Zhonghui Tang

Oscar Junhong Luo

Xingwang Li

Meizhen Zheng

Jacqueline Jufen Zhu

Przemyslaw Szalaj

Pawel Trzaskoma

Adriana Magalska

Jakub Wlodarczyk

Blazej Ruszczycki

Paul Michalski

Emaly Piecuch

Ping Wang

Danjuan Wang

Simon Zhongyuan Tian

May Penrad-Mobayed

Laurent M Sachs

Xiaoan Ruan

Chia-Lin Wei

Edison T Liu

Grzegorz M Wilczynski

Dariusz Plewczynski

Guoliang Li

Yijun Ruan
email: yijun.ruan@jax.org.

CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

The human genome consists of more than 3 billion nucleotides, spanning over 2 meters in length. Packaging this genomic material within the micrometer-sized nuclear space requires extensive folding (Bickmore, 2013). Such folding is presumed to be both specific and functional [START_REF] Ong | CTCF: an architectural protein bridging genome topology and function[END_REF]. However, details regarding general folding principles, distinct topologies and/or relationships to gene activity are still largely unknown.

Current technologies in studying 3-dimensional (3D) structures of the human genome include 3D-FISH nuclear imaging and 3D genome mapping. 3D-FISH (fluorescence in situ hybridization) can visualize realistic chromosome conformation and individual contacts within nucleus [START_REF] Cremer | Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes[END_REF]. However, it lacks sufficient genomic detail and accuracy. The core strategy in 3D genome mapping is nuclear proximity ligation [START_REF] Cullen | Interaction between transcription regulatory regions of prolactin chromatin[END_REF], which allows detection of distant genomic segments residing in close spatial proximity to one another, yet are linearly far away. Using this strategy, a number of highthroughput methods have been developed for genome-wide chromatin interaction mapping, including ChIA-PET and Hi-C [START_REF] Fullwood | An oestrogen-receptor-alpha-bound human chromatin interactome[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]. ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag sequencing) was designed to detect genome-wide chromatin interactions mediated by specific protein factors, whereas Hi-C (High-throughput Chromosome Conformation Capture) was developed to capture all chromatin contacts. Hi-C has been proven effective for mapping large-scale structures, such as topologically associated domains (TAD) [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF]; however, it lacks the resolution to detect precise functional interactions mediated by proteins. In contrast, by inclusion of ChIP (chromatin immunoprecipitation), ChIA-PET is unique in detecting protein factor mediated chromatin interactions and is capable of generating high-resolution (~100bp) genome-wide chromatin interaction maps with binding-site specificity among functional elements in human and mouse [START_REF] Li | Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation[END_REF][START_REF] Zhang | Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations[END_REF].

To comprehensively characterize the 3D topology of chromatin interactions between functional elements and higher-order organization in the human genome, we applied ChIA-PET, targeting on two protein factors, CTCF (CCCTC-binding zinc finger protein) and RNAPII (RNA polymerase II) in a number of human cell lines. CTCF is the master weaver of genome organization [START_REF] Ong | CTCF: an architectural protein bridging genome topology and function[END_REF], and Hi-C studies further correlated CTCF binding at TAD boundaries [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. RNAPII is involved in transcription of all protein-coding and many non-coding genes [START_REF] Sims Rj 3rd | Recent highlights of RNA-polymerase-II-mediated transcription[END_REF].

Therefore, comprehensive analyses of chromatin interactions mediated by these two factors have the potential to reveal the overall relationship between organizational structure and transcriptional function. Herein, we demonstrate that ChIA-PET is inclusive for mapping both ChIP-enriched and non-enriched chromatin contacts with haplotype specificity and nucleotide resolution, and we uncovered detailed chromatin topology that provide the framework for regulating transcriptional activity.

Results

I. ChIA-PET is multifaceted for chromatin interaction mapping

In addition to the original ChIA-PET data deposited in the ENCODE project (ENCODE Project Consortium, 2012), we have generated new CTCF-and RNAPII-mediated chromatin interaction datasets using an improved ChIA-PET protocol (Figure S1A) for longer reads (2x150bp). Altogether, we collected 364 million uniquely mapped ChIA-PET reads in 12 ChIA-PET libraries from four human cell lines: GM12878, HeLa, K562 and MCF7 for analysis (Table S1).

A ChIA-PET experiment delivers paired-end-tag (PET) sequencing data from self-ligation and inter-ligation products (Figure 1A, S1B). The self-ligation PET data identify ChIPenriched protein-binding sites. The clustered inter-ligation PET data detect enriched interactions mediated by the ChIP targeted protein factor, whereas the singleton interligation data reflects higher-order topological proximity, similar to Hi-C data (Figure S1B-E). Therefore, in theory, the multifaceted ChIA-PET data is ideal for comprehensive 3D genome mapping.

Recently, a study using in situ Hi-C generated 4.9 billion contact reads in GM12878 cells and found the majority of chromatin interaction loops to be associated with CTCF-binding sites [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. We compared this dataset with our CTCF ChIA-PET and demonstrate that the two datasets displayed very similar contact patterns (Figure 1B). In addition, ChIA-PET identified specific CTCF loops with binding-site resolution at 100s bp level. Importantly, only a single HiSeq 2500 rapid mode run or even a MiSeq run was sufficient for ChIA-PET to reach the same output as in situ Hi-C, plus 10-fold higher resolution. Thus, ChIA-PET is cost-effective, inclusive and reproducible (Data S1, I; Extended Experimental Procedures) of generating multi-layer mapping data, capturing protein binding sites and enriched functional chromatin interactions, as well as non-enriched chromatin contacts for higher-order chromosomal conformations.

II. CTCF organizes chromatin contact domain into CTCF foci

CTCF-mediated chromatin interactions are pervasive across the entire genome in the four tested human cell lines (Figure S2A). Based on the CTCF interaction clustering scheme, we identified 53,741 high quality CTCF-mediated interactions in GM12878 cells (Figure S2B-E; Extended Experimental Procedures). Considering that cohesin protein complex is highly associated with CTCF in chromatin biology [START_REF] Ong | CTCF: an architectural protein bridging genome topology and function[END_REF], we examined the CTCF-anchor sites for co-occupancy by cohesin using ChIP-Seq data of subunits RAD21 and SMC3. The vast majority (99%) of the CTCF interactions had cohesion co-occupancy in either one (n=10,952) or both anchors (n=42,297). Moreover, interactions with cohesin support on both anchors had significantly higher contact frequency than those with cohesin only on one anchor (Figure 2A,left). Since the sites with co-occupancy of CTCF and cohesin represent the most biologically relevant regions in our study, we focused further analyses on this subset.

We investigated the CTCF-motifs in anchors of CTCF loops identified in this study. Of the 42,297 interactions, ~83% (n=35,230) had CTCF motifs in both anchors with unique orientation. Among these, 33.1% were in tandem (i.e. tandem loop) and 64.5% (n=22,709) in convergent orientation (i.e. convergent loop) (Figure 2A right; Figure S2F; Table S2; Extended Experimental Procedures). Thus, CTCF-mediated chromatin loops have a clear orientation preference (convergent) that represents strong interactions with high contact frequency (Figure 2A, right), in agreement with in situ Hi-C data [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. In addition, tandem loops were present in significant numbers with intermediate interacting strength and span (Figure 2A, S2G), and most of them (82%) were positioned within convergent loops, perhaps representing a subgroup with possible supplemental function. A possible reason for the tandem loops discovered in our study but not in Hi-C is likely due to the power of specific enrichment in CTCF ChIA-PET experiments (Table S3; Extended Experimental Procedures).

To further understand how cohesin cooperates with CTCF in chromatin biology, we analyzed ChIP-Seq data of RAD21 and SMC3 and suggest that cohesin may surround the CTCF occupancy but have a preference toward the 3' side of CTCF motif (Data S1, II). To more precisely define CTCF/cohesin co-occupancy, we performed ChIP-nexus [START_REF] He | ChIP-nexus enables improved detection of in vivo transcription factor binding footprints[END_REF] to identify the specific borders of DNA footprints bound by cohesin in relation to CTCF. The RAD21 ChIP-nexus data detected one 5' border that is very close to the 5' border of CTCF, and two 3' border sites with the weak one matching exactly to the CTCF 3' border and the stronger one being 40 bp downstream (Figure 2B; Data S1, II). Similar patterns were observed for SMC3 (Data S1, II). Collectively, these results suggest part of the cohesin ring complex directly overlap with the CTCF binding around the motif and embrace additional space downstream in the 3' direction.

Many CTCF/cohesion-mediated loops often interconnect and continuously cover large chromatin segments (Figure 2C). Based on connectivity and contact frequency (Figure S3A; Extended Experimental Procedures), we identified 2,267 CTCF-mediated chromatin contact domains (CCDs) in GM12878 cells (Figure 2D, S3B). Comparison to Hi-C data showed that CTCF ChIA-PET and non-selective Hi-C were highly concordant in detecting chromatin domain structures (Figure 2C-D, S3C-D), indicating that CTCF-associated chromatin interactions are abundant in human 3D nucleome.

At the 4,534 boundaries of the 2,267 CCDs, the majority contained inward-facing CTCFbinding motifs (Figure 2E,S3E). Many CCD boundaries contained multiple CTCF-binding peaks with inward-facing motifs (Figure 2C inserts; Figure S3F), suggesting a possible double-knot-tie mechanics for tightening domain end structures (Figure S3G-I; Extended Experimental Procedure). In contrast, tandem loops were evenly distributed within the CCD space (Figure S3I), and showed high consistency in motif orientation (Figure 2F,S3J).

Interconnected CTCF binding and looping with convergent and tandem motif orientations constitute the finer details in chromatin topology. Figure 2G illustrates a CCD composed of multiple CTCF loops. Theoretically, CTCF dimerization with motifs could be either symmetric or asymmetric. Since the cohesin ring complex is most likely bound toward the 3' downstream of CTCF motif (Figure 2B), we speculate that CTCF dimerization with two interacting motifs favors symmetric conformation. Therefore, a pair of convergent motifs would form a "hairpin loop", while a pair of tandem motifs form a "coiled loop" (Figure 2H). This principle may have critical topological implications for the 3D structure of CCD and the overall chromosomal topology and genome organization. The example CCD in Figure 2G shows all 8 CTCF anchors (a-h) to be interconnected, thus, suggesting anchors in this CCD form an aggregated scaffold of "CTCF/cohesin focus" (Figure 2I). Consequently, the overall properties of genome organization would be collectively shaped by the 2,267 CTCF/cohesin-foci (2,267 CCDs) in GM12878 cells.

III. RNAPII transcription factories are spatially associated with CTCF/cohesin foci

To functionally characterize CTCF/cohesin-mediated chromatin topology, we overlaid functional genomics data (RNAPII ChIA-PET, chromatin state and RNA-Seq) with CCD footprint on the genome landscape (Figure 3A). The results indicated that most transcription activity occurs within CTCF-looped chromatin structures (Figure S4A). Most of the RNAPII-associated loops are smaller than CTCF loops (Figure S4B), and the vast majority of RNAPII-looping structures are included within CCD-defined genomic space (Figure S4B-C; Table S2).

To dissect the associations with transcription, we divided CTCF/cohesin-bound chromatin loop structures into "anchor" and "loop" regions, and then examined their epigenomic and transcriptional features (Extended Experimental Procedures). Unlike the loop regions, the anchors were enriched with active epigenomic markers, RNAPII occupancy and the presence of TSS (transcription start site) (Figure 3B), suggesting that CTCF-anchor regions are the foci for transcriptional activity. Unexpectedly, further detailed analyses focusing on anchors uncovered structure-function features related to transcription activity and directionality at three distinct levels. First, signals of active epigenomic markers tend to be higher towards 3' direction of CTCF-motif for both convergent and tandem loops (Figure 3C). More strikingly, TSS at anchor regions showed clear strand-specificity and directional enrichment along with CTCF motif orientation, indicating that a sub-group of genes are prepositioned within the CTCF-defined anchor regions with their promoters in harmony with CTCF motif orientation, thus dictating the directionality of transcription. Second, active epigenomic markers, RNAPII and TSS densities were highly enriched around the anchors of tandem loops compared to the convergent loops (Figure 3C). The same trend was also observed for B-cell specific transcription factors (TFs), e.g. ELF1 and ZEB1, but not for chromatin structural proteins CTCF, RAD21 and ZNF143 (Figure 3D). Third, the paired anchors involved in tandem loops also exhibited directionality: the "head" anchor tends to have higher signal intensities of active epigenomic markers, RNAPII and B-cell specific TF binding than the "tail" anchor (Figure 3C-D, S4D), while there was no such notable difference for the anchors of convergent loops. It implies that the head anchor in tandem loops may have more promoter property, whereas the tail anchor could possess more enhancer potential. To test this, H3K4me1 (an enhancer marker) and H3K4me3 (a promoter marker) ChIP-Seq data were used to assess the relative strength of promoter and enhancer. The ratio of H3K4me1/H3K4me3 at the tail anchor of tandem loops was significantly higher than the head anchor (Figure 3E right), indicating that the tail anchor is more likely involved in enhancer function, whereas the head anchor is more related to promoter. In contrast, no difference was observed for the paired anchors of convergent loops (Figure 3E left). Collectively, these data suggest that tandem loops possess distinctive features important for organizing transcription activity.

In GM12878 cells, thousands of genes and enhancers were found proximal to CTCF/cohesin anchors (i.e. anchor-genes/enhancers), and the rest were scattered within the CTCF/cohesin loop regions (i.e. loop-genes/enhancers) (Extended Experimental Procedures). We examined the anchor-and loop-genes based on their expression profiles in 56 different human tissues. Gene expression breadth analysis (Extended Experimental Procedures) indicated that anchor-genes were significantly less tissue-specific than loop-genes (Figure 3F left). Further analysis revealed that active anchor-genes were almost exclusively housekeeping (Figure 3F right; Figure S4E-F), emphasizing the notion that CTCF interaction anchors selectively enrich for constitutively expressed genes.

To investigate how active anchor-genes relate to active loop genes and enhancers, we examined their connectivity by RNAPII ChIA-PET, as described previously [START_REF] Li | Extensive promoter-centered chromatin interactions provide a topological basis for transcription regulation[END_REF]. Using the newly generated RNAPII ChIA-PET data (Table S1), most active loopgenes were found connected to anchor-genes and/or anchor-enhancers (Table S2; Extended Experimental Procedures) through RNAPII-mediated interactions. Therefore, most active genes are connected, either directly or indirectly, to the anchors of CTCF loops (Figure 3G), suggesting that anchor-genes and anchor-enhancers could serve as nucleation points to aggregate related loop-genes towards corresponding CTCF/cohesin anchors for coordinated transcription. Figure 3H illustrates an example CCD with seven interconnecting CTCF anchors and a number of CTCF anchor-genes/enhancers and loop-genes/enhancers. RNAPII interactions indicated that these genes and enhancers were interconnected, which could be viewed (spatially) as a transcription factory docked to the chromatin structural base of CTCF focus. More examples are shown in Figure S4G-H.

Together, CTCF and RNAPII ChIA-PET analyses, along with chromatin state and RNA readout data, revealed that the basic topological units of chromatin looping structures and transcriptional function are highly cooperative for housekeeping functions and cell-type specificity.

IV. Haplotype variants alter CTCF-mediated chromatin structure and function

Allelic differences between two homologous chromosomes can influence inheritance characteristics in the human genome [START_REF] Mcdaniell | Heritable individual-specific and allele-specific chromatin signatures in humans[END_REF]. The means by which allelespecific genetic variation affects chromatin organization has become an intriguing question [START_REF] Leung | Integrative analysis of haplotype-resolved epigenomes across human tissues[END_REF]. Hi-C analyses have demonstrated that nuclear proximity ligation is an efficient genome-wide approach for haplotype mapping of chromatin interactions [START_REF] Selvaraj | Whole-genome haplotype reconstruction using proximity-ligation and shotgun sequencing[END_REF]. Here, we sought to use ChIA-PET to investigate haplotype-specific chromatin interactions and subsequent structural and functional consequences.

We identified 65,718 phased PET reads mapped intra-chromosomally in the phased GM12878 genome, of which the vast majority (n=64,805, 98.6%) were cis-interacting PETs (Figure 4A, S5A-B; Extended Experimental Procedures). To investigate haplotype-specific chromatin interactions mediated by CTCF and RNAPII, we focused on phased PETs that were clustered to represent interactions enriched by these factors. Using this criterion, we identified 350 haplotype-biased anchors and 1,728 interactions mediated by CTCF, and 299 haplotype-biased anchors and 1,322 interactions mediated by RNAPII (Figure 4B-C; Extended Experimental Procedures).

Among the identified haplotype-biased chromatin interactions mediated by CTCF was the well-studied H19-IGF2 locus [START_REF] Nativio | Disruption of genomic neighbourhood at the imprinted IGF2-H19 locus in Beckwith-Wiedemann syndrome and Silver-Russell syndrome[END_REF] involved in genomic imprinting of autosomes (Figure S5C left, and additional example in Figure S5C right) thus demonstrating the robustness of our approach. We then explored whether allelic variations alter chromatin 3D structure between homologous chromosomes. Indeed, we found paternally biased superlong interactions (13Mb) mediated by CTCF on ChrX connecting the DXZ4 and FIRRE loci (Figure 4D), which has been reported previously [START_REF] Horakova | The macrosatellite DXZ4 mediates CTCF-dependent long-range intrachromosomal interactions on the human inactive X chromosome[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. In addition, we identified another super long-range interaction between FIRRE and G6PD (23Mb) exclusively on the same haplotype as indicated by both contact heatmaps and CTCF-enriched interactions. This interaction was further validated by DNA-FISH (Figure 4E).

Next, we investigated whether SNPs could directly alter chromatin topology and function at a finer scale (e.g. domain structure and individual loop). Others have demonstrated deletion and inversion of DNA fragments containing CTCF/cohesin binding sites could disrupt the nearby TAD structure and alter associated gene transcription [START_REF] Dowen | Control of cell identity genes occurs in insulated neighborhoods in mammalian chromosomes[END_REF][START_REF] Guo | CRISPR Inversion of CTCF Sites Alters Genome Topology and Enhancer/Promoter Function[END_REF]. Despite the success, CRISPR/Cas9-engineered regions in these studies involved from 100s to 1,000s bp, which could contain other sequence elements of unknown function. To avoid potential "collateral damage", we exploited the SNP "genotype" as single nucleotide "perturbation" and evaluated the corresponding CTCF binding/looping property as the "phenotype" in different human individuals (cell lines) with either heterozygous or homozygous allele composition (Figure 5A). We first focused on the 39 allele-biased CTCF interaction anchors (i.e., 39 loci with phased SNPs as "naturally occurred" single nucleotide "perturbation") located at CCD boundary regions. As shown in Figure 5B, a heterozygous SNP (maternal "T" and paternal "A") was located on the 3' boundary of a CCD. The maternal "T" exhibited strong CTCF binding (i.e. functional allele) while the paternal "A" allele showed weak binding (i.e. dysfunctional allele). We hypothesize that the loss of CTCF binding at the "A" allele in this locus would cause loss of CTCF-mediated looping and, in turn, alter CCD structure. To test this, we examined other individuals (cell lines) with homozygous "A/A" genotype at this locus. Indeed, in HeLa and MCF7 cells, no CTCFmediated interactions originated from this locus and the corresponding CCD structures in these two cell lines were drastically different from GM12878 (See another case in Figure S6A). Together, these analyses validated that functional CTCF sites are necessary to maintain the proper CCD boundaries, and suggest that single nucleotide variations in CTCF binding sites could alter chromatin topology.

We then explored if "naturally occurred SNP perturbation" could alter CTCF tandem loops and consequently impact transcription inside CCDs. We identified 50 loci where allelespecific tandem loops were associated with genes harboring phased SNPs in the gene body, thus, testable for allelic expression bias. From them, 22 (44%) displayed significant allelespecific expression (P < 0.05; See examples in Figure S6B). In a particular example, the promoters of DENND2D and CHI3L2 reside at the opposite anchors of a CTCF tandem loop that is paternal-specific (Figure 5C). Consistently, the RNAPII occupancy and associated chromatin loops in this region are also paternal-biased, indicating this CTCF tandem loop is functionally involved in a paternal-specific transcriptional regulation. However, only CHI3L2 exhibited paternal-biased gene expression (see also Figure S6C) but not DENND2D. This example supports that, in an allele-specific manner, the enhancer at the tail anchor of a CTCF tandem loop could interact with gene promoters proximal to the head anchor of the loop in concordance with the motif orientation for transcription regulation (Figure 3C-E). Additionally, the phased SNP with allele-biased CTCF-binding coverage was located inside the CTCF-binding motif, and this SNP was the only nucleotide difference between the two homologs in kilobase-wide span. This observation impelled us to systematically examine how SNPs in the CTCF-motif would subsequently influence CTCF binding and looping.

Of the SNPs mapped within the 350 allelic-specific anchors bound by CTCF (Figure 4C), 70 reside in the core motif (Figure S6D). The alignment of each of the heterozygous SNPs within the CTCF-motif showed that alleles having strong CTCF-binding possess canonical motif consensus, whereas alleles with weak or no binding had deviated motif sequences, especially with position 14 ("G") being the most affected (Figure 5D, S6E), suggesting that this "G" is critical for CTCF-binding affinity. For example, at position 14 of a CTCF motif in Chr17q21, the maternal and paternal alleles are G|C, respectively (Figure 5E). Despite this SNP being the only variable site in this locus and the surrounding kilobase region, the CTCF binding and interaction in maternal allele is 68-fold stronger than paternal, indicating that nucleotide "C" in the motif is disruptive for CTCF to bind to the paternal allele in this locus. Since SNP variation in CTCF motif could lead to such drastic alteration in CTCF binding and looping, we sought to determine whether changes in chromatin topology would link to human diseases.

We systematically assessed disease association of the disrupted CTCF-mediated interactions by examining the linkage disequilibrium (LD) between the 70 SNPs residing in CTCF motif (i.e. CTCF-SNPs) and GWAS identified disease associated SNPs (Extended Experimental Procedures). We hypothesized that disease-associated SNPs and dysfunctional CTCF-SNPs would be linked if they reside in strong LD blocks. In this setting, 32 of the 70 CTCF-SNPs were documented in dbSNP database, and 8 showed LD with disease associated SNPs in the tested populations (Data S1, IV; Table S4). Since GM12878 originated from an individual of European ancestry, we further focused on the CTCF-SNPs with disease association by LD in CEU population. One of the CTCF-SNPs found associated with asthma is SNP rs12936231 (Figure 5E). Of the two alleles (G|C), the dysfunctional "C" has been documented as a highrisk allele for asthma and autoimmune diseases, and suggested to alter chromatin remodeling and domain-wide transcription of certain genes (e.g. ZPBP2, GSDMB and ORMDL3; [START_REF] Verlaan | Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ ORMDL3 locus associated with the risk of asthma and autoimmune disease[END_REF]. Separate eQTL data also suggested that this allele alters the expression level of GSDMB and ORMDL3 [START_REF] Montgomery | Transcriptome genetics using second generation sequencing in a Caucasian population[END_REF][START_REF] Stranger | Population genomics of human gene expression[END_REF]. Interestingly, six additional asthma-associated SNPs were found in the same LD block (209kb) with this CTCF-SNP (rs12936231) in CEU having high pairwise correlation (D'>0.7), and the haplotype comprising these 7 risk alleles was frequently (0.422) observed in the CEU population (Figure 5E). Collectively, these results suggest that the disruption of CTCF motif by this "C" allele, which abrogates CTCF binding, looping, and chromatin topology, may be the primary molecular event leading to disease susceptibility, while the other 6 asthma-SNPs were likely non-causative "bystanders". Moreover, these findings highlight the potential of allelic chromatin topology analyses to infer mechanisms by which SNPs are associated with disease and traits.

V. RNAPII-mediated chromatin interactions regulate allele-specific transcription

To investigate whether allelic variations affect RNAPII-mediated chromatin interaction and/or result in functional consequences, we identified 1,322 haplotype-specific RNAPII interactions that involved 299 haplotype-specific RNAPII interaction anchors in GM12878 (Figure 4C; Extended Experimental Procedures). Our haplotype analyses showed significant ChrX maternal-specificity of RNAPII interactions and gene expression (Figure 4C), consistent with the fact that paternal-X in GM12878 is imprinted [START_REF] Rozowsky | AlleleSeq: analysis of allele-specific expression and binding in a network framework[END_REF]. Thus, at chromosomal scale, the connection between allele-specific RNAPII interaction and gene transcription is broadly established. To further validate that haplotype-specific chromatin interaction could lead to allele-specific transcription regulation (Figure 6A), we examined 40 TFs for their allelic binding specificity and found that the vast majority (95%) of the TF binding allele-biases were consistent with the RNAPII allele-specificity (Figure 6B; Extended Experimental Procedures). Furthermore, we identified 89 genes with allelebiased expression involved in allele-specific RNAPII interactions, and the majority (n=79) displayed the same allele-specificity in transcription as the allele-biased RNAPII interactions (Figure 6C-D; Table S5). For example, RNAPII binding and interactions over the SNPs at the XIST (X inactive specific transcript) locus showed highly consistent allele bias with haplotype-specific expression of XIST (Figure S5D). Such observations at the XIST locus demonstrate the accuracy of our haplotype chromatin interaction analysis.

Our data also reveals the haplotype effect of long-range enhancer-promoter interactions. For example, the promoters of LOC374443, CLEC2D and CLECL1 were in contact with an RNAPII-associated multi-gene complex (Figure 6E). It is observed RNAPII occupancy in the paternal allele at the upstream enhancer and promoter sites (300kb apart) were approximately 3-fold and 2.5-fold higher, respectively, than the matched maternal allele. In addition, the distal enhancer exhibited more than 3-fold higher paternal-biased binding by 3 B-cell specific TFs: BCL3, EBF1 and TCF12. We also observed 3.5-fold higher paternally biased expression in these three genes (Figure 6E). In contrast, nearby genes not directly involved in the RNAPII mediated interactions showed balanced expression between homologs. This example supports the notion that allele-specificity at distant enhancers is also effective in regulating allele-specific RNAPII activity at the target genes with high specificity.

VI. 3D genome models elucidate the human genome structure and function

Using an integrated 3D NucleOme Modeling Engine (3D-NOME) that builds a hierarchical tree structure to represent the 3D genome with increasing resolution (Figure S7A-C) (Szalaj et al., In preparation), we simulated the 3D genome models from the combined CTCF and RNAPII ChIA-PET data derived from GM12878. In these models, several known chromosomal features were captured, e.g. at the whole nucleome level, large chromosomes were preferentially projected in the periphery of nucleus, and small chromosomes were positioned in the inner nuclear space (Figure S7D-E; Extended Experimental Procedures). To gain further specificity at the level of individual chromosomes, we modeled Chr1 at different resolutions, and observed a putative conformation, whereby its two chromosomal arms bend and extend in the same direction (Figure 7A). Since our mapping data were derived from millions of cells, the predicted 3D model would reflect an average representation. It is possible that the body of Chr1 is fluidly changing between "open" and "closed" conformations (Figure 7B). Our 3D DNA-FISH results supported such speculation (Figure 7C; Extended Experimental Procedures).

From 3D genome simulation and DNA-FISH nuclear imaging, as well as the growing knowledge of chromatin topology [START_REF] Boyle | Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis[END_REF][START_REF] Kalhor | Genome architectures revealed by tethered chromosome conformation capture and population-based modeling[END_REF], a general feature of chromosome topology starts to emerge. Although much less condensed, chromosomes in interphase still maintain their core axes, comprised of mostly condensed heterochromatin segments (probably other matrix-filling material as well) (Figure 7D). The loosely organized "open" chromatin segments could extend laterally outward (as chromatin loops) around the chromosome axis, similar to the morphology of lampbrush chromosome [START_REF] Morgan | Lampbrush chromosomes and associated bodies: new insights into principles of nuclear structure and function[END_REF]. We envision CTCF and cohesin (possibly with other factors e.g. Topoisomerase II, [START_REF] Liang | Chromosomes Progress to Metaphase in Multiple Discrete Steps via Global Compaction/Expansion Cycles[END_REF] localizing on the surface of the core chromosome axis and defining the interface between the inner condensed (inside the chromosome axis core) and the outer loose domains. The condensed chromosome axis core could help to maintain the desired shape and physical properties for the overall chromosome territories, which are also impermeable as repressive or inactive compartments. On the contrary, the loose domains are open and permissive to molecules mediating nuclear functions.

A key component in our model is the spatial overlap of RNAPII associated transcription factories with the CTCF/cohesin contributed structural foci. To test this, we conducted super-resolution structured illumination microscopic (SIM) analysis of immunostaining using antibodies against CTCF and RNAPII in GM12878 cells (Figure 7E; Extended Experimental Procedures). To further increase the detection resolution, we performed Förster resonance energy transfer (FRET) assay using fluorescence lifetime imaging microscopy (FLIM) to detect the co-localization of CTCF and RNAPII (Figure 7F; Extended Experimental Procedures). Together, the SIM and FRET-FLIM analyses validated that the CTCF-and RNAPII-foci are indeed in close distance in the human nucleome.

Lastly, to test if CTCF prevalently locates along the chromosomal axes, we exploited lampbrush chromosomes. Although not ideal, lampbrush chromosome is still considered the classic model for chromatin looping morphology. We examined the lampbrush chromosomes isolated from newt nuclei using immunostaining to CTCF, and showed the CTCF signals predominantly along chromosome axes instead of the laterally extended chromatin loops (Figure 7G; Extended Experimental Procedure).

Discussion

The inclusiveness of ChIA-PET for enriched specific chromatin interactions and nonenriched higher-order chromatin contacts, and the high degree of data correlation between ChIA-PET and Hi-C demonstrated here are extremely encouraging to the field of 3D genome biology. In addition, the high accuracy of haplotype-resolved chromatin contact mapping by ChIA-PET and Hi-C, in agreement with the concept of chromosome territory established by DNA-FISH, further collectively validates the primary principles of our strategies in characterizing genuine events of chromatin interaction and 3D genome topology. The immediate technical challenges are to further improve the efficiency, accuracy, specificity, and throughput of our technologies for 3D genome mapping in mixed and individual cells.

TAD is an important concept in chromatin biology established recently. However, the detailed structures and associated functions are still to be uncovered. Our analytic strategy focusing on CTCF and RNAPII represents a highly practical, efficient and comprehensive solution to provide mechanistic insights on sub-TAD structures and the embedded functions for transcription regulation. The high degree alignment of CCDs identified by CTCF ChIA-PET to TADs identified by Hi-C further support the idea that CTCF, along with cohesin, is a major contributor that shape chromatin topology in the human nucleome. As indicated in this study, most of the convergent CTCF loops (which probably require more energy and are more stable once established) are engaged to define the CCD/TAD structures and boundaries, whereas the tandem loops (which possibly require less energy and are more dynamic) are involved inside CCD/TAD for transcription and regulatory functions. The discovery of orientational alignment between CTCF motifs and large portion of genes proximal to chromatin interaction anchors elucidates an interesting directional framework of chromatin topology for coordinated transcription regulation between the interplays of CTCF foci and RNAPII machinery. Although we provided initial nuclear imaging evidence to support the proximity of CTCF-and RNAPII-foci within the nuclear space, additional validations are expected in near future.

Given the structural importance of CTCF to chromatin configuration, we anticipate that strong CTCF binding sites would be the candidate mutational targets, where single nucleotide changes may have dramatic consequences. Conveniently, our strategy using SNP-based haplotype mapping of chromatin interactions and allelic-biased chromatin structure features enabled us to use "naturally occurred point mutations" as effective means for "naturally occurred single nucleotide perturbation" in human individuals to study genetic effects on chromatin structures and consequent gene expression possibly linked to disease susceptibility. Together, several lines of evidence provided in this study suggest that genetic variations that affect CTCF/cohesin-mediated chromatin topology could lead to changes in gene expression, thus, laying the molecular foundation for genome topology change, disease susceptibility and evolutionary adaptation. With further detailed comprehension of the chromatin-organizing role played by CTCF, RNAPII and other protein factors, a clearer view of 3D genome topology and associated nuclear function will soon emerge.

Experimental Procedures

Long read ChIA-PET Instead of using MmeI digestion as in the original ChIA-PET protocol [START_REF] Fullwood | An oestrogen-receptor-alpha-bound human chromatin interactome[END_REF], long read ChIA-PET uses Tn5 tagmentation to generate random size of PET templates for long tag sequencing reads (2X150bp) by HiSeq2500 (Extended Experimental Procedures).

ChIA-PET data processing

Short-read ChIA-PET data was obtained from ENCODE (Table S6) and processed using the original ChIA-PET Tool [START_REF] Li | ChIA-PET tool for comprehensive chromatin interaction analysis with pairedend tag sequencing[END_REF]. Long-read ChIA-PET data was processed by a customized ChIA-PET data processing pipeline. All data has been submitted to the NCBI GEO database (GEO accession: GSE72816).

ChIP-nexus

ChIP-nexus on RAD21 and SMC3 in GM12878 cells was performed as described in [START_REF] He | ChIP-nexus enables improved detection of in vivo transcription factor binding footprints[END_REF].

3D DNA-FISH

DNA-FISH of GM12878 cells was performed according to [START_REF] Cremer | Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes[END_REF] with minor modifications. The 3D images of all-chromosome painting were acquired with the Zeiss LSM 780 confocal microscope. The 3D Chr1 territory was obtained using Imaris (Bitplane) and Amira (FEI).

Immunostaining and SIM super-resolution microscopy

The CTCF and RNAPII immunofluorescence staining in GM12878 was performed according to routine procedures [START_REF] Hall | Localization and regulation of PML bodies in the adult mouse brain[END_REF]. Specimens were analysed using a Zeiss ELYRA PS.1 super-resolution system. The super-resolved images were generated using Zeiss Zen 2012 black edition software.

Co-localization detection of CTCF and RNAPII by FRET-FLIM

The FRET-FLIM analysis of the same specimen was performed following the same immunocytochemistry protocol described above. The measurement of fluorescence lifetime of the donor was performed on a Picoquant PicoHarp 300 Time-Correlated Single Photon Counting (TCSPC) system attached to Leica Sp8 confocal microscope, using 63x oil immersion objective (NA 1.4).

Lampbrush chromosome

Ovarian biopsies were performed on adult female newts. Germinal vesicles (nuclei) from stage V-VI oocytes were manually isolated. Lampbrush chromosomes were prepared as previously described [START_REF] Penrad-Mobayed | Tips and tricks for preparing lampbrush chromosome spreads from Xenopus tropicalis oocytes[END_REF], and then immunostained with CTCF [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. Total numbers of sequence reads generated for the in situ Hi-C data, and the CTCF ChIA-PET data are given at the bottom. See also Figure S1. F. Cumulative density of the consistency of motif orientation for tandem loops resided within the same CCD unit (red). The rewired data (grey) refers to the tandem looping directions randomly assigned (either left or right). It showed that the observed tandem loops within a given CCD have significantly higher directional consistency than random chance. P-value is calculated by Wilcoxon test. See also Figure S3J. G. An example CCD of 2 simple-convergent (not cross other loop anchor, red), 5 tandem (blue) and 3 complex-convergent (cross other loop anchor, purple) loops from 8 anchors (ah). The motifs in the 5 tandem loops are all in the rightward direction. Numbers in brackets depict the contact frequency. H. Proposed models, hairpin for convergent and coiled for tandem loops. I. Simulated 3D model of chromatin looping structure using the contact frequency and genomic span in G based on the folding principles proposed in H. This model is an average representation of data derived from millions of cells. The simulation is detailed in Extended Experimental Procedures. See also Figure S2 andS3. F. Expression breadth (number of tissues a gene is expressed in) of CTCF anchor-genes and loop-genes in GM12878 (left). Anchor-genes (yellow) are significantly less represented as tissue-specific than loop-genes (green) (P < 2.2e-16, nonparametric Kolmogorov-Smirnov test). Anchor-genes are further divided as active (red) and inactive (yellow) for analysis (right). The expression breadth of all genes (grey) is included as reference. G. Proposed chromatin model. Top: A schematic CCD with anchor-gene/enhancer and loopgene/enhancer associated with RNAPII interactions. g: gene; e: enhancer. Bottom left: CTCF-mediated loop model shows relative anchor and loop positions. Dotted arrow lines indicate the connectivity brought by RNAPII. Bottom right: RNAPII-participated model shows that RNAPII draws loop-genes/enhancers towards the CTCF anchors, docking the RNAPII foci onto the CTCF-foci. H. Browser view of a CCD with complex sub-domain structures. It involves a numbers of anchor-genes/enhancers and loop-gene/enhancers, which are also connected by RNAPIImediated loops. Orange and purple vertical bars highlight the promoters of anchor-gene and loop-gene, respectively. Right: A simulated 3D model for this topological domain mediated by CTCF/cohesin and the embedded transcriptional complex. This model is an average representation of data obtained from millions of cells. See also Figure S4 and Extended Experimental Procedures. A. Schematic of using SNP as single nucleotide "perturbation" for validation of CTCFmediated chromatin interactions. In individual 1, phased SNP and allele-specific CTCF binding are used to determine the functional and dysfunctional alleles for CTCF interaction. However, it is not immediate ready to extrapolate "no binding = no looping". In individual 2 and 3, homozygous alleles at the corresponding SNP location, possessing either the functional or the dysfunctional CTCF interaction allele, were analyzed for the presence or absence of CTCF binding and looping, respectively, thus, validating the function of CTCF in mediating chromatin interaction.

B. An example using data from GM12878, HeLa and MCF7 shows CCD structures perturbed by SNP. A phased SNP (maternal "T", paternal "A") is identified at the right boundary of a CCD in GM12878. Differential strength of CTCF binding (169:37) was detected and the CTCF loops were extrapolated based on the biased binding. At this SNP locus, both HeLa and MCF7 were of homozygous "A/A" (dysfunctional CTCF allele), had no CTCF binding, and no chromatin contact originated from. *: P << 0.05; Binomial test. See also Figure S6A. C. An example in GM12878 illustrating CTCF tandem loop with allele-specificity and consequent impact on allele-biased transcription. A phased SNP (rs599134) is located in the CTCF-motif (dashed box highlighted) of a "tail" anchor of a tandem loop with the "head" anchor and CTCF-motif (highlighted in orange) proximal to the promoter of CHI3L2. The CTCF binding and looping in this region are paternal-specific, and the RNAPII binding and interactions are significantly paternal-biased as indicated by multiple heterozygous SNPs in this region. The expression of CHI3L2 also exhibited significant paternal-bias. In contrast, the genes (CEPT1 and DENND2D) immediately upstream of the tandem loop showed balanced expression. Nucleotide sequences of the highlighted CTCF-binding site are shown at the bottom with the motif underlined. *: P << 0.05, Binomial test. D. Logos from 70 CTCF-motifs with allelic SNP disruption on CTCF interaction. Haplotype motifs with strong CTCF bindings had canonical consensus (top), motifs with weak CTCF binging displayed deviated consensus (down), especially at position 14. Examples of SNPs in CTCF-motif disrupting CTCF-binding and looping patterns are shown in Data S1, III. E. CTCF-motif disrupted by SNP is linked to disease susceptibility. Top: An example of allele-specific disruption on CTCF-interaction by having SNP within a CTCF motif. SNP (rs12936231) resides at motif position 14 of a CTCF-interaction site. Middle: Linkage disequilibrium between this CTCF-SNP (rs12936231, in red box) and the other 6 asthma associated SNPs (in orange boxes) in the CEU population. These seven SNPs are identified in a significant LD block (D' value > 0.5 and LOD ≥ 3) as highlighted in black triangle. Bottom: Haplotypes of these seven SNPs associated with asthma in the CEU population. The dysfunctional "C" allele of the CTCF-SNP (rs12936231) is frequently (0.422) associated with the risk alleles of the other 6 SNPs in CEU. See also Figure S6. C. Genes involved in allele-specific RNAPII-mediated interactions with phased transcripts. Eighty-nine (89) genes showed significant allele-biased gene expression D. Left: Boxplot of the expression levels of genes with (red box) and without (grey box) allele-bias. Genes with allele-bias (n=89) are of significantly higher expression than the none-biased (n=393) (P = 1.9e-06). Right: MA-plot of the allele-biased gene expression levels. X-axis measures expression abundance, Y-axis indicates differential expression between the two haplotypes. M: maternal-biased, red, n=61; P: paternal-biased, blue, n=18; C: contradictively biased with the corresponding haplotype-biased RNAPII interaction, black, n=10; N: No bias, grey, n=393. See also Table S5. E. An example shows allele-biased RNAPII binding/looping, and the regulatory effect on the associated genes. Left: Haplotype contact heatmaps (M, maternal; P, paternal) of the genomic segment indicated paternal haplotype-specific long-range chromatin interactions (blue arrows). Right: Loop/peak browser view. On the left side, an enhancer was identified. This enhancer overlaps with a phased SNP (maternal "T", paternal "C") and connects downstream to an RNAPII-mediated interaction complex involving 3 genes (LOC374443, CLEC2D, CLECL1). There are 11 phased SNPs in the multi-gene complex. Both of the enhancer and the gene complex exhibited paternal-biased RNAPII binding and interactions. The expression of the 3 genes is also paternal-biased. In addition, the enhancer also showed paternal-biased binding by 3 B-cell specific TFs BCL3, EBF1 and TCF12. All allelespecific sequence reads coverage by RNAPII and TF binding and transcripts are shown in aggregate numbers in the parentheses. *: P<<0.05, Binomial test.

Figure 1 .

 1 Figure 1. Characteristics of ChIA-PET data for 3D genome mapping A. Graphic of ChIA-PET mapping properties including binding peaks, enriched chromatin interactions, and non-enriched singleton PETs inferring topological neighborhood proximity. B. Comparison between CTCF ChIA-PET and in situ Hi-C data (GM12878). Left: loop/ peak map views of CTCF ChIA-PET data at different zoom-in scopes. For each data track, loop view is at top, peak view at bottom; Y-axis indicates the contact frequency of loops (log10 scale) and intensity of binding peaks (linear scale). The maximum frequency and

Figure 2 .

 2 Figure 2. CTCF-defined chromatin looping topology A. Characterization of CTCF-mediated loops in relation to cohesin binding and CTCF-motif orientation. See also Figure S2E-G. B. CTCF and RAD21 binding patterns centered on CTCF-motif. ChIP-Exo data of CTCF (Rhee and Pugh, 2011) and ChIP-nexus data of RAD21 were plotted as density curve around CTCF-motif sites. Borders of DNA footprints were identified by occupancy peaks. The green peaks depict the two borders of CTCF occupancy. The dashed blue line shows the peak position depicting 5' border of RAD21 footprint. The solid blue line shows bimodal peaks from the 3' border of RAD21 occupancy. See also Data S1, II. C. A mapping browser screenshot shows the CTCF-defined chromatin interactions and contact domains. Hi-C determined TADs (Dixon et al., 2012) and in situ Hi-C identified loops (Rao et al., 2014) are also shown. CTCF-motif position and orientation at the corresponding interaction anchors are shown as red arrows. Insert: zoom-in regions highlight CCD boundaries having multiple CTCF-binding peaks and motifs with inwardfacing orientation. D. Cumulative density plot shows the genomic span distribution of individual CTCF loops, CCDs, in situ Hi-C loops and Hi-C TADs. See also Figure S3C-D. E. Statistics of CTCF-motif orientation at CCD boundaries. See also Figure S3E.

Figure 3 .

 3 Figure 3. Relationship between CTCF/cohesin-mediated chromatin structure and RNAPIIassociated transcriptional function A. Browser view of a 5Mb genomic segment with 4 CCDs showing overlapped CTCF, RNAPII ChIA-PET data along with chromatin state (ChromHMM) and RNA-Seq data. In the ChromHMM track, red for active promoter, yellow for enhancer and green for transcribed region. See Extended Experimental Procedures for the detailed color code. B. Aggregation density plots showing histone modification (top), RNAPII binding and TSS (bottom) distribution profiles around the CTCF anchors and the loop regions. X-axis: CTCFanchors were taken from the anchor center with ±10% extension proportional to the enclosed loop regions. Y-axis: Intensity (FPKM). C. Similar to B, but only around the anchor centers (±2kb). Anchors of convergent and tandem loops are analyzed separately. D. Similar to C, but ChIP-Seq data of selected TFs are plotted. Upper: ELF1 and ZEB1. Lower: CTCF, RAD21 and ZNF143. E. Boxplots for the ratio of H3K3me1/H3K4me3 ChIP-Seq at the anchors of convergent and tandem loops. High ratio suggests enhancer potential, low value indicates promoter function. F. Expression breadth (number of tissues a gene is expressed in) of CTCF anchor-genes and loop-genes in GM12878 (left). Anchor-genes (yellow) are significantly less represented as

Figure 4 .

 4 Figure 4. Haplotype mapping of chromatin interaction A. Statistics of phased PETs in GM12878. Intra-chromosomal PETs were distinguished as cis-PETs and trans-PETs. A cis-PET has the two tags mapped to phased SNPs with the same haplotype (M-M or P-P); a trans-PET has the two tags mapped to phased SNPs in the opposite haplotypes (M-P or P-M). B. Identification of haplotype chromatin interactions. Top: Schematic of the haplotype phasing of ChIA-PET mapping. Phased SNPs with CTCF or RNAPII binding were first identified. Interaction anchors overlapping with phased SNPs are referred as "Phased anchors" (vertical bar indicates the phased SNP). Interaction loops originating from paired phased anchors are "Phased interactions" (red). Interactions with only one side originating from phased anchors are "Extended phased interactions" (yellow). All other interactions that cannot be reliably determined are "Unphased interactions" (grey). Bottom: An example CCD, where three phased SNPs are identified with significant haplotype bias in CTCFbinding. The SNP nucleotides are color coded for their haplotypes (red: maternal, blue:

Figure 5 .

 5 Figure 5. SNPs altering allelic CTCF chromatin interaction and the functional implication

Figure 6 .

 6 Figure 6. Allele-biased chromatin interactions mediated by RNAPII A. Schematic of using SNPs to investigate allelic-effects of transcription regulation via haplotype-biased occupancy and interaction mediated by RNAPII and TFs. B. Profile of allele-biased binding by 40 TFs at the allele-biased anchors (maternal 81, paternal 56) of RNAPII interactions. Each row represents an allele-biased RNAPII anchor with allele-biased binding by at least one TF. Each column represents one of the 40 tested TF. Each colored tile indicates TF binding bias: maternal, red; paternal, blue.

Figure 7 .

 7 Figure 7. Chromatin model of CTCF foci and RNAPII transcription factories A. 3D models of Chr1 at 3 resolutions (16Mb, 2Mb, 100bp) with views from different angles. The color bar indicates the proportional genomic coordinates of 3D models. B. An ensemble model of Chr1 folding dynamics in GM12878. C. 3D images of DNA-FISH for the two copies of Chr1 (red) in a nucleus from different angles. The positional patterns of the two probes (green and blue) indicate two chromosomal conformations, "open" and "close".

Cell. Author manuscript; available in PMC 2016 December 17.

Acknowledgements

Y.R. is supported by the Director Innovation Fund of The Jackson Laboratory, NCI R01 CA186714, NHGRI R25HG007631, NIDDK U54DK107967 (4DN), and the Roux family as the Florine Roux Endowed Chair in Genomics and Computational Biology. X. L. is supported in part by China "111 project" (B07041). Polish National Science Centre supports G.M.W. [UMO-2012/05/E/NZ4/02997]; D.P. and P.S. [2014/15/B/ST6/05082; UMO-2013/09/B/NZ2/00121]; and J.W. [DEC-2012/06/M/NZ3/00163]. D.P. and P.S. are also supported by National Leading Research Centre in Bialystok and the European Union under the European Social Fund. The authors thank CZ Zhang for initial DNA-FISH, Agnieszka Walczak and Katarzyna Krawczyk for FISH discussion, Rafael Casellas, Michael Stitzel and Duygu Ucar for manuscript discussion, and Gosia Popiel for help on preparing Figure S7. The HeLa genome sequence described/used in this research was derived from a HeLa cell line (http:// www.ncbi.nlm.nih.gov/gap). This study was reviewed by the NIH HeLa Genome Data Access Working Group.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


O Sandre

R E Rosensweig

J.-C Bacri

V Cabuil

R Perzynski

NEW FERROFLUID COMPOSITES : MAGNETISM AND OPTICS EXPERIMENTS

An ionic ferrofluid is an aqueous colloidal suspension of surface charged magnetic nanoparticles. Dispersion of the particles is ensured by the Coulomb forces. The effect of an added electrolyte is the screening of the electrostatic repulsions. Above a threshold ionic strength, a separation occurs between one phase rich in particles and another poor in particles [1]. Near the threshold, this ferrofluid composite consists of highly magnetic droplets (a few micrometers in diameter) surrounded by a dilute weakly magnetic medium. The nanoparticles are made of γ-Fe 2 O 3 (maghemite) and their polydispersity is high : their diameter follows a log-normal distribution with parameters D 0 =6.8nm and σ=0.5. Hence relatively large particles are present which favors phase separation. Iron titration of the concentrated phase gives a volume fraction of particles Φ c =0.31.To characterise the microdroplets we use the method [2] which consists in measuring their ellipsoidal deformation (axisymmetric prolate ellipsoids with long semi-axis a and short semi-axis b) as a function of the magnetic field. The equilibrium shape results from the competition between magnetic and interfacial energy. Figure (1) shows the aspect ratio a/b for a droplet of given volume versus field intensity. The best fit gives the magnetic susceptibility χ=63 of the concentrated phase and the interfacial tension σ=7.5x10 -7 N.m -1 between the two phases. Because of the high value of χ and the small value of σ a magnetic induction so small as a few 10 -4 T induces a large deformation of the droplets.

(2) Picture of an assembly of microdroplets in a magnetic field with intensity H=1280A.m -1 rotating at frequency Ω=3.45Hz.

We study this magnetic composite material which consists in an assembly of magnetic and deformable microdroplets. We present here two complementary experiments in order to show the competition between the magnetic torque created by a field and the viscous torque due to a local vorticity. In the first experiment the assembly of microdroplets is submitted to a rotating magnetic field at frequency Ω and observed under an optical microscope. Figure ( 2) is a typical view for a field intensity H=1280A.m -1 and a frequency Ω=3.45Hz. The rotation is counter clockwise and the picture is taken at a moment when the field is along the vertical.

Thus there is a visible phase lag θ between the field and the droplets. The highest values of θ are found for the most elongated droplets. For a given droplet, θ increases with the frequency Ω. At a critical value Ω c the droplet breaks into two parts or more in order to minimize the total dissipation [3]. Figure (3) shows the mean aspect ratio a/b of the droplets distribution as a function of Ω at constant field intensity and for several values of the field. The decreasing of the average aspect ratio with increasing Ω is a consequence of the break up of the droplets. Simultaneously the mean volume of the droplets decreases and the number of droplets in the screen increases to keep the total volume of concentrated phase constant. The second experiment consists in a rotating sample at frequency Ω in a static field H (  H ⊥  Ω ).The solid rotation of the cell under a constant field is equivalent to the rotation of the field with a static sample. We study the diffraction pattern of a laser beam parallel to  Ω in order to obtain a direct measurement of the phase lag θ averaged on a huge amount of droplets. Thus we deduce that the population of droplets is bi-modal, which can be a consequence of the break up of the droplets caused by the viscous torque. In a simple model for large aspect ratio and large χ, the balance between magnetic and viscous torques scales as :

ηa 3 Ω ∝ m 0 χab 2 H 2 sin2θ
where η is the viscosity of the dilute phase.

As it is expected the upper limit of θ is 45°. The plot on figure (5) of sin2θ versus Ω/H 2 allows to superpose all the data on only two branches.

( 1 )

 1 Aspect ratio a/b versus magnetic Bond number B m for a droplet of initial radius R 0 =6.65mm : the best fit gives χ=63 and σ=7.5x10 -7 N.m -1 .

( 3 )

 3 Aspect ratio a/b averaged on approximately 100 droplets versus Ω.

  Figure (4) is a picture of the scattering pattern for H=980A.m -1 and Ω=0.23Hz. There is a wide range of H and Ω where two scattering lines corresponding to two different values of θ are observed simultaneously. (4) Scattering pattern of an assembly of microdroplets in a sample rotating counter clockwise submitted to a static field.

  Plot of sin2θ versus Ω/H 2 for several H.