Cajal bodies are linked to genome conformation

The mechanisms underlying nuclear body (NB) formation and their contribution to genome function are unknown. Here we examined the non-random positioning of Cajal bodies (CBs), major NBs involved in spliceosomal snRNP assembly and their role in genome organization. CBs are predominantly located at the periphery of chromosome territories at a multi-chromosome interface. Genome-wide chromosome conformation capture analysis (4C-seq) using CB-interacting loci revealed that CB-associated regions are enriched with highly expressed histone genes and U small nuclear or nucleolar RNA (sn/snoRNA) loci that form intra- and inter-chromosomal clusters. In particular, we observed a number of CB-dependent gene-positioning events on chromosome 1. RNAi-mediated disassembly of CBs disrupts the CB-targeting gene clusters and suppresses the expression of U sn/snoRNA and histone genes. This loss of spliceosomal snRNP production results in increased splicing noise, even in CB-distal regions. Therefore, we conclude that CBs contribute to genome organization with global effects on gene expression and RNA splicing fidelity.

(CTs), CB interacting gene locus (HIST1) detected by DNA-FISH and CB localization visualized by CB marker protein coilin in aneuploid HeLa (CB-positive) and CB-deficient (siTCAB1) cells, (b) Box and whisker plot of DNA-FISH loci-CB association frequencies determined by manual scoring DNA FISH foci proximity to CBs using 6-color DNA FISH fluorescent microscopy images. Chromosome location is indicated below the X-axis. Black = non-CB interacting gene loci. Data represents assessment of at least 150 HeLa cells, (c) Validation of highly CB-interacting trans-chromosomal genomic regions identified by 4C analysis with the RNU1 bait by using 6-color DNA FISH fluorescent microscopy analysis of CB-interacting loci (various) by DNA-FISH and coilin (blue) immunostaining in HeLa cells targeting the HLB marker protein NPAT (red) and CB marker coilin (blue), respectively, (d) Microscopic fluorescent analysis of the role of major histone locus (HIST1) on chromosome 6 and minor histone locus (HIST2) in the formation of CBs and HLBs in HeLa cells. HIST1 (green) and HIST2 (white) DNA FISH foci denote the major and minor histone gene cluster loci on chromosomes 6 and 1, respectively. Immunofluorescent detection of HLBs and CBs was achieved using specific antibodies, (e) Cajal body numbers in siControl and siTCAB1-treated HeLa cells. N=2 (400 cells), **=p<0.01, significance was assessed by Student's T-test. Error bars represent SEM, (f) Representative TCAB1 and USPL1 immunoblots using extracts from HeLa cells exposed to indicated siRNA for up to 72 hours.
GAPDH was used as a loading control, (g) (h) Quantitative assessment of HIST1 localization in control HeLa cells (red) and CB-deficient cells (siTCAB1, blue). Scoring reflected the relative position within the CT6 territory, from completely internal (inside), to positions reflecting more active states either within or on the outside edge of the CT (inside edge & outside edge, respectively) as well as gene completely looped out of the CT (Outside).
(a) Representative 4C-seq contact profile of intra-chromosomal, CB-dependent interactions using a RNU1 bait sequence for HeLa WT (pink) and siTCAB1 (blue) treatments. A number of small U RNA genes were enriched within this region (dotted lines) which were analyzed following siTCAB1 and siUSPL1 (green) treatment by small RNA-seq. The expression of these genes within human primary cervical epithelial cells is also shown (purple). A number of small U genes were determined to be insensitive to TCAB1 or USPL1kd, possibly distinguished from sensitive genes by their higher expression in primary epithelial cervical cells. The Y-axis displays the read counts/genome fragment, (b) Expression of histone genes in D551 primary diploid fibroblasts following depletion of TCAB1 by siRNA. N=2, error bars represent SEM. D551 siControl = orange, D551 siTCAB1 = purple, (c) Representative 4C-seq contact profile validations of non-CB-interacting (4C negative contact) loci used in DNA-FISH experiments as negative control. Profiles display RNU1 bait to negative 4C region contacts on chromosome 1 and 17 in control (pink) and siTCAB1-treated (blue) HeLa cells. The Y-axis displays the read counts/genome fragment.
(a)(b) Genome-wide RNU1 4C-seq physical contact profile (red, log10 scale) in WT HeLa cells following CB disassembly using TCAB1 siRNA (B). For comparison, active gene density is shown 6 adjacently (blue, log10 scale), as well as small U RNA gene and histone gene expression (green and red, respectively), (c) 4C-seq replicate reproducibility for RNU1 and H3F3A bait analyses in HeLa cells treated with either control or TCAB1 siRNA treatments (siControl or siTCAB1, respectively).

Supplementary Figure 5
Karyogram depicting the locations of RNA expression changes, small U gene expression levels and alternative splicing events in HeLa cells following CB disassembly (siTCAB1/siUSPL1). Up-regulated genes (total RNA-seq) are denoted by green chromosome bands (non-concordant = dark, concordant = light green) and down-regulated by red (non-concordant = dark, concordant = light red). Baseline Represented here are seven nuclear-related functional groups of potential CB targets in HeLa cells which show a significant (p<0.01) reduction in RNA expression (identified by total RNA-seq) following TCAB1 or USPL1 siRNA treatment by CuffDiff or DEXseq analysis. Red or yellow arrows respectively identify TCAB1-or USPL1-only expression changes. Black arrows depict genes whose expression changed under both knockdown conditions. Genes that displayed alternative pre-mRNA splicing (assessed by Spanki and DEXseq) are italicized.