along periphery.

Gene-rich MHC can be fond on large chromatin loops. DNA extends outwards from CT.  Transcriptional upregulation of MHC increases frequency this cluster was found in chromatin loop extending out of CT.  Surface of CT can be increased by looping out DNA into interchromosomal compartment and infoldings of interchromosomal compartment into CTs.

Studies show that chromatin fibres from periphery of CTs are intermingled in interphase nuclei. Blocking transcription can change pattern of intermingling of CTs without changing general properties of CTs.

Looped out activated gene locus of Hox B has increased interchromosomal interactions  Inactive locus favour intrachromosomal interactions with other loci on same chromosome.

Intrachromosomal interactions favour compactness of CT. Interchromosomal favours intermingling.

Homologous interchromosal interactions
On onset of X choromosome inactivation, X chromosomes move from nuclear periphery. Come into close proximity. Xic regions are juxtaposed.  During pairing, both X interact, allowing cross-talk. After they dissociate, inactive X, Xi is targeted to perinucleolar compartment.

Nonhomologous interchomosomal interactions
Flavell et al found genes on diff chromosomes associating physically to coordinate their expression. In naive T cells, Ifng and Th2 locus are localised together in a region in nucleus poised for gene expression. When native T cells are stimulated, Ifng (if T cells differentiate into TH1 cells) or Th2 locus (if they differentiate into TH2 cells) become activated. Other gene becomes inactive.  Activated gene keeps its nuclear position. It is poised for activation. Silent one is moved to a more repressive region of nucleus.


A three-dimensional model of the yeast genome by Duan et al


Our method relies on the 4C procedure by using cross-linking, two rounds of alternating restriction enzyme (RE) digestion (6-bp-cutter RE1 for the 3C-step digestion and 4-bp-cutter RE2 for the 4C-step digestion) and intra-molecular ligation. At step 7, each circle contains the 6-bp restriction enzyme recognition site originally used to link the two interacting partner sequences (RE1). Diverging from 4C, we relinearize the circles using RE1, then sequentially insert two sets of adaptors, one of which permits digestion with a type IIS or type III restriction enzyme (such as EcoP15I). Following EcoP15I digestion, fragments are produced that incorporate interacting partner sequence at either end, which can be rendered suitable for deep sequencin

Chromosomes III (ab) and XII (cd) are shown. The heat maps (ac) and Circos diagrams (bd) were generated using the intra-chromosomal interactions identified from the HindIII libraries at an FDR threshold of 1%. In the heat maps (ac), the chromosomal positions of centromeres (dashed pink lines), telomeres (pink hatches), mappable (green hatches) and un-mappable (black hatches) HindIII fragments are indicated. Circos diagrams (bd) depict each chromosome as a circle. Each arc connects two HindIII fragments and represents a distinct interaction. The shade of each arc, from very light grey to black, is proportional to the negative log of the P-value of the interaction. The chromosomal positions of centromeres (red rectangles), telomeres (red coloured areas), tRNA genes (blue outer hatches), mappable (green inner hatches) and un-mappable (black inner hatches) HindIII fragments are indicated. Black outer hatches and numbers mark genomic positions. Note that the two ends of chromosome XII (cd) exhibit extensive local interactions, but very little interaction with each other. Separating the ends of chromosome XII are 100–200 rDNA repeats, of which only two copies are depicted here (from coordinates 450 to 470kb). Additional heat maps and Circos diagrams for all chromosomes are shown in Supplementary Fig. 8.

a, Circos diagram showing interactions between chromosome I and the remaining chromosomes. All 16 yeast chromosomes are aligned circumferentially, and arcs depict distinct inter-chromosomal interactions. Bold red hatch marks correspond to centromeres. To aid visualization of centromere clustering, these representations were created using the overlap set of inter-chromosomal interactions identified from both HindIII and EcoRI libraries at an FDR threshold of 1%. Additional heat maps and Circos diagrams are provided in Supplementary Fig. 9b, Circos diagram, generated using the inter-chromosomal interactions identified from the HindIII libraries at an FDR threshold of 1%, depicting the distinct interactions between a small and a large chromosome (I and XIV, respectively). Most of the interactions between these two chromosomes primarily involve the entirety of chromosome I, and a distinct region of corresponding size on chromosome XIV. c, Inter-chromosomal interactions between all pairs of the 32 yeast chromosomal arms (the 10kb region starting from the midpoint of the centromere in each arm is excluded). For each chromosome, the shorter arm is always placed before the longer arm. Note that the arms of small chromosomes tend to interact with one another. The colour scale corresponds to the natural log of the ratio of the observed versus expected number of interactions (see Supplementary Materials). d, Enrichment of interactions between centromeres, telomeres, early origins of replication, and chromosomal breakpoints. To measure enrichment of strong interactions with respect to a given class of genomic loci, we use receiver operating curve (ROC) analysis.


Two views representing two different angles are provided. Chromosomes are coloured as in Fig. 4a (also indicated in the upper right). All chromosomes cluster via centromeres at one pole of the nucleus (the area within the dashed oval), while chromosome XII extends outward towards the nucleolus, which is occupied by rDNA repeats (indicated by the white arrow). After exiting the nucleolus, the remainder of chromosome XII interacts with the long arm of chromosome IV.
A culture of a bar1 derivative of Saccharomyces cerevisiae BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 bar1::KanMX) was crosslinked with 1% formaldehyde for 10min. Two sequential rounds of alternating restriction enzyme digestion, intra-molecular ligation, biotin-streptavidin-mediated purification, linear PCR amplification, and gel purification were carried out before construction of paired-end libraries. Libraries were paired-end sequenced using the Illumina Genome Analyzer 2, and sequence reads were mapped to the S. cerevisiae reference genome. To identify signal from background noise, we performed statistical confidence estimation. To estimate a false discovery rate (FDR), we eliminated self-ligations, ligations between adjacent restriction fragments, and ligations between restriction fragments separated by less than 20kb at their midpoints. To account for the strong influence of genomic proximity on ligation frequency, we subdivided the remaining intra-chromosomal interactions into 5kb bins as measured by the genomic distance between the midpoints of the two ligated fragments. Inter-chromosomal interactions were placed into a separate bin. In each bin, the observed interactions were ranked according to their sequence frequency and assigned a P-value relative to all other possible interactions in the same bin. Lastly, the P-value of each interaction was converted into a q value (defined as the minimal FDR threshold at which the interaction is deemed significant), and we used these values to rank interactions library-wide. After the true interaction sets were derived, further computational analyses were performed as described in detail in the online supplementary information.

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