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SUMMARYEukaryotic chromosomes compact during mitosis and meiosis into elongated cylinders – and not the spherical globules expected of self-attracting long flexible polymers. This process is mainly driven by condensin-like proteins. Here, we present Brownian-dynamics simulations involving two types of such proteins. The first anchors topologically-stable and long-lived chromatin loops to create bottlebrush structures. The second forms multivalent bridges between distant parts of these loops without entrapping them. We show bridging factors lead to the formation of shorter and stiffer mitotic-like cylinders, without requiring any energy input. These cylinders have several features matching experimental observations. For instance, the axial condensin backbone breaks up into clusters as found by microscopy, and cylinder elasticity qualitatively matches that seen in chromosome pulling experiments. Additionally, simulating global condensin depletion or local faulty condensin loading gives phenotypes in agreement with experiments, and provides a mechanistic model to understand mitotic chromatin structure at common fragile sites.

The genomes of mammalian cells exist as chromatin—a complex and dynamic structure that both serves as the background and actively participates in all fundamental nuclear processes such as transcription, replication, and DNA repair. Chromatin is characterized by multiple levels of organization: at the primary level, DNA is wrapped around a set of proteins called histones; interactions between histones promote further folding of the nucleoprotein fiber into a 30-nm structure with an unknown mechanical composition. The 30-nm fibers are then additionally folded into higher-order domains with differing structural and functional properties, which are then arranged in the nucleus in a probabilistic manner, with gene-poor regions preferring the periphery and gene-rich regions accumulating in the interior. Every level of chromatin organization has relevance for genome stability. At the 30-nm fiber level, the chromatin response to DNA damage is driven by the “access, repair, restore model,” while higher levels of organization determine the frequency and nature of chromosomal translocations. A modern view of genome stability aims to integrate the influence of fundamental cellular processes such as transcription and replication with chromatin context to give a better understanding of the processes that shaped our genomes.

Human cells lacking RIF1 are highly sensitive to replication inhibitors, but the reasons for this sensitivity have been enigmatic. Here, we show that RIF1 must be present both during replication stress and in the ensuing recovery period to promote cell survival. Of two isoforms produced by alternative splicing, we find that RIF1-Long alone can protect cells against replication inhibition, but RIF1-Short is incapable of mediating protection. Consistent with this isoform-specific role, RIF1-Long is required to promote the formation of the 53BP1 nuclear bodies that protect unrepaired damage sites in the G1 phase following replication stress. Overall, our observations show that RIF1 is needed at several cell cycle stages after replication insult, with the RIF1-Long isoform playing a specific role during the ensuing G1 phase in damage site protection.

Summary Cells coordinate interphase-to-mitosis transition, but recurrent cytogenetic lesions appear at common fragile sites (CFSs), termed CFS expression, in a tissue-specific manner after replication stress, marking regions of instability in cancer. Despite such a distinct defect, no model fully provides a molecular explanation for CFSs. We show that CFSs are characterized by impaired chromatin folding, manifesting as disrupted mitotic structures visible with molecular fluorescence in situ hybridization (FISH) probes in the presence and absence of replication stress. Chromosome condensation assays reveal that compaction-resistant chromatin lesions persist at CFSs throughout the cell cycle and mitosis. Cytogenetic and molecular lesions are marked by faulty condensin loading at CFSs, a defect in condensin-I-mediated compaction, and are coincident with mitotic DNA synthesis (MIDAS). This model suggests that, in conditions of exogenous replication stress, aberrant condensin loading leads to molecular defects and CFS expression, concomitantly providing an environment for MIDAS, which, if not resolved, results in chromosome instability., Graphical Abstract, Highlights • Cytogenetic lesions appear at common fragile sites (CFSs) after replication stress • CFSs have impaired chromatin folding visible with molecular FISH probes • CFS lesions are marked by faulty condensin loading • Condensin depletion increases the frequency of CFS lesions, Common fragile sites are genomic regions that exhibit chromatin-folding defects in conditions of exogenous replication stress. Using FISH, Boteva et al. show those sites also have altered chromatin architecture in unperturbed conditions and show faulty condensin loading, which leads to chromatin-folding defects and mitotic DNA synthesis.

The onset of random X inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. Here we show that RIF1 and KAP1 are two fundamental factors for the definition of the asymmetry. Our data show that at the onset of mESC differentiation, upregulation of the long non-coding RNA Tsix weakens the symmetric RIF1 association with the Xist promoter, and opens a window of opportunity for a more stable association of KAP1. KAP1 is required to sustain high levels of Tsix, thus reinforcing and propagating the asymmetry, and, as a result, marking the future active X chromosome. Furthermore, we show that RIF1 association with the future inactive X chromosome is essential for Xist upregulation. This double-bookmarking system, based on the mutually exclusive relationships of Tsix and RIF1, and RIF1 and KAP1, thus coordinates the identification of the inactive and active X chromosomes and initiates a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X chromosome state.

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterised by increased expression of type I interferon (IFN)-regulated genes and a striking sex imbalance towards females. Through combined genetic, in silico, in vitro, and ex vivo approaches, we define CXorf21, a gene of hitherto unknown function, which escapes X-chromosome inactivation, as a candidate underlying the Xp21.2 SLE association. We demonstrate that CXorf21 is an IFN-response gene and that the sexual dimorphism in expression is magnified by immunological challenge. Fine-mapping reveals a single haplotype as a potential causal cis-eQTL for CXorf21. We propose that expression is amplified through modification of promoter and 3′-UTR chromatin interactions. Finally, we show that the CXORF21 protein colocalises with TLR7, a pathway implicated in SLE pathogenesis. Our study reveals modulation in gene expression affected by the combination of two hallmarks of SLE: CXorf21 expression increases in a both an IFN-inducible and sex-specific manner., Systemic lupus erythematosus (SLE) shows a striking bias towards higher prevalence in females. Here, the authors perform fine-mapping of an SLE-associated locus at Xp21.2 and characterise a candidate gene, CXorf21, as IFN-responsive in immune cells that shows sexually dimorphic expression.

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA

ObjectivesSystemic lupus erythematosus (SLE) is a chronic multisystem genetically complex autoimmune disease characterised by the production of autoantibodies to nuclear and cellular antigens, tissue inflammation and organ damage. Genome-wide association studies have shown that variants within the major histocompatibility complex (MHC) region on chromosome 6 confer the greatest genetic risk for SLE in European and Chinese populations. However, the causal variants remain elusive due to tight linkage disequilibrium across disease-associated MHC haplotypes, the highly polymorphic nature of many MHC genes and the heterogeneity of the SLE phenotype.MethodsA high-density case-control single nucleotide polymorphism (SNP) study of the MHC region was undertaken in SLE cohorts of Spanish and Filipino ancestry using a custom Illumina chip in order to fine-map association signals in these haplotypically diverse populations. In addition, comparative analyses were performed between these two datasets and a northern European UK SLE cohort. A total of 1433 cases and 1458 matched controls were examined.ResultsUsing this transancestral SNP mapping approach, novel independent loci were identified within the MHC region in UK, Spanish and Filipino patients with SLE with some evidence of interaction. These loci include HLA-DPB1, HLA-G and MSH5 which are independent of each other and HLA-DRB1 alleles. Furthermore, the established SLE-associated HLA-DRB1*15 signal was refined to an interval encompassing HLA-DRB1 and HLA-DQA1. Increased frequencies of MHC region risk alleles and haplotypes were found in the Filipino population compared with Europeans, suggesting that the greater disease burden in non-European SLE may be due in part to this phenomenon.ConclusionThese data highlight the usefulness of mapping disease susceptibility loci using a transancestral approach, particularly in a region as complex as the MHC, and offer a springboard for further fine-mapping, resequencing and transcriptomic analysis.

Genetic variants resulting in non-expression of complement C4A and C4B genes are common in healthy European populations and have shown association with a number of diseases, most notably the autoimmune disease, systemic lupus erythematosus. The most frequent cause of a C4 “null” allele, following that of C4 gene copy number variation (CNV), is a non-sense mutation arising from a 2 bp CT insertion into codon 1232 of exon 29. Previous attempts to accurately genotype this polymorphism have not been amenable to high-throughput typing, and have been confounded by failure to account for CNV at this locus, as well as by inability to distinguish between paralogs. We have developed a novel, high-throughput, paralog-specific assay to detect the presence and copy number of this polymorphism. We have genotyped healthy cohorts from the United Kingdom (UK) and Spain. Overall, 30/719 (4.17%) individuals from the UK cohort and 8/449 (1.78%) individuals from the Spanish cohort harboured the CT insertion in a C4A gene. A single Spanish individual possessed a C4B CT insertion. There is weak correlation between the C4 CT insertion and flanking MHC polymorphism. Therefore it is important to note that, as with C4 gene CNV, disease-association due to this variant will be missed by current SNP-based genome-wide association strategies.

International audience; The complement C4 locus is in the class III region of the MHC and exhibits copy number variation. Complement C4 null alleles have shown association with a number of diseases including systemic lupus erythematosus (SLE). However, most studies to date have used protein immunophenotyping and not direct interrogation of the genome to determine C4 null allele status. Moreover, a lack of accurate C4 gene copy number (GCN) estimation and tight linkage disequilibrium across the disease-associated MHC haplotypes has confounded attempts to establish whether or not these associations are causal. We have therefore developed a high through-put paralog ratio test (PRT) in association with two restriction enzyme digest variant ratio tests (REDVRs) to determine total C4 GCN, C4A GCN and C4B GCN. In the densely genotyped CEU cohort we show that this method is accurate and reproducible when compared to gold standard Southern blot copy number estimation with a discrepancy rate of 9%. We find a broad range of C4 GCNs in the CEU and the 1958 British Birth Cohort populations under study. In addition, SNP-CNV analyses show only moderate levels of correlation and therefore do not support the use of SNP genotypes as proxies for complement C4 GCN.

Background Systemic lupus erythematosus (SLE) is a complex genetic autoimmune disease characterised by B cell hyperactivity and up-regulation of type I IFN. The DRB1*03:01 extended haplotype confers the greatest genetic risk for SLE in Europeans, but the molecular mechanisms underlying this association are unclear. Both coding and non-coding genetic variants arising from this haplotype may contribute to disease risk, implicating a role for gene regulation. Genetic effects on gene expression are context-specific, depending on factors such as cell type and cell state. Objectives To investigate the effect of (i) IFN-α stimulation, (ii) DRB1*03:01 haplotypes and (iii) the haplotype-environment interaction between DRB1*03:01 and IFN-α on differential gene expression in ex vivo B cells. Methods RNA was extracted from ex vivo CD19+ B cells from 50 healthy European women harbouring DRB1*03:01 homozygous (n=17), heterozygous (n=3) and non- DRB1*03:01 (n=30) haplotypes. Gene expression from cells at rest (n=49) and after stimulation with IFN-α for 6 hours (n=33) was quantified using the Affymetrix Human Exon 1.0 ST array. The individual and interactive effects of IFN-α stimulation and the DRB1*03:01 haplotype on gene expression were analysed using a two-way ANOVA (FDR 5%) using Partek Genomics Suite. Pathway and disease enrichment were analysed using Ingenuity Pathway Analysis. Results 6,906 out of 15,468 genes are significantly differentially expressed in IFN-α-treated compared to resting cells, of which 1,263 have fold changes >2. As expected, the data is enriched for the IFN-α pathway (P=3.4x10 -2 ) and B cell receptor signalling genes (P=2.1x10 -12 ). Approximately 60% of SLE- and rheumatoid arthritis-associated genes from GWAS are significantly enriched in this dataset (P SLE =4.4x10 -2 , P RA =6.1x10 -6 ), in comparison to the non-autoimmune disease osteoarthritis (20%; P OA =0.6). Interestingly, IFN-α affects SLE-associated genes outside canonical type I IFN and B cell signalling pathways, such as ITGAM (P=1.9x10 -10 , ↓) and PTPN22 (P=4.5x10 -10 , ↓). Genes implicated in monogenic SLE such as TREX1 and DNASE1L (DNA repair) and C1R (complement pathway) are also differentially expressed on IFN-α stimulation. The only gene significantly differentially expressed between DRB1*03:01 and non- DRB1*03:01 samples is BTN3A2 , which is downregulated in DRB1*03:01 haplotypes in both resting (P=2.3x10 -2 ) and IFN-α-treated cells (P=1.2x10 -2 ). Furthermore, BTN3A2 levels differ between DRB1*03:01 and non- DRB1*03:01 individuals in response to IFN-α stimulation, implicating haplotype-environment interactions (P int =1.4x10 -7 ). Conclusions Stimulation of B cells with IFN-α influences the expression of genes associated with complex and monogenic forms of SLE not known to be involved in type I IFN or B cell signalling pathways. Individuals harbouring DRB1*03:01 haplotypes demonstrate downregulation of BTN3A2 , implicating effects independent of HLA alleles for this risk haplotype. Additionally, we show that IFN-α and DRB1*03:01 interactively downregulate BTN3A2 , shedding some light into the mechanism behind the known association of IFN-α exposure with SLE risk. Investigating the effects of disease-associated haplotypes on gene expression in relevant contexts such as IFN-α stimulation may help in resolving genetic associations and identifying candidate genes underlying susceptibility to complex diseases. Disclosure of Interest None declared

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease. Complete deficiency of complement component C4 confers strong genetic risk for SLE. Partial C4 deficiency states have also shown association with SLE, but despite much effort over the last 30 years, it has not been established whether this association is primarily causal or secondary to long-range linkage disequilibrium. The complement C4 locus, located in the major histocompatibility complex (MHC) class III region, exhibits copy-number variation (CNV) and C4 itself exists as two paralogs, C4A and C4B. In order to determine whether partial C4 deficiency is an independent genetic risk factor for SLE, we investigated C4 CNV in the context of HLA-DRB1 and MHC region SNP polymorphism in the largest and most comprehensive complement C4 study to date. Specifically, we genotyped 2,207 subjects of northern and southern European ancestry (1,028 SLE cases and 1,179 controls) for total C4, C4A, and C4B gene copy numbers, and the loss-of-function C4 exon 29 CT indel. We used multiple logistic regression to determine the independence of C4 CNV from known SNP and HLA-DRB1 associations. We clearly demonstrate that genetically determined partial C4 deficiency states are not independent risk factors for SLE in UK and Spanish populations. These results are further corroborated by the lack of association shown by the C4A exon 29 CT insertion in either cohort. Thus, although complete homozygous deficiency of complement C4 is one of the strongest genetic risk factors for SLE, partial C4 deficiency states do not independently predispose to the disease.

The onset of random X chromosome inactivation in mouse requires the switch from a symmetric to an asymmetric state, where the identities of the future inactive and active X chromosomes are assigned. This process is known as X chromosome choice. Here, we show that RIF1 and KAP1 are two fundamental factors for the definition of this transcriptional asymmetry. We found that at the onset of differentiation of mouse embryonic stem cells (mESCs), biallelic up-regulation of the long non-coding RNA Tsix weakens the symmetric association of RIF1 with the Xist promoter. The Xist allele maintaining the association with RIF1 goes on to up-regulate Xist RNA expression in a RIF1-dependent manner. Conversely, the promoter that loses RIF1 gains binding of KAP1, and KAP1 is required for the increase in Tsix levels preceding the choice. We propose that the mutual exclusion of Tsix and RIF1, and of RIF1 and KAP1, at the Xist promoters establish a self-sustaining loop that transforms an initially stochastic event into a stably inherited asymmetric X-chromosome state.