Abstract
Background
X chromosome inactivation, the mechanism used by mammals to equalise dosage of X-linked genes in XX females relative to XY males, is triggered by chromosome-wide localisation of a cis-acting non-coding RNA, **st. The mechanism of **st RNA spreading and **st-dependent silencing is poorly understood. A large body of evidence indicates that silencing is more efficient on the X chromosome than on autosomes, leading to the idea that the X chromosome has acquired sequences that facilitate propagation of silencing. LINE-1 (L1) repeats are relatively enriched on the X chromosome and have been proposed as candidates for these sequences. To determine the requirements for efficient silencing we have analysed the relationship of chromosome features, including L1 repeats, and the extent of silencing in cell lines carrying inducible **st transgenes located on one of three different autosomes.
Results
Our results show that the organisation of the chromosome into large gene-rich and L1-rich domains is a key determinant of silencing efficiency. Specifically genes located in large gene-rich domains with low L1 density are relatively resistant to **st-mediated silencing whereas genes located in gene-poor domains with high L1 density are silenced more efficiently. These effects are observed shortly after induction of **st RNA expression, suggesting that chromosomal domain organisation influences establishment rather than long-term maintenance of silencing. The X chromosome and some autosomes have only small gene-rich L1-depleted domains and we suggest that this could confer the capacity for relatively efficient chromosome-wide silencing.
Conclusions
This study provides insight into the requirements for efficient **st mediated silencing and specifically identifies organisation of the chromosome into gene-rich L1-depleted and gene-poor L1-dense domains as a major influence on the ability of **st-mediated silencing to be propagated in a continuous manner in cis.
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Background
Classical studies on X; autosome rearrangements have demonstrated that X inactivation propagates in cis from a single locus on the X chromosome, the X inactivation centre (** of ** of each transgene (Tg) by DNA FISH, indicating position of nearest BAC probes in Mb. (b) RNA FISH analysis illustrating ** by fluorescence in situ hybridization. Proc Natl Acad Sci USA. 1990, 87: 7757-7761. 10.1073/pnas.87.19.7757." href="/article/10.1186/1756-8935-3-10#ref-CR24" id="ref-link-section-d190910995e749">24]. HL1 domains are often gene poor but are enriched for specific classes of genes, for example olfactory and vomeronasal receptor genes (Additional file 2). LL1 domains on the other hand are highly enriched for genes and also for CpG islands. The extent of HL1 and LL1 domains ranges from <500 Kb through to several Mb. The X chromosome is exceptional in having relatively high L1 and FL-L1 density throughout with only a few gene-rich LL1 domains that are in turn atypically small (Additional file 3).
As illustrated in Figure 3, downregulated genes were located along the entire length of the transgene-bearing chromosomes, chromosome 3, 12 and 17 regardless of their overall length (approximately 160, 120 and 100 Mb, respectively). Their distribution broadly mirrors overall gene density and distance from the transgene integration site does not appear to affect the probability of silencing. These results substantiate RNA FISH data indicating that ** X inactivation. Trends Genet. 2003, 19: 432-438. 10.1016/S0168-9525(03)00177-X." href="/article/10.1186/1756-8935-3-10#ref-CR26" id="ref-link-section-d190910995e913">26]. In the case of Is1ct discontinuous inactivation is at least in part attributable to 'spread and retreat' of X inactivation where chromosome 7 genes are initially silenced and then progressively reactivated [3]. In this study discontinuous inactivation is apparent when ** region (as well as a common Pvu II site in exon 5), using PGK-strain kidney cDNA as PCR template. The RT-PCR products were cloned into pBSX1 by coligation, using the Pvu II and Eco RI sites at exons 5 and 7, respectively, giving pBSX5. Finally, an 8.9 kb Sac II-Pac I exon 1 genomic DNA fragment (also from 129 strain background) was cloned into pBSX5 at the Pac I site, giving pBS** transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc Natl Acad Sci USA. 1997, 94: 3789-3794. 10.1073/pnas.94.8.3789." href="/article/10.1186/1756-8935-3-10#ref-CR35" id="ref-link-section-d190910995e1025">35] as described previously [9]. To test the efficiency of transgene induction doxycycline (1 μg/ml) was added to the medium and 24 h later the cells were screened for transgene expression by RT-PCR across ** data from Ensembl (version 46.36 g), up/downregulated probe sets were defined as those which were mapped to unique locations in the genome and with a FDR ≤ 5%. Downregulated genes were defined as those represented by at least one downregulated probe set and no upregulated probe set at FDR ≤ 5%. The expression value of a downregulated gene was calculated as the average of all associated downregulated probe sets. See Additional file 10 for further details.
Statistical modelling was used to determine whether there was a specific subset of genomic features that explained the observed patterns of gene expression. We employed non-parametric methods in order to model the conditional distribution of gene expression given the genomic features while also determining the importance of each feature and how the salient features interact with each other. Classification and regression trees (CART) models were fit to the experimental data because of their ability to model complex relationships between features, possibly measured on different scales, and deliver compact statistical representations or rules that can be easily visualized and interpreted. Parameter estimation was carried out using the recently developed conditional inference framework for fitting tree-based models based on permutation tests. This framework allowed us to perform feature selection using hypothesis testing procedures. See Additional file 10 for further details.
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Acknowledgements
We would like to thank the members of the CSC microarray facility for technical assistance and members of the lab for helpful discussion. This work was funded by the MRC UK and the Wellcome Trust.
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Authors' contributions
YAT carried out most of the experimental work, contributed to the bioinformatic and statistical analysis and to the study design and manuscript preparation. DH and GM contributed to the bioinformatic and statistical analysis and manuscript preparation. AC contributed to the experimental work and manuscript preparation. TBN and NB contributed to the study design and manuscript preparation. All authors read and approved the final manuscript.
Electronic supplementary material
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Additional file 1: Examples of L1 domain organisation. (a) Genome Environment Browser (GEB) histogram plot of mouse chromosome 4 illustrating the frequency of genes (green bars), CpG islands (lilac bars), full length L1 (FL-L1) elements (light blue bars), L1 homology sequences (yellow bars) and short interspersed nuclear elements (SINEs, grey bars) within 1-Mb intervals. The 25 Mb regions highlighted by a red box are shown in GEB detailed view in (b). (b) The 25 Mb region 1 (top panel) with boxed areas illustrating small (left) and moderate (right) sized gene-rich domains that are depleted for FL-L1. Note that gene density (green boxes) is higher in these domains. The 25 Mb region 2 (bottom panel) illustrates a large low L1 density (LL1) domain (approximately 22 Mb) flanked on each side by a small high L1 density (HL1) domain. (TIFF 4 MB)
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Additional file 2: Olfactory receptor (OR) gene clusters localise to high L1 density (HL1) domains. (a) Top panel: Genome Environment Browser (GEB) histogram display for mouse chromosome 9 illustrating one of the largest OR clusters in the mouse genome (marked by a red box), showing high gene density, low CpG island density and high full length L1 (FL-L1) density. Red arrows indicate other gene-rich regions on the chromosome that, as is generally the case, do not show FL-L1 enrichment. The lower panel shows the GEB detailed view of the OR cluster, black dotted lines marking the boundaries. (b) GEB detailed view illustrating a 6-Mb region covering the major histocompatibility complex (MHC) and an OR cluster on mouse chromosome 17 (34.0-40.0 Mb). Boxed areas mark the boundaries of the gene clusters. The OR genes and the MHC are in contrasting HL1 and low L1 density (LL1) domains, respectively. (c) GEB detailed view illustrating a 6-Mb region covering Hist1 histone (H) and vomeronasal receptor (VR) domains on mouse chromosome 13 (19.5-25.5 Mb). Boxed areas mark the boundaries of the gene clusters. Histone gene clusters are L1 depleted whereas VR, like OR, are in an HL1 domain. (d) Scatter plots illustrating L1 density of OR gene clusters in mouse and human (blue). Hist1 histone clusters are included for comparison (red). (TIFF 6 MB)
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Additional file 3: Unique organisation of high L1 density (HL1) and low L1 density (LL1) domains on the mouse X chromosome. (a) Genome Environment Browser (GEB) histogram display of features on the mouse X chromosome as described for Additional file 1. Boxed 25 Mb regions 1-4 that have relatively high gene density are shown as GEB detailed view in (b). (b) Detailed views of 25 Mb domains. Note that LL1 regions in which genes are concentrated are very small compared with typical autosomes and overall L1 density is high across the entire chromosome. (TIFF 7 MB)
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Additional file 5: Supplementary table 2. File contains Ensembl coordinates (version 46.36 g) for the position of high L1 density (HL1) and low L1 density (LL1) domains on chromosomes 3, 12 and 17. (DOC 438 KB)
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Additional file 6: Classification trees for downregulated genes on chromosome 17. Classification trees comparing (a) 10% most downregulated genes on chromosome 17 with all other genes. The first split divides the chromosome into two parts, proximal and distal of 35 Mb, based on the distance between the gene and a unique Lx5 element at 9.5 Mb (×82). The left side then shows that for the proximal 35 Mb of the chromosome downregulation is associated with location within a high L1 density (HL1) domain truncated by 500 kb at both ends (×37 >0). For genes distal to 35 Mb downregulation is either associated with location in a low L1 density (LL1) domain less than 1.3 Mb in size (×30), or in larger LL1 domains associated with L1 density downstream of a gene (×25) >3.7%. For trees comparing (b) 30%, (c) 40% and (d) 50% most downregulated genes on chromosome 17 with all other genes, the first split from the top node divides the chromosome into two parts, proximal and distal of approximately 70 Mb, based on the distance between the gene and the nearest full-length (FL) L1_Mus2 element (×44) which can be found at four locations on chromosome 17 (20.7, 31.6, 41.2 and 54.5 Mb). In the proximal 70 Mb of the chromosome, the models suggest that genes less susceptible to silencing are located centrally in large LL1 domains, not associated with large HL1s, and/or surrounded by a low (≤ 12.77%) local L1 density. ×5, overlaps (1) or not (0) with an HL1 domain; ×25, L1 density 100 kb upstream of the gene; ×31, size of HL1 domain with which the gene is associated; ×33 and ×34 whether a gene overlaps (1) or not (0) with a core LL1 domain (LL1 truncated by 250 kb or 500 kb, respectively, at its proximal and distal boundaries). Y-axis denotes probability of a gene being silenced (black fill). (TIFF 492 KB)
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Additional file 7: Genes located in relatively large low L1 density (LL1) domains on chromosome 3 have a lower probability of being silenced. This effect was seen across the first five deciles, in classification trees comparing (a) 10%, (b) 20%, (c) 30%, (d) 40% and (e) 50% most downregulated genes against all other genes. For every decile, the tree estimated from the data only contained one split, which is either based on a LL1 domain size measure (×30), or on the location of the gene with respect to large LL1 domains (×33 and ×34). Generally, genes located in LL1 domains >1.31 Mb in size or those associated with 'core' LL1 regions (×33 or ×34 >0) have approximately 50% less chance of being silenced. Additional files 4 and 9 give further details on feature definition. (TIFF 565 KB)
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Additional file 8: Lower local short interspersed nuclear elements (SINE) density may be associated with gene silencing on chromosome 12. Classification trees for chromosome 12 data identified a reciprocal relationship between i**st mediated silencing and SINE density. The effect was only observed when we compared the 10% (a) and 20% (b) most strongly downregulated genes on the chromosome to all other genes. ×26, SINE density in the 100 kb region upstream of the gene. (TIFF 268 KB)
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Additional file 9: Regression trees for chromosome 12. (a) The first split (×80) measures the distance between the gene to the nearest full length (FL) Lx4B element (which is uniquely located at approximately 6.5 Mb), followed by the second split (×53) which records the orientation of the nearest FL L1 Md_F2 to the gene (1 = upstream of the gene, -1 = downstream). (There are 35 copies of FL L1 Md_F2 on chromosome 12 altogether.) The regression tree suggests that the gene silencing effect would be the strongest for a given gene not more than 18.89 Mb away from the FL Lx4B element, (in other words, in the proximal 25.5 Mb of the chromosome), with the nearest FL L1 Md_F2 element being downstream of the gene. (b) When estimated on a subset of genomic features, the regression tree modelled the gene silencing effect differently. Here the gene silencing effect is the strongest for genes with a relatively low local SINE density (≤ 14.5%) and not associated with the core of a large low L1 density (LL1) domain (×34 = 0). Note that genes located in the centre of a large LL1 domain have a median downregulation level of just 10% (node 4 of the tree), which is a very mild effect. Additional file 4 gives details on genomic feature definition. (TIFF 291 KB)
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Additional file 10: Supplementary methods. File includes supplementary methods for RNA and DNA fluorescence in situ hybridisation (FISH) analysis, immunofluorescence, detailed description of statistical analysis, genomic data analysis and supplementary references. (PDF 110 KB)
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Tang, Y.A., Huntley, D., Montana, G. et al. Efficiency of **st-mediated silencing on autosomes is linked to chromosomal domain organisation. Epigenetics & Chromatin 3, 10 (2010). https://doi.org/10.1186/1756-8935-3-10
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DOI: https://doi.org/10.1186/1756-8935-3-10