An ectopic LCR does not activate a natural target gene on the homologous chromosome. (a) Schematic representation of the endogenous mouse and human β-globin loci. Below each globin gene, gene activity in (transgenic) mice is indicated. (b) Targeting strategy for the insertion of the human β-globin LCR and a human Aγ-globin–green fluorescent protein (GFP) reporter gene into the 8C3–C4 locus on mouse chromosome eight. (c) RT–qPCR of Aγ-globin transcript levels, normalized to Hprt1 transcript levels. Data are from at least two independent samples. (d) Representative examples of DNA FISH showing co-localized and separate 8C3–C4 alleles. DAPI, 4,6-diamidino-2-phenylindole. Scale bar: 2 μm. (e) Co-localization frequencies of 8C3–C4 alleles. Significance levels are indicated above the graph (G-test).

Contacts of 8C3–C4 with and without the LCR are similar. (a) Intrachromosomal DNA interactions of 8C3–C4 with (top) and without (bottom) an integrated β-globin LCR are essentially similar, as determined by 4C analysed with a running-mean analysis of microarray data (average probe spacing: 7 kb). (b) Intrachromosomal DNA interactions of 8C3–C4 with (top) and without (bottom) an integrated β-globin LCR are essentially similar, as determined by 4C analysed with domainograms that visualize probability scores (P-values indicated with colour codes) for the clustering of positive 4C signals over windows ranging in size from 1 to 200 probes. (c) Interchromosomal 4C data for two chromosomes (7 and 11), analysed as described above. (d) Validation of 4C results by cryo-FISH; examples of results. Scale bar: 2 μm. (e) Interaction frequencies with a series of genomic regions, measured by cryo-FISH in wild-type and LCR transgenic fetal livers. The number of cells analysed (n) is indicated. Colour codes indicate the significance of the 4C signal (probe clustering), with green referring to P <0.01 and red referring to P ≥0.01.

Within a predetermined genomic environment the ectopic LCR shows preferential interactions with specific genes. (a) 4C results (running median over sliding windows of 21 probes) for 8C3–C4 with (blue) and without (red) the integrated LCR, at the endogenous β-globin locus on chromosome 7 (left) and the α-globin locus on chromosome 11 (right). (b) Probes were binned according to increasing difference in (LCR – wild-type) 4C signal and characterized depending on their location relative to highly expressed and GATA-1- and EKLF-regulated genes. The yellow dashed line represents the expected frequency on the basis of all probes. (c) Venn diagram showing the number of, and overlap between, probes captured more frequently in the integrated LCR 4C experiment for each category of genes analysed in the population marked with an asterisk in b.

The ectopic LCR on chromosome 8 enhances the expression of the endogenous βh1 gene on chromosome 7. (a) Affymetrix gene-expression data for all probe-sets analysing βh1 transcripts (n = 3). (b) RT–qPCR comparison of βh1 gene expression between multiple wild-type and homozygous LCR-AS littermates, normalized to Hprt1 transcript levels and to own wild-type littermates. (c) RT–qPCR analysis showing that the insertion at 8C3–C4 of a neomycin selection cassette instead of the hLCR does not lead to upregulation of the βh1 gene. Data from two independent samples. (d) RT–qPCR analysis of expression of β- and α-globin genes in wild-type and homozygous LCR littermates. Error bars: standard error on the basis of 3 littermates (n).

Increased βh1 mRNA levels in cells showing interchromosomal LCR–βh1 interactions. (a) RNA FISH on E14.5 fetal liver cells, with one cell showing strongly increased βh1 mRNA levels in the cytoplasm (‘jackpot cell’). Scale bars: 2 μm. (b) Enlargement of the ‘jackpot cell’, revealing an interchromosomal interaction between the endogenous β-globin locus on chromosome 7 and the ectopic LCR on chromosome 8. (I–IV) Probes from one focal plane are shown separately and merged. (V) Z stack showing all RNA signals for β^maj and 8C3–C4. (c) Quantification of RNA FISH. Determining interchromosomal interaction frequencies between the endogenous β-globin locus and 8C3–C4 without (wild type) and with an integrated LCR (hLCR-AS), in all red blood cells and in ‘βh1 jackpot cells’. The number of cells analysed (n) is indicated. NS: no significant difference.

Increased βh1 and β-major mRNA levels in cells showing interchromosomal LCR–βh1 interactions. (a,b) Automated RNA-FISH image-analysis (see Methods) results, showing that cells in which the ectopic LCR interacts in trans with the endogenous β-globin locus more often have high βh1 (a) or β-major (b) transcript levels than cells that have the loci apart. (c) Cells in which the ectopic LCR interacts in trans with the endogenous α-globin locus do not differ in their levels of mRNA for α-globin from cells without this interchromosomal interaction. The probability score for the difference in distributions for interacting and non-interacting cells is calculated by a one-sided Kolmogorov–Smirnov test. (d) SEV: variegated expression among otherwise identical cells caused by cell-specific long-range DNA interactions (intra- or interchromosomal) that are relatively stable during interphase.