Interchromosomal association and gene regulation in trans

Abstract

The nucleus is an ordered three-dimensional entity, and organization of the genome within the nuclear space might have implications for orchestrating gene expression. Recent technological developments have revealed that chromatin is folded into loops bringing distal regulatory elements into intimate contact with the genes that they regulate. Such intrachromosomal contacts appear to be a general mechanism of enhancer-promoter communication in cis. Tantalizing evidence is emerging that regulatory elements might have the capacity to act in trans to regulate genes on other chromosomes. However, unequivocal data required to prove that interchromosomal gene regulation truly represents another level of control within the nucleus is lacking, and this concept remains highly contentious. Such controversy emphasizes that our current understanding of the mechanisms that govern gene expression are far from complete.

Figure 1

3C based methodologies. Development of chromosome conformation capture (3C) technologies has enabled detailed analysis of chromosome conformations for the first time. (a) 3C begins with the formaldehyde treatment of living cells, which cross-links DNA sequences in close proximity at the time of fixation. Cross-linked DNA is purified and subjected to restriction digest, in this case HindIII (H). Fragments are diluted and incubated with DNA ligase resulting in intramolecular-ligation of cross-linked fragments. Ligation of pre-selected genomic regions is quantified using locus specific PCR primers, . 3C is thus limited to the analysis of pre-determined regions of interest. (b) 4C is a variation of 3C. Cross linked and ligated templates are generated as in 3C. Cross-links are then reversed and the template is digested with a second ‘frequent cutting’ restriction enzyme such as DpnII (D) to reduce fragment sizes before a second round of ligation. This generates small DNA circles that form the template for inverse PCR using primers designed within the bait region (green). The resulting PCR products are hybridized to custom arrays (or sequenced), allowing interrogation of genome wide associations for a single pre-selected bait sequence. (c) Hi-C is the most recent adaptation of 3C and potentially allows analysis of all associations genome wide. In Hi-C cross-linked, digested 3C material is tagged with biotin and the ‘sticky ends’ generated by restriction digestion are filled in. The material is ligated and then sheared to generated small fragments, which are captured using streptavidin beads. Linkers are added and the material is subjected to paired end sequencing.

Figure 2

Cis looping: the active chromatin hub (ACH). (a)In fetal brain, where the β-globin genes are not expressed, the locus adopts a linear conformation. (b) In mature erythroid cells, where the β-globin gene is expressed, the locus forms chromatin loops which bring the LCR and distal hypersensitive sites (upstream HS-85/HS-62.5/HS-60.7 and downstream HS1) into proximity with the expressing globin gene, to form an active chromatin hub. Intervening sequences and olfactory receptor genes are looped out of this complex.

Figure 3

Interchromosomal associations of cytokine genes. (a) In murine naïve CD4⁺ T cells the TH2 locus (containing Il4, Il5, Il13 and Rad50) on chromosome 11 associates with the Ifng gene on chromosome 10. Differentiation into TH1 or TH2 effector cell results in loss of interchromosomal associations concomitant with induction of Ifng and TH2 cytokine expression respectively. (b) Representative three-dimensional fluorescence in situ hybridization (3D-FISH) and confocal analysis of associations in naïve CD4⁺ T cells. Red color represents a TH2 BAC probe, and green represents an Ifng BAC probe. DNA staining with DAPI is shown in blue. Each spot represents one allele. The upper panel shows a representative nucleus displaying an interchromosomal between a single allele of Ifng and a single allele of the TH2 locus. The lower panel shows a representative example nucleus displaying no interchromosomal associations between the Ifng and TH2 loci.

Figure 4

Dynamics of chromosomal association. (a) If associations are stable but present in only a proportion of cells, these cells might generate different responses (response 1 or 2). (b) If associations are dynamic all cells within a population have the potential to form associations. Most probably each cell would generate a similar response (response 3). However, it is also possible that cells with or without associations at the moment of stimulation might respond differently as in (a).

Figure 5

Three types of Interchromosomal association. (a) Positioning of loci is probabilistic, determined by the sum of properties of neighboring sequences and the chromosome as a whole. Associations have no obvious functional significance. (b) Co-transcribed genes coalesce in and around nuclear bodies such as transcription factories and splicing speckles. As such bodies might be specialized in transcribing similar genes, these associations could help in coordinating gene expression and increase efficiency of transcription. Alternatively, these associations may be probabilistic as in (a) and have no functional significance. (c) Sequences regulate gene expression in trans through interchromosomal contacts.