Depletion of the Chromatin Looping Proteins CTCF and Cohesin Causes Chromatin Compaction: Insight into Chromatin Folding by Polymer Modelling
(A, B) The genomic context of analysed regions of the q-arms of human chromosome 1 (A) and chromosome 11 (B) is illustrated by the human transcriptome map. Vertical lines represent protein coding genes, the length depicting the average transcription level in a number of human tissues and cell lines . The location of BAC probes used for FISH labelling are indicated above the maps. Arrows above the maps designate the regions where spatial distances between pairs of BAC probes were measured; the start of the arrow indicating the position of the reference BAC probe. On chromosome 1q three regions were analysed. Two 3 Mb regions were selected in a gene-rich and transcriptionally active region (green box) and in a region characterized by low gene density and low transcription (red box). (C–F) Graphs show the mean square physical distances <R2> between the FISH probes as a function of the genomic distance g for a 3 Mb gene-rich (C) and a gene-poor (D) region on chromosome 1 q-arm. Longer regions of chromosome 1 (30 Mb) (E) and of chromosome 11 (76 Mb) (F) were also analysed. Each data point represents an average of 30–150 distance measurements in at least two independent experiments. Error bars represent standard errors. Graphs are shown for control cells (black dots) and cells after knockdown of CTCF (green diamonds), of cohesin subunit Rad21(blue triangles) and of CTCF and Rad21 simultaneously (red squares). Results show that chromatin becomes more compact after knockdown of CTCF and Rad21 (cohesin). (G, H) Radial positions of BAC probes in nuclei for chromosome 1q (G) and chromosome 11q (H). Radial nuclear position of a BAC probe was calculated the distance between the centre of gravity of a FISH labelled genomic site and the centre of gravity of the nucleus and dividing that distance with the length of a straight line drawn from the nuclear centre to the nuclear envelope through the centre of the gravity of the labelled site. Error bars represent standard errors. Note that some chromatin regions were poorly labelled in CTCF and/or cohesin knock down conditions and therefore omitted from the graph.