Article
Reference
Local chromatin interactions contribute to expression of the fibrinogen gene cluster
ESPITIA JAIMES, Cindy, FISH, Richard, NEERMAN ARBEZ, Marguerite
Abstract
Essentials: The fibrinogen gene cluster is flanked by CCCTC-binding factor (CTCF) interaction sites. Chromatin looping of the fibrinogen cluster was demonstrated by chromosome conformation capture. Deleting a CTCF interaction site alters chromatin looping and halves fibrinogen expression. Looping of the human fibrinogen locus is functionally linked to fibrinogen gene expression.
ESPITIA JAIMES, Cindy, FISH, Richard, NEERMAN ARBEZ, Marguerite. Local chromatin interactions contribute to expression of the fibrinogen gene cluster. Journal of Thrombosis and Haemostasis, 2018, vol. 16, no. 10, p. 2070-2082
PMID : 30039577
DOI : 10.1111/jth.14248
Available at:
http://archive-ouverte.unige.ch/unige:115122
Disclaimer: layout of this document may differ from the published version.
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Local chromatin interactions contribute to expression of the fibrinogen gene cluster
C. Espitia Jaimes*, R. J. Fish* and M. Neerman-Arbez.*,†
*Department of Genetic Medicine and Development, University of Geneva Faculty of Medicine and †Institute of Genetics and Genomics in Geneva (iGE3), Geneva, Switzerland.
To whom correspondence should be addressed:
Marguerite Neerman-Arbez
Department of Genetic Medicine and Development, University of Geneva Faculty of Medicine, 1, rue Michel Servet, 1211 Geneva, Switzerland.
Tel: ++41223795655 Fax: ++41223795707
Email: Marguerite.Neerman-Arbez@unige.ch
2 Essentials
• The fibrinogen gene cluster is flanked by CCCTC-Binding Factor (CTCF) interaction sites.
• Chromatin looping of the fibrinogen cluster was demonstrated by chromosome conformation capture.
• Deleting a CTCF interaction site alters chromatin looping and halves fibrinogen expression.
• Looping of the human fibrinogen locus is functionally linked to fibrinogen gene expression.
3 Summary
Background: The coordinately regulated genes encoding human fibrinogen are clustered.
This evolutionarily conserved configuration provides a possible mechanism for co-regulation whereby regulatory elements influence gene expression locally. The cluster is flanked by CCCTC-Binding Factor (CTCF) interaction sites which are candidate insulator regions mediating chromatin looping.
Objectives: To further our understanding of fibrinogen gene regulation, we aimed to investigate whether interactions exist between parts of the fibrinogen locus and how these contacts contribute to fibrinogen expression.
Methods: We used chromosome conformation capture in cultured cell lines to detect chromatin interactions at the fibrinogen gene cluster.We generated clonal cell lines where two CTCF interaction sites at one end of the locus were deleted using CRISPR-Cas9-mediated genome editing. Fibrinogen expression and protein production were measured using qRT- PCR and ELISA, respectively.
Results: We detected proximity between the ends of the fibrinogen locus, regardless of whether cells express fibrinogen. An interaction between the FGA promoter and the edge of the locus was more frequent in fibrinogen-expressing cells. Deletion of a CTCF site at one edge of the cluster altered chromatin interactions, reduced steady-state expression of FGB and FGG mRNA, and led to a halving of secreted fibrinogen. These phenotypes were completely restored by re-introduction of the CTCF interaction motif in previously motif- deleted clones.
Conclusions: Chromatin interactions are important for the coordinated regulation of the human fibrinogen genes. This finding furthers our comprehension of how fibrinogen is produced and identifies a possible source of variability in plasma fibrinogen levels seen in populations.
Keywords: CCCTC-Binding Factor; CRISPR-Cas Systems; fibrinogen; gene expression regulation; multigene family.
4 Introduction
Fibrinogen, the soluble precursor of fibrin, is a coagulation factor with a central role in blood clotting, platelet aggregation and thrombus formation [1]. Fibrinogen is abundant in plasma, and varies from about 1.5 to 3.5 g/L in the healthy population [2]. A small number of genetic variants have been linked to plasma fibrinogen levels [3]. Understanding the source of plasma fibrinogen variability is a clinically relevant goal because high circulating fibrinogen levels are associated with increased risk of cardiovascular events [4, 5].
Fibrinogen is an acute phase protein synthesized by hepatocytes as a hexameric protein assembled from two copies of three polypeptides (Aα, Bβ, γ)2. These are encoded by three genes (FGB-FGA-FGG), that are clustered within a 65 kilobase region on human chromosome 4 (4q23-q32)[6]. Remarkably, this configuration is conserved among tetrapods. Expression of the fibrinogen genes is coordinated [2, 6], and the mechanism for this control appears to reside in the activity of proximal promoters and local enhancer elements. Four enhancers, CNC12, PFE2, E3 and E4, have been identified within the fibrinogen gene cluster [3, 7]. These regulatory sequences bind common transcriptional regulators and are delineated by active chromatin histone marks in fibrinogen-expressing cells. Interestingly, the fibrinogen cluster is flanked by CCCTC-binding factor (CTCF) interaction sites. CTCF is a broadly expressed zinc- finger protein which binds DNA, and has a role in insulating genomic regions [8, 9].
Therefore, the fibrinogen gene cluster could represent a regulatory unit where regulatory elements interact and contribute to coordinated expression.
Chromatin structure plays a pivotal role in the regulation of gene expression in eukaryotes [10]. Chromatin is not static in structure, but can fold to bring regulatory elements
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(enhancers, insulators) into proximity with target genes or other regulatory elements.
Chromatin loops are the result of these long-range interactions [11, 12].
Mammalian genomes are folded in a hierarchy of compartments: topologically associating domains (TADs), subTADs and looping interactions [13]. Mammalian TADs are, on average, a megabase long and represent looped chromosomal units within which sequences can preferentially contact each other, favoring common transcriptional regulation [14-16].
Contact maps of the human genome have allowed the annotation of nearly 10,000 loops which typically lie between CTCF motifs found in a convergent orientation, as predicted by the loop extrusion model [17]. This finding highlights a role for CTCF in chromosomal structure and chromatin folding.
Looping of the human genome has been demonstrated using chromosome conformation capture (3C)-based technologies. They enable detection of physical proximity between segments of chromatin. The original 3C technology interrogates chromatin interactions between selected pairs of sequences in a region-of-interest [18] [19]. If regulatory element interactions, for example promoter-enhancer proximity, are to be interrogated, prior knowledge of these sequences in the selected genomic region is required[16]. 3C-derived genomics methods [20] have provided detailed evidence of chromosome territories [21], the role of CTCF and cohesin in mediating TAD looping [22, 23] and regulatory element contact [24]. Furthermore, they are now used in molecular medicine, helping interpretations of disease-associated variants [25] and improving our ability to predict the possible functional consequences of genetic variation on chromosomal architecture [26].
The impact of deletion of CTCF interactions has been addressed. In locus-specific studies, for example the β-globin locus, CTCF was deleted in fetal liver cells in a conditional knockout
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mouse model. This demonstrated that CTCF is involved in chromatin architecture [22].
Recently, using an auxin-inducible degron system in mouse embryonic stem cells, it was shown that CTCF is required for looping between CTCF sites and for the maintenance of TADs [27].
In the present study we performed loss-of-function and restoration-of-function experiments in HepG2 cells, using CRISPR-Cas9 genome editing to delete and re-introduce CTCF interaction sites at one boundary of the fibrinogen cluster. 3C was used to examine the spatial organization of chromatin in the human fibrinogen locus in cells that express or do not express fibrinogen and in CTCF site-edited cell lines. We investigated if local chromatin architecture influences fibrinogen gene expression and fibrinogen protein production. We show that fibrinogen gene expression relies on precise chromatin interactions. Our results provide a new mechanism to understand coordinate fibrinogen gene expression that may also control the expression of other clustered genes.
7 Methods
Chromosome conformation capture
1x107 HepG2, HEK-293T, CTCF3 deleted (∆CTCF3), CTCF4 deleted (∆CTCF4), double deleted (∆CTCF3+4), ∆CTCF3 rescue (∆CTCF3R), ∆CTCF4 rescue (∆CTCF4R) and double rescue (∆CTCF3+4R) cell lines were treated, and data analyzed, according to the 3C-qPCR method [28], with small modifications. Cells were crosslinked with 1.5% formaldehyde (Sigma) for 10 min at room temperature and lysed in 5 ml of cold lysis buffer (10 Mm Tris-HCl, pH 7.5; 10 mM NaCl; 0.2% NP-40; 1X complete protease inhibitor (Roche)). Chromatin fragmentation was achieved by digesting nuclei with 400 U of NlaIII (New England Biolabs) to give an average fragment size of about 500 bp. Digestion efficiency was assessed using primers that amplify across each NlaIII site of interest (Table S1). % restriction = 100-100/2^ ((CtR -CtC) Digested - (CtR -CtC) Undigested). Interacting fragments were ligated under diluted conditions with 200 U of T4 DNA ligase (New England Biolabs) for 4 h at 16°C and de- crosslinked at 65°C overnight. 3C templates were purified by phenol-chloroform extraction.
Real-time PCR quantifications of ligation products were performed in a StepOnePlus™ Real- Time PCR System using the KAPA SYBR FAST qPCR Master Mix and specific primers for every fragment of interest (Table S2). For each ligation product, a digested and re-ligated bacterial artificial chromosome (BAC RP11-111A7), covering the fibrinogen gene cluster and control regions analyzed, was used as a control template. A constant primer and a test primer were used in each reaction. Standard curves using serial dilutions of control templates were performed to test amplification efficiency. 3C-qPCR data was normalized to a loading control using internal primers amplifying a region of the GAPDH gene. For each 3C assay, using a pair
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of test/constant primers localized in a locus that contains a ubiquitously expressed gene, ERCC3 [22], control interaction frequencies were also assessed (Table S3).
CRISPR-Cas9-mediated genome editing
CTCF3 and CTCF4 interaction sites flanking the telomeric end of the human fibrinogen gene cluster were edited using CRISPR-Cas9-based genome editing. They were identified by factorbook [29] and the UCSC genome browser display (http://genome.ucsc.edu) of ENCODE consortium ChIP-seq and ChIA-PET datasets [30]. ENCODE datasets for CTCF and RAD21 ChIP-seq in HepG2 cells, and CTCF ChIA-PET in other cells, were contributed by the Bernstein, Myers, Stamatoyannopoulos, Iyer, Ruan and Snyder laboratories. Single guide RNAs (sgRNAs) were designed to direct the Cas9 nuclease to two sequences for each CTCF site (Figs. S1 and S2). Plasmids based on pSpCas9(BB)-2A-GFP (Addgene plasmid:48138, contributed by the Zheng laboratory) [31] were developed for expression of sgRNAs, along with a Cas9-2A-EGFP fusion transcript, leading to separate expression of Cas9 and EGFP from the same transcript. HepG2 cells were co-transfected with combinations of plasmids, using Fugene HD reagent (Roche), to drive target site cleavage in the presence of ssDNA oligonucleotides as homology-directed repair (HDR) templates (Figs. S1 and S2). EGFP- positive cells were clonally sorted into 96-well plates by flow cytometry 2 days post- transfection, and cultured. Cell lines were genotyped by PCR, using the Guide-it Mutation Detection Kit (Clontech). Clones with bi-allelic edits, demonstrating the expected 35 and 31 bp deletions for CTCF3 and CTCF4 sites respectively, were confirmed by Sanger sequencing.
To re-introduce the CTCF interaction sites of CTCF3 and CTCF4, further pSpCas9(BB)-2A-GFP- based plasmids were developed to target the previously edited region, with ssDNA
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oligonucleotides for HDR. Transfections and clone identification were performed as for deletion clones.
qRT-PCR and fibrinogen protein production
Total RNA from 1x106 HepG2 cells, HepG2 ∆CTCF3/∆CTCF4 deleted clones or HepG2
∆CTCF3R/∆CTCF4R rescued clones was extracted using TRI Reagent® (Sigma) and 1 µg DNase-treated (Ambion) RNA reverse transcribed (SuperScript II; Invitrogen). mRNA levels of the fibrinogen genes were measured by qRT-PCR with KAPA SYBR FAST qPCR Master Mix.
Copy number quantification was inferred using standard curves of a single-stranded oligonucleotide template for each amplicon. Each gene was amplified in 3 technical replicates per sample, and expression was normalized with β-actin [32] (Table S4).
Fibrinogen protein production was measured as a ratio of fibrinogen secreted into cell- conditioned medium (ELISA) divided by total cell protein measured with a small-scale Bradford protein assay, as described previously [33].
Interleukin-6, tumor necrosis factor alpha and lipopolysaccharide treatment
HepG2 and HepG2 ∆CTCF4 cells were cultured for 18 h post-seeding. Recombinant human interleukin-6 (IL-6) (Gibco) was added to the cell media at a concentration of 50 ng/ml, tumor necrosis factor alpha (TNF-α) (R & D Systems) at 10 ng/ml and lipopolysaccharide (LPS) (Sigma) at 100 ng/ml. Non-treated and treated cells were collected 24 h following exposure to IL-6, TNF-α or LPS prior to the 3C protocol and RNA extraction and quantification. In TNF-α-treated cells β-actin levels increased. We therefore used SCLY gene expression [34] as a control for samples treated with IL-6, TNF-α and LPS (Table S4). From our SCLY qPCR standard curve, TNF-α treatment gave a 1.4 fold increase in SCLY (less fold- change than β-actin). Normalization of fibrinogen expression using this control is not
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optimal, but the decrease in fibrinogen expression seen with TNF-α treatment cannot be explained by a normalization artefact of increased SCLY.
Results
Chromatin interactions occur at the edges and within the human fibrinogen gene cluster
Functionally related genes and their regulatory sequences tend to be arranged in the same regulatory domain [35]. Chromatin looping between the extremities of the fibrinogen gene cluster is inferred by CTCF interaction sites detected by ChIP-seq (Chromatin immunoprecipitation-sequencing) and CTCF ChIA-PET (Chromatin Interaction Analysis by Paired-End Tag Sequencing) signals [30].
Knowing the regulatory landscape of the fibrinogen locus (Fig. 1A) and in order to explain the co-regulation of the three fibrinogen genes, we aimed to detect chromatin interactions between regulatory elements of the gene cluster using chromosome conformation capture (3C) in a fibrinogen-expressing hepatocellular carcinoma cell line (HepG2) and a non- expressing human embryonic kidney cell line (HEK-293T).
A representation of the genomic region targeted for 3C is shown in Figure 1B. Numbers represent regulatory fragments that were obtained after NlaIII fragmentation of cross-linked chromatin. The CTCF0-bearing restriction fragment, found upstream of FGB, was used as the
“bait” for this analysis, and qPCRs were used to detect proximity between it and 29 restriction fragments across the gene cluster, concentrating on known enhancers, CTCF interaction sites and control fragments. As expected, self-ligation products and close linear proximity contribute to a peak found at and around the CTCF0 position in HepG2 and HEK- 293T cells. A clear interaction is also detected between CTCF0 and CTCF4 at the other end of
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the locus in both cell types (Fig. 1C and 1D). This is reminiscent of interactions described by ChIA-PET in other cell types [30], and suggests a looping out of the fibrinogen gene cluster.
In HepG2 cells, which produce fibrinogen, we also detected an interaction between CTCF0 and the FGA promoter. This interaction was detectable but of lower frequency in HEK-293T cells (Fig. 1C and 1D), suggesting that frequency of this internal interaction correlates with fibrinogen expression.
Targeted disruption of a chromatin interaction affects fibrinogen expression
To investigate whether the fibrinogen locus architecture is important for gene expression, we designed a loss-of-function experiment in which CTCF3 and CTCF4 sites, flanking one edge of the fibrinogen gene cluster, were deleted using CRISPR-Cas9 genome editing [31]
(Figs. 2A, S1 and S2). We reasoned that this could disrupt the sub-TAD-like structure of the fibrinogen locus, reduce the frequency of the looped configuration and affect coordinated fibrinogen gene expression. We transfected HepG2 cells with plasmids that lead to expression of sgRNAs and the Cas9 nuclease in order to introduce double-stranded DNA breaks and specific HDR-directed deletions of the CTCF factorbook motif sequences [29] of CTCF3 (ΔCTCF3) and CTCF4 (ΔCTCF4). Double CTCF site deletion cell lines were also generated (ΔCTCF3/ ΔCTCF4) (Figs. S1 and S2).
We measured chromatin interactions in the CTCF site-deleted clones, using 3C and the CTCF0 site as bait. A ΔCTCF3 clone showed a similar interaction frequency profile compared to HepG2 cells. However, in ΔCTCF4 and ΔCTCF3/ΔCTCF4 clones, the HepG2 cluster looping interaction of CTCF0 with CTCF4 was lost (Fig. 2B). With loop disruption, the CTCF0 to FGA promoter interaction was less frequently detected, and a previously undetected interaction between CTCF0 and PFE2 was observed. In ΔCTCF4 cells, an interaction between CTCF0 and
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CTCF3 was also detected, suggestive of cluster loop maintenance and re-organization.
However, this was not sufficient to sustain the CTCF0 to FGA promoter interaction, pointing towards the CTCF4 site having a determinant role in the relative frequency of the CTCF0 to FGA promoter interactions.
We studied the effect of CTCF site deletion on fibrinogen expression. Fibrinogen mRNAs were quantified by qRT-PCR in HepG2 cells and CTCF site deletion clones (Fig. 2C). The ΔCTCF3 cells showed mRNA levels of the three fibrinogen genes that were comparable with HepG2 cells. In ΔCTCF4 and ΔCTCF3/ΔCTCF4 clones, FGB and FGG mRNA levels were reduced compared to HepG2 cells. FGA mRNA levels remained unaffected in all deleted clones (Fig.
2C).
As transcripts to sub-units of secreted hexameric fibrinogen, lowered mRNA levels of any of the fibrinogen transcripts should reduce fibrinogen protein production. We measured fibrinogen levels in cell-conditioned medium from HepG2 cells and CTCF site deletion clones, using data from a fibrinogen ELISA assay normalized to total cellular protein. Fibrinogen protein output was halved in ΔCTCF4 and ΔCTCF3/ΔCTCF4 clones, compared to HepG2 cells.
ΔCTCF3 cells were phenotypically similar to HepG2 cells in chromatin interactions, mRNA levels and protein production (Fig. 2D). This data indicates that disruption of the fibrinogen locus CTCF4 interaction site, that is detected in proximity to CTCF0, affects FGB and FGG expression, and lowers fibrinogen protein production.
Introducing a CTCF interaction site at the CTCF4 position rescues fibrinogen expression in ΔCTCF4 or ΔCTCF3/ΔCTCF4 cells
After detecting a loss-of-function effect for deletion of the fibrinogen locus CTCF4 site, we tested the specificity of this effect by using CRISPR-Cas9 tools to re-introduce CTCF3 and
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CTCF4 interaction motifs, in the previously engineered cell lines where they were deleted.
Deletion of these sites produced unique sequences in the genome to which we designed sgRNAs to guide Cas9-mediated dsDNA cleavage, in the presence of a HDR oligonucleotide template for repair. We did not re-introduce the original HepG2 genomic sequence but rather each complete factorbook [29] interaction motif with 4 nucleotides on each side (Fig.
3A). This enabled unambiguous identification of re-introduced motifs in potential phenotypic
“rescue” clones, while re-establishing the predicted functional CTCF interaction sequences. A description of this targeting is given in Figs. S1 and S2.
3C experiments on CTCF site-rescued clones showed a locus boundary interaction between CTCF0 and CTCF4 in both ΔCTCF4R (R for rescue) and ΔCTCF3R/ΔCTCF4R cells (Fig. 3B). The CTCF0 to FGA promoter interaction was also restored, with the overall 3C data resembling that of HepG2 cells. Fibrinogen mRNA and protein production were then assessed in the CTCF interaction site-rescued cells. By qRT-PCR, FGB, FGA and FGG mRNA were at HepG2 cell levels in ΔCTCF4R and ΔCTCF3R/ΔCTCF4R cells (Fig. 3C). Fibrinogen protein production from these cells was also restored to HepG2 levels (Fig. 3D).
Using loss- and restoration-of-function approaches, our results demonstrate that a chromatin interaction between the edges of the fibrinogen gene cluster, mediated by the CTCF4 interaction motif and the CTCF0 region, contribute to coordinated expression of the fibrinogen genes and ultimately the quantity of fibrinogen protein production.
Local chromatin interactions of the fibrinogen locus are not affected by IL-6 treatment of HepG2 or ΔCTCF4 cells
Inflammatory stimuli can modulate fibrinogen expression. Previous studies have focused on cytokine-mediated transcriptional regulation of fibrinogen genes [36]. IL-6 can increase
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fibrinogen gene expression [37, 38]. We aimed to assess chromatin interactions and fibrinogen expression under inflammatory conditions. We treated HepG2 cells and ∆CTCF4 cells with IL-6, TNF-α and endotoxin (LPS). Chromatin interactions at the fibrinogen locus were similar in IL-6-treated and untreated. This was true for HepG2 and ΔCTCF4 cells (Fig.
4A-B). Both cell types responded to IL-6 treatment, with increases in fibrinogen mRNA and protein levels compared to untreated cells (Fig 4C-D). We conclude that changes in local chromatin interactions are not a major feature of the fibrinogen locus response to IL-6 signalling. We did not detect significant differences in chromatin interactions in either cell type after treatment with TNF-α or LPS. However, mRNA and protein levels were reduced after treatment with TNF-α (Fig 4C-D), in agreement with previous studies [39].
Discussion
The major finding of our study is that the human fibrinogen locus has a looped configuration that is functionally linked to fibrinogen gene expression. Using 3C methodology, we detected local chromatin interactions at the fibrinogen locus in HepG2 cells which express fibrinogen and HEK-293T cells which do not. The fibrinogen cluster is a regulatory unit where flanking CTCF sites are in proximity, creating a cluster length chromatin loop. Using one of these sites as a constant anchoring fragment for our analyses (CTCF0), we were able to identify a loop interaction with the other site (CTCF4) in both cell types. However, we detected a difference in an internal chromatin interaction between fibrinogen-expressing (HepG2) and non- expressing cells (HEK-293T), with a higher frequency looping-in of the FGA promoter region towards CTCF0 in cells that produce fibrinogen. We interpret this as an interaction that facilitates the co-regulation of the fibrinogen genes, bringing regulatory elements into proximity. In a configuration where the FGA promoter is near CTCF0, it is also presumably
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closer to the E4 enhancer element. Even without the FGA promoter to CTCF0 interaction, the larger cluster loop (CTCF0 to CTCF4) “pulls” the E3 and E4 enhancers closer together than in the linear configuration, and leaves them well-placed to influence expression from nearby FGB and FGG promoters.
The cluster looping we describe occurs in non-fibrinogen-expressing cells. Therefore it is not the cause of expression and more likely represents a structural chromatin unit. The expression of fibrinogen presumably relies on the complement of transcription factors and histone modifications at the locus in hepatocytes. However, we were intrigued to know what would happen to fibrinogen expression if the frequency of the cluster looping were lessened, would fibrinogen gene expression be sensitive to the structural configuration of this chromatin unit?
We used CRISPR-Cas9 technology to delete two CTCF interaction sites at one end of the fibrinogen locus in HepG2 cells. These CTCF sites were chosen because they were seen as interaction partners for the other end of the cluster in data from the ENCODE project [30], albeit in non-fibrinogen-expressing cells. As expected, deleting CTCF4’s CTCF motif disabled its interaction with CTCF0, but also led to a novel interaction of CTCF0 with CTCF3 and lowering of the CTCF0 to FGA promoter interaction frequency. Despite the new loop between CTCF0 and CTCF3, fibrinogen expression was halved, suggesting that the CTCF0 to CTCF4 contact contributes more productively to fibrinogen expression, perhaps with a more optimal configuration of regulatory elements. The source of the lower fibrinogen protein production in ΔCTCF4 cells appears to come from lower steady-state expression of FGB and FGG transcripts, with FGA expression unaffected. FGA expression does not seem reliant on FGA promoter to CTCF0 interactions. This data does not exclude the possibility that the
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proximity of CTCF0 to the 5’ of FGA influences coordinated expression of FGB and FGG, perhaps with the FGA promoter contributing to a local transcription machinery or transcription factor “sink”, in conjunction with the FGB and FGG promoters, and the E3 and E4 enhancers.
Interestingly, RAD21, a component of the cohesin complex involved in the formation of local chromatin interactions, is one of the transcription factors that bind the FGA promoter but not the FGB or FGG promoters [23, 40]. This could explain why the FGA promoter is looping with CTCF0 and CTCF4.
Deletion of nucleotides in our ΔCTCF motif cell lines was aimed at revealing a loss-of- function phenotype. Engineering these cells by re-introducing the CTCF interaction sites, gave us clear restoration-of-function effects; rescue of the CTCF0 to CTCF4 interaction and HepG2 cell fibrinogen expression levels. By manipulating CTCF3 and CTCF4 individually or together, our data unambiguously demonstrate the importance of the CTCF0 to CTCF4 interaction for fibrinogen expression. Despite subtle differences in the chromatin interaction profile of the fibrinogen locus in ΔCTCF4 versus ΔCTCF3/ΔCTCF4 cells, fibrinogen expression was similarly affected and no effect was detected in ΔCTCF3 cells.
Our data has limitations. 3C informs on contact frequencies between known chromatin segments in cell populations, but does not inform about the situation in individual cells. Also, the high number of cells needed for 3C experiments makes it unfeasable to use primary hepatocytes whose expansion in vitro is challenging [41]. Figure 5 summarizes, using graphical representations, an overview of our interpretation of the chromatin interactions we have measured in HepG2 cells and after CTCF motif deletion or restoration, based on the frequency of interactions. In order to address this dynamic aspect of chromosome
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organization, single cell analyses would be pertinent. Also, thus far, we have not assessed chromatin interactions of the fibrinogen locus in an intact organism; rather we focused on the human gene cluster in a cellular model which may show subtle mechanistic differences to hepatocytes in vivo. We did not functionally assess all the interactions we measured. Our data highlight an interaction between CTCF0 and CTCF6, a site telomeric to CTCF4 which has an interaction frequency with CTCF0 generally lower than with CTCF4 in the cell lines described. We did not disrupt CTCF6 as its orientation with respect to CTCF0 does not fit the criteria for an interaction with CTCF0. The vast majority of CTCF site interaction pairs detected genome-wide have their CTCF motifs in the convergent orientation as for CTCF0 and CTCF4 [12, 42]. CTCF0 and CTCF6 do not show this orientation. The effect of CTCF6 disruption may be additive to loss of the CTCF0 to CTCF4 interaction. While we have measured lower fibrinogen gene expression in ΔCTCF4 cells that can be attributed to changes in chromatin architecture, this effect may also relate to an altered balance of active and repressive histone marks, as described for the β-globin locus upon conditional deletion of CTCF [22]. To better understand how the 3D architecture of chromatin is associated with the mechanism of gene expression, changes in chromatin organization must be correlated with other epigenetic effects, such as binding of transcription factors, architectural proteins or histone modifications.
Finally, we examined chromatin interactions and fibrinogen expression in HepG2 cells and
∆CTCF4 cells after inflammatory stimuli. In both cell types, mRNA levels of the three fibrinogen genes were significantly increased after treatment with IL-6. This is in agreement with previous studies in which IL-6 stimulates a coordinate increase in fibrinogen mRNA [36, 43]. Fibrinogen was decreased after TNF-α treatment, as previously reported [39]. LPS treatment did not affect fibrinogen levels.
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We did not detect major changes in the chromatin architecture of the fibrinogen gene cluster in response to IL-6 or TNF-α, in HepG2 or ∆CTCF4 cells. Therefore, at the resolution of our assays, cytokines that increase or decrease fibrinogen production via changes in steady- state mRNA levels do not have their effects mediated by modification of chromatin interactions.
We have demonstrated that local chromatin contacts occur at the fibrinogen gene cluster and contribute to fibrinogen gene expression and protein output. This links chromatin architecture to the production of a clinically important blood clotting factor, revealing a regulatory level that may play a role in the determination of plasma fibrinogen levels.
Addendum
C. N. Espitia Jaimes performed the experiments, analyzed and interpreted the data and wrote the paper. R. J. Fish designed the experiments, analyzed and interpreted the data and wrote the paper. M. Neerman-Arbez designed the research and wrote the paper. All authors reviewed and approved the final manuscript.
Acknowledgements
We are very grateful to Corinne Di Sanza for expert technical assistance. This work was supported by a Swiss National Science Foundation grant [#31003A_152633] to M. Neerman- Arbez.
Disclosure of Conflict of Intersts
The authors state that they have no conflict of interest.
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Figure legends
Figure 1. Chromatin interactions of the fibrinogen gene cluster. (A) Representation of the human fibrinogen gene cluster (~73 kb shown here), with the three genes (FGB, FGA and FGG) and their transcriptional orientation denotated with arrows. Enhancers (E4, CNC12, PFE2 and E3) are annotated as green boxes and CTCF sites flanking the cluster (0-4) are represented with red boxes. CTCF Motif orientations are represented with red arrows. (B) Representation of the chromatin fragmentation at the fibrinogen gene cluster after NlaIII digestion for the described 3C experiments. The majority of the interrogated fragments are shown and are numbered below for clarity, either side of CTCF0 (0). Control regions are labeled (C a-e) and represented with orange triangles. Chromatin interactions were assesed for all the fragments with respect to CTCF0. (C) Chromatin interactions are defined by an interaction peak that represents physical proximity of two fragments in HepG2 cells that express fibrinogen or in (D) HEK-293T cells that do not. Clear differences at the interaction between CTCF0 and FGA promoter can be observed between cell types. Vertical lines help visualize equivalent positions on the two graphs and the dashed line box highlights the interaction signal seen at CTCF0 due to fragment self-ligations and linear proximity to the constant fragment (CTCF0). Data were normalized with the GAPDH gene for each ligation
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product. Error bars show SEM, n=5 in HepG2 cells and n=3 in HEK-293T cells. Each ligation product was analyzed in triplicate.
Figure 2. Deletion of CTCF4 affects fibrinogen gene cluster architecture and fibrinogen expression. (A) CTCF3 and CTCF4 deletion. Two sgRNAs were delivered into expression plasmids in HepG2 targeting the CTCF factorbook motif (green box) of CTCF3 or CTCF4, resulting in 35 or 31 nucleotide deletions respectively. (B) Chromatin interactions measured by 3C in CTCF motif deleted clones (ΔCTCF3, ΔCTCF4 and ΔCTCF3+4). The HepG2 data is that shown in Figure 1C, copied here to help compare interaction plots. Clones in which the CTCF4 interaction motif was deleted show a less frequent interaction between CTCF0 and the FGAp, although the overall chromosome domain was maintained with a novel interaction detected between CTCF0 and CTCF3. Data were normalized with the GAPDH gene for each ligation product. Error bars show SEM, n=5 in HepG2 cells and n=3 for each motif-deleted clone analyzed in the study. Each ligation product was analysed in triplicate.
(C) Steady-state mRNA levels of fibrinogen gene expression (FGB, FGA and FGG) quantified in HepG2 cells and CTCF motif-deleted clones by qRT-PCR. Every data set represents a unique clone (∆CTCF3= 1, ∆CTCF4= 3, ∆CTCF3+4=3). Data were normalized with expression of β- Actin. Note the reduction in FGB and FGG expression in all clones lacking CTCF4 (ΔCTCF4 and ΔCTCF3/ΔCTCF4 cells). Two-tailed students t-test *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 and
****P ≤ 0.0001. Error bars show SEM, n=3. (D) ELISA quantification of fibrinogen production in HepG2 cells and CTCF motif-deleted cells, demonstrating a halving of fibrinogen levels in ΔCTCF4 cells with respect to HepG2 cells. Data were normalized to HepG2 fibrinogen levels.
Two-tailed students t-test *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001. Error bars show SEM, n=3.
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Figure 3. Introducing a CTCF interaction site rescues chromatin interactions and fibrinogen expression in ΔCTCF4 or ΔCTCF3/ΔCTCF4 cells. (A) CTCF3 and CTCF4 rescue. A sgRNA, Cas9, and a HDR template were was used to re-introduce the CTCF factorbook motif of CTCF3 or CTCF4 in previously deleted clones. We designed a repair oligo template containing 15 nucleotides corresponding to the factorbook motif and 4 extra nucleotides on each side.
Obtained rescue sequences are unique in the genome (*). (B) Chromatin interactions were measured by 3C in CTCF motif-deleted cells after re-introduction of the CTCF interaction site(s), using CTCF0 as the constant fragment. Interactions between CTCF0 and CTCF4 resemble closely those in HepG2 cells when the CTCF motif was re-introduced at the CTCF4 position. The chromatin interaction between CTCF0 and the FGA promoter was also rescued.
Data were normalized with the GAPDH gene for each ligation product, error bars show SEM, n=5 in HepG2 cells and n=3 for each rescued clone analyzed in the study. Each ligation product was analysed in triplicate. Fibrinogen mRNA (C) and protein production (D) were restored to HepG2 levels when the CTCF motif was re-introduced at the CTCF4 position (ΔCTCF4R or ΔCTCF3R/ΔCTCF4R). For ease of comparison, control HepG2 data from Figures 1 and 2 are copied into this figure for the chromatin interations detected in HepG2 cells and the mRNA expression and protein production data for HepG2 cells. Error bars show SEM, n=3.
Figure 4. Chromatin interactions and fibrinogen expression after treatment with IL-6, TNF- α and LPS. Chromatin interactions at the fibrinogen gene cluster without inflammatory stimuli and after treatment with IL-6, TNF-α and LPS in (A) HepG2 cells and (B) ∆CTCF4 cells.
(C) FGB, FGA and FGG mRNA levels are significantly increased after treatment with IL-6 in HepG2 and ∆CTCF4 cells. In contrast, mRNA levels of the three fibrinogen genes are significantly reduced in HepG2 and ∆CTCF4 cells after treatment with TNF-α. Treatment with
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LPS did not show any significant changes in fibrinogen mRNA levels. Two-tailed students t- test *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001 compared to non-treated HepG2 cells; Two-tailed students t-test #P ≤ 0.05; ##P ≤ 0.01; ###P ≤ 0.001 and ####P ≤ 0.0001, compared to non-treated ∆CTCF4 cells. Error bars show SEM, n=3. (D) Fibrinogen protein levels were significantly increased in HepG2 and ∆CTCF4 cells after treatment with IL-6. In agreement with mRNA levels, protein levels were also significantly reduced in HepG2 and
∆CTCF4 after treatment with TNF-α. Treatment with LPS did not show significant differences.
Two-tailed students t-test *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 and ****P ≤ 0.0001, compared to non-treated cells. Error bars show SEM, n=3.
Figure 5. Proposed chromatin looping scenarios of the fibrinogen gene cluster
The models presented here are possible scenarios to explain the global interaction frequencies we have detected by 3C in populations of cells, and represent potential chromatin folding of single fibrinogen gene clusters, i.e. parts of a single chromosome in one cell. The sketches are not shown to scale and are annotated with detected interaction sites and the approximate position of the three fibrinogen proximal promoters for clarity. Models with a gray background, on the left, represent a proposed most frequent interaction seen for each cell type in our study. A simple linear configuration without looping cannot be excluded using our analysis but is not shown for each cell type to avoid repetition. From the top, in HepG2 cells our data are consistant with CTCF0 to CTCF4 and CTCF0 to FGA promoter interactions together (left image) or individually (centre and right images). HEK-293T cells also have the CTCF0 to CTCF4 interaction, but the CTCF0 to FGA promoter interaction is less frequent. In the absence of CTCF4 (ΔCTCF4), additional CTCF0 to CTCF3 and CTCF0 to PFE2 interactions are detected and fibrinogen expression is reduced. These interactions may or
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may not occur together. When both CTCF3 and 4 are deleted, CTCF0 to FGA promoter or PFE2 interactions are possible (ΔCTCF3/ΔCTCF4) and a linear configeration may be more frequent, but there is no further impact on fibrinogen expression compared to ΔCTCF4 cells.
Finally, rescue or re-insertion of the CTCF interaction motif at the CTCF4 postion in ΔCTCF4 or ΔCTCF3/ΔCTCF4 cells (ΔCTCF4R or ΔCTCF3R/ΔCTCF4R) restores the chromatin folding seen in HepG2 cells, and the HepG2 fibrinogen expression level.
