Positioning of human chromosomes in murine cell hy

Authors: Karen J Meaburn Robert F Newbold Joanna M Bridger

Publish Date: 2008/07/24

Volume: 117, Issue: 6, Pages: 579-

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Abstract

Chromosomes occupy nonrandom 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 chromosomesWe thank Dr Paul Perry and Prof Wendy Bickmore MRC HGU Edinburgh for use of the erosion analysis script and Mrs Alison Marriott Brunel University for providing the initial cell cultures We are grateful to Dr Tom Misteli for critical reading of the manuscript and Dr Ian Kill for helpful suggestions concerning the inception of this project For help in editing the manuscript we thank Dr Sara Snyder This work was supported by awards to JMB from Brunel UniversityDistribution of human proteins in hybrid nuclei Indirect immunofluorescence was performed on hybrid nuclei containing HSAs 1 19 and 13 on which LMNA LMNB2 or RB1 genes are located respectively The antibodies used recognise specifically the human forms of these proteins Parental A9 and a hybrid cell line lacking the human chromosome containing the gene of interest were used as negative controls and human fibroblasts HDF as a positive control JPG 846 MBRadial positioning of human chromosomes in hybrid nuclei in the presence or absence of selection antibiotic A92 A93 A918 and A9X hybrid cell lines were grown for 3 weeks in the absence of selection antibiotic hygromycin B After 2D FISH 50–60 nuclei per chromosome were submitted to erosion analysis The normalised chromosome signal mean of probe signal/ of DAPI signal for each of the five shells was plotted as a histogram Red bars denote the distribution of the human chromosome in hybrid cells grown in the absence of hygromycin B Black bars denote hybrid cells grown in the presence of hygromycin B data from Fig 2 Error bars show standard error of the mean The number or letter above a graph denotes the human chromosome represented No statistical differences were found for any chromosome when grown with or without hygromycin B as assessed by unpaired unequal variances twotailed Student’s t test at the 95 confidence level GIF 561 MB

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