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. 2014 Aug 28;10(8):e1004544.
doi: 10.1371/journal.pgen.1004544. eCollection 2014 Aug.

Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome

Jutta Vogelmann  1 , Antoine Le Gall  1 , Stephanie Dejardin  1 , Frederic Allemand  1 , Adrien Gamot  2 , Gilles Labesse  1 , Olivier Cuvier  2 , Nicolas Nègre  3 , Martin Cohen-Gonsaud  1 , Emmanuel Margeat  1 , Marcelo Nöllmann  1
Affiliations

Chromatin insulator factors involved in long-range DNA interactions and their role in the folding of the Drosophila genome

Jutta Vogelmann et al. PLoS Genet. .

Abstract

Chromatin insulators are genetic elements implicated in the organization of chromatin and the regulation of transcription. In Drosophila, different insulator types were characterized by their locus-specific composition of insulator proteins and co-factors. Insulators mediate specific long-range DNA contacts required for the three dimensional organization of the interphase nucleus and for transcription regulation, but the mechanisms underlying the formation of these contacts is currently unknown. Here, we investigate the molecular associations between different components of insulator complexes (BEAF32, CP190 and Chromator) by biochemical and biophysical means, and develop a novel single-molecule assay to determine what factors are necessary and essential for the formation of long-range DNA interactions. We show that BEAF32 is able to bind DNA specifically and with high affinity, but not to bridge long-range interactions (LRI). In contrast, we show that CP190 and Chromator are able to mediate LRI between specifically-bound BEAF32 nucleoprotein complexes in vitro. This ability of CP190 and Chromator to establish LRI requires specific contacts between BEAF32 and their C-terminal domains, and dimerization through their N-terminal domains. In particular, the BTB/POZ domains of CP190 form a strict homodimer, and its C-terminal domain interacts with several insulator binding proteins. We propose a general model for insulator function in which BEAF32/dCTCF/Su(HW) provide DNA specificity (first layer proteins) whereas CP190/Chromator are responsible for the physical interactions required for long-range contacts (second layer). This network of organized, multi-layer interactions could explain the different activities of insulators as chromatin barriers, enhancer blockers, and transcriptional regulators, and suggest a general mechanism for how insulators may shape the organization of higher-order chromatin during cell division.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Protein constructs, protein purification, and genomic localization of insulator proteins and associated factors.
(A) Description of protein constructs used in this study. C2H2 zinc-finger motifs (Zn-Fn) are shown as vertical rectangles, BESS motifs as a vertical red line, BTB/POZ domains as rounded boxes and chromo-domains (ChD) as ellipses. N-terminal domains (N-) are always on left. Lengths of each domain or fragment is indicated in number of amino-acids from the N-terminal end. His indicates a 6-Histidine tag, and MBP the maltose binding-protein. (B) Purity of purified BEAF32, CP190, CP190-C, Chromator, and Chromator-C was assessed by poly-acrylamide gel electrophoresis (PAGE, Coomassie blue staining, top panel), and resulted in single bands (>95% purity, see arrows). Molecular weight ladder is shown on the left. Western-blot analysis (bottom panel) of each purified protein shows the specific recognition by each of the antibodies developed. (C) Binding profile of insulator-associated proteins (BEAF32, Chromator, CP190, dCTCF, Su(HW)) and epigenetic marks (H4K27m3, and H3K9AcS10) in chromosome 3L from ModEncode data (S2 cells; Generic Genome Browser version 2.40). Tracks used are described in Supplementary Table S6. For each protein, the track depicts the MAT score of each probe plotted on the y-axis versus chromosomal position plotted along the x-axis. The genomic region used for EMSA-analysis (DNAtudor, part of the Tudor-SN lucus) is highlighted in pink (3L: 264375–264822). DNAtudor contains six CGATA binding motifs.
Figure 2
Figure 2. Binding of insulator factors to DNA.
(A) Electric mobility shift assay (EMSA) of BEAF32-DNAtudor complexes. A plasmid containing DNAtudor was digested resulting in three linear fragments of size 750, 4025 and 1627 bp (the fragment containing DNAtudor, red). Addition of BEAF32 (200 nM) leads to the preferential disappearance of the band containing CGATA motifs. (B) Scheme representing the experimental setup for fluorescence anisotropy measurements of BEAF32-DNA binding equilibrium. Binding of BEAF32 to DNAS (short DNA fragment containing three CGATA motifs) leads to an increase in the size of the complex that can be detected by an increase in the fluorescence anisotropy signal. KD represents the apparent equilibrium dissociation constant of the complex. (C) BEAF32 binding isotherms for DNAS (red circles) and DNANS (DNA fragment of the same size as DNAS but with no CGATA motif, green triangles). Solid lines represent fits to a single-site binding (green) or a Hill model (red). (D) EMSA of CP190-DNAtudor complexes show no specificity of DNA binding for CP190 at this genomic locus. In contrast to BEAF32, CP190 shifted the three DNA fragments with similar efficiency even at high protein concentrations (400 nM). Concentrations used were: 0, 100, 200, 300 and 400 nM, respectively. The decrease in the intensity of the top band is less pronounced due to intensity saturation. (E) CP190 binding isotherms for DNAS (red circles) and DNANS (green triangles). Solid lines represent fits to a Hill model. CP190 binds both fragments with no specificity and equal affinity. (F) EMSA of Chromator-DNAtudor complexes show no specific binding for Chromator at this genomic locus. Concentrations used were: 0, 450, and 900 nM, respectively. The intensity of all bands is decreased to the same extent by the binding of Chromator, reflecting non-specific binding to these DNA fragments. (G) Chromator binding isotherms for DNAS (red circles) and DNANS (green triangles). Solid lines represent fits to a Hill model. Consistent with (F), Chromator binds both fragments with no specificity. (H) CP190-C (light blue) and Chromator-C (green) binding isotherms for DNAS. Addition of large protein concentration does not lead to detectable changes in fluorescence anisotropy.
Figure 3
Figure 3. Interactions between insulator factors.
(A) Co-immunoprecipitation pulldown assay (co-IP) with heterologously purified BEAF32 and Chromator. Goat-IgG or purified guinea-pig polyclonal antibodies against Chromator were covalently coupled to agarose beads. BEAF32 and Chromator were incubated and analyzed by SDS-PAGE followed by Western-Blot-analysis. Lane 1 (input) shows the presence of both BEAF32 and Chromator in the mix. Both proteins are retained by an anti-Chromator column, but not by an anti-goat-IgG column. (B) Co-IP of purified BEAF32 and Chromator-C. Chromator antibody recognizes Chromator and Chromator-C equally well (Materials and Methods). A mix of BEAF32/Chromator was incubated and analyzed by PAGE/Western blotting as before. Both BEAF32 and Chromator-C remain bound to an anti-Chromator column, consistent with the interaction between BEAF32 and Chromator being mediated by its C-terminal domain. (C) Co-IP of purified BEAF32 and CP190. A mix of BEAF32/CP190 was incubated and analyzed by PAGE/Western blotting. Both BEAF32 and CP190 remain bound to a rabbit anti-CP190 column, suggesting a direct interaction between these proteins. (D) BEAF32/CP190 interactions are mediated by CP190-C. A mix of BEAF32/CP190-C was incubated and analyzed by PAGE/Western blotting. Both BEAF32 and CP190-C remain bound to an anti-CP190 column, but not to the control anti-IgG column. (E) A mix of BEAF32, Chromator, and CP190 was incubated and analyzed by PAGE/Western blotting. The three proteins are bound to an anti-Chromator column, but not to the control anti-IgG column. (F) S2 nuclear extracts were incubated in an anti-Chromator or anti-IgG column and analyzed by PAGE/Western blotting. Both BEAF32 and Chromator remain bound to the anti-Chromator column, suggesting that these proteins interact in vivo. (G) S2 nuclear extracts were incubated in an anti-CP190 or anti-IgG column and analyzed by PAGE/Western blotting. Consistent with previous results, BEAF32, CP190 and Chromator remain bound to the anti-CP190 column, suggesting that these proteins are part of the same complex in vivo.
Figure 4
Figure 4. Insulator factors interact upon DNA binding.
(A) Native agarose band-shift assay of BEAF32-DNAtudor, and higher order complexes. Lane 1, 476 bp DNAtudor; lane 2, DNAtudor incubated with BEAF32; lane 3, DNAtudor incubated with CP190; lane 4, DNAtudor incubated with BEAF32 and CP190; lane 5, DNAtudor incubated with Chromator; lane 6, DNAtudor incubated with BEAF32 and Chromator; lane 7, DNAtudor incubated with CP190-C; lane 8, DNAtudor incubated with BEAF32 and CP190-C; lane 9, DNAtudor incubated with Chromator-C; lane 10, DNAtudor incubated with BEAF32 and Chromator-C; lane 11, DNAtudor incubated with CP190 and Chromator; lane 12, DNAtudor incubated with CP190-C and Chromator-C; lane 13, DNAtudor incubated with BEAF32, CP190 and Chromator. Band 1 represents DNAtudor. Band 2 represents the complex between BEAF32 and DNAtudor. Band 3 represents the BEAF32/DNAtudor complex super-shifted by binding of CP190, Chromator, Chromator-C, or the addition of both CP190 and Chromator. The shift of band 1 requires the presence of BEAF32. Protein concentrations used: BEAF32 (400 nM), CP190 (50 nM), CP190-C (50 nM), Chromator (100 nM), Chromator-C (100 nM). (B) Native agarose band-shift assay of MBP-DNAtudor. This experiment used the same protein mixes and concentrations as those used in (A) but replacing BEAF32 by MBP. No shifted band is apparent.
Figure 5
Figure 5. Formation of long-range interactions by insulator proteins.
(A) Scheme depicting a typical fluorescence fluctuation spectroscopy configuration. Fluorescently-labeled dsDNA fragments (cyan ribbon with green star) diffuse in and out of an excitation volume (red gradient) producing a time-dependent fluctuation in the fluorescence signal. Binding of protein (red cylinder) to DNA lead to a larger molecular complex, with a corresponding increase in its diffusion time. (B) Normalized auto-correlation of DNAS-Cy3B (2.5 nM, black), and shift in the auto-correlation curve due to BEAF32 binding (400 nM, B32S complex, red) or CP190 binding (50 nM, blue). No noticeable change in the diffusion time is observed when adding CP190-C (50 nM, light blue) to DNAS-Cy3B. (C) Normalized auto-correlation of DNAS-Cy3B (black), B32S (red), and a complex of B32S with CP190 (violet) or CP190-C (yellow). Addition of CP190 (50 nM) to B32S (400 nM) considerably increased the diffusion time, consistent with direct BEAF32/CP190 interactions leading to the formation of a higher molecular mass complex. Inset shows the two possible models that could lead to this increase in diffusion time. (D) Scheme presenting the two models tested by fluorescence cross-correlation spectroscopy. The formation of long-range interactions between B32S complexes (with either a Cy3B- or an atto655-labeled DNAS fragment) lead to a cross-correlation signal between these two colors. In contrast, the absence of cross-correlation signal implies no long-range interaction between B32S complexes. BEAF is shown in red, and CP190 in blue. DNAS is represented by a cyan ribbon with a star representing the fluorophore at its 5′-end. (E) Cross-correlation between the two fluorophores was only observed in the presence of CP190 (50 nM) and B32S (400 nM), and not when DNA alone, CP190+DNAS, or CP190-C+B32S were used at the same concentrations. (F) CP190 (50 nM) was pre-incubated with B32S (400 nM BEAF32), leading to a complex with a large cross-correlation signal in which CP190 forms long-range contacts between CGATA motifs (see inset scheme). The titration of this complex with CP190-BTB/POZ leads to the disappearance of the cross-correlation signal, consistent with the CP190-BTB/POZ domain being responsible for the CP190-CP190 interactions required for establishing long-range interactions. (G) Normalized auto-correlation of DNAS-Cy3B (2.5 nM, black), and shift in the auto-correlation curve due to BEAF32 binding (800 nM, B32S complex, red) or Chromator binding (100 nM, green). No noticeable change in the diffusion time is observed when adding Chromator -C (100 nM, light green) to DNAS-Cy3B. (H) Normalized auto-correlation of DNAS-Cy3B (black), B32S (red), and a complex of B32S with Chromator (dark yellow) or Chromator-C (yellow). Addition of Chromator (100 nM) to B32S (800 nM) considerably increased the diffusion time, consistent with direct BEAF32/Chromator interactions leading to the formation of a higher molecular mass complex. The small decrease in diffusion time observed upon addition of Chromator-C to B32S was not due to BEAF32 dissociating from DNA (Supplementary Figure S4), but probably due to a change in the translational diffusion of the complex triggered by a rearrangement of BEAF32 on DNAS upon interaction with Chromator-C , . (I) Cross-correlation signal was only observed in the presence of Chromator (100 nM) and B32S (800 nM BEAF32), but not when DNA alone, Chromator+DNAS, or Chromator-C+B32S were used at the same concentrations.
Figure 6
Figure 6. Structure of CP190-BTB/POZ, genome-wide localization of insulator factors at long-range contacts, and models.
(A) CP190-BTB/POZ crystallizes as a homo-dimer. The secondary structure of one monomer is shown in green and the surface of the second monomer is color-coded by conservation (pink: high, green: low). Monomers are mainly held together by interactions between helices α1 and α2. The peptide binding groove and the N-terminal domains are highly conserved and may participate in protein-protein interactions (discussed in text). (B) Schematic model for the formation of long-range interactions by CP190. The BTB/POZ domains of CP190 (blue) interact to form a dimer. Contacts between the C-terminal domain of CP190 and BEAF32 (red) or other insulator binding proteins (Su(HW), blue, dCTCF, brown) can lead to the formation of hybrid long-range contacts. (C) Venn diagram showing the genome-wide overlap between BEAF32, CP190 and Chromator in S2 cells calculated from publicly available modENCODE ChIP-chip data. (D) Schematic model highlighting the possible roles of Chromator/BEAF32 interactions. Chromator (green) could act as a LRI-forming protein bridging BEAF32 (red) binding sites, as well as serve to recruit the JIL-1 kinase (blue box) to regions of active chromatin. (E) Aggregation analysis was performed on Hi-C data to identify proximity correlations and functional relationships between anchors (BEAF32 binding sites) and signals (CP190/Chromator binding sites). Aggregation profiles are built by aligning and aggregating the Hi-C signals of CP190/Chromator peaks at a certain genomic distance d (15<d<60 kbp) from BEAF32 binding sites. BEAF32 is used as the anchor and CP190/Chromator signals are aligned (at d = 0) and summed together. The y-axis shows the number of interactions every 500 bp normalized by the total number of sites (interacting and not interacting) that localize at the same distances from the anchor. Normalized aggregation Hi-C profiles for CP190 + Chromator are shown as blue solid lines, whereas control regions with no anchor are shown in grey.
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