Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells

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Abstract

Gene expression states influence the 3D conformation of the genome through poorly understood mechanisms. Here, we investigate the conformation of the murine HoxB locus, a gene-dense genomic region containing closely spaced genes with distinct activation states in mouse embryonic stem (ES) cells. To predict possible folding scenarios, we performed computer simulations of polymer models informed with different chromatin occupancy features that define promoter activation states or binding sites for the transcription factor CTCF. Single-cell imaging of the locus folding was performed to test model predictions. While CTCF occupancy alone fails to predict the in vivo folding at genomic length scale of 10 kb, we found that homotypic interactions between active and Polycomb-repressed promoters co-occurring in the same DNA fiber fully explain the HoxB folding patterns imaged in single cells. We identify state-dependent promoter interactions as major drivers of chromatin folding in gene-dense regions.

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Figure 1: Classification of gene promoter states across the HoxB locus in mouse ES cells.
Figure 2: The SBS polymer model predicts folding of the HoxB locus under different biological scenarios.
Figure 3: Genomic regions chosen for testing model predictions of HoxB locus folding by FISH in ES cells.
Figure 4: Imaging the extended HoxB locus by cryoFISH identifies a folding pattern dependent on homotypic contacts between active and poised promoters, as predicted by the SBS model.
Figure 5: Agreement between cryoFISH imaging of the HoxB locus and the SBS homotypic interaction model extends to the distributions of inter-locus distances.
Figure 6: Homotypic interactions between active and poised genes coincide with their respective colocalization with RNAPII-S2p and Polycomb complexes.
Figure 7: Model representing HoxB locus folding in mouse ES cells.

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Acknowledgements

We thank K.J. Morris for help growing the mouse ES cells, E. Brookes, R.A. Beagrie and C. Ribeiro de Almeida for help and advice, and W. Bickmore for providing the ESC-OS25 cell line (MRC Human Genetic Unit, Edinburgh, UK). The work was supported by the Medical Research Council, UK (A.P., S.Q.X., I.d.S., M.R.B., D.R.), the Helmholtz Foundation (A.P., M.B., E.T.T.), and the Berlin Institute of Health (A.P., M.B., M.N.). This work was supported by grants to M.N. from CINECA ISCRA ID HP10CYFPS5. M.N. also acknowledges computer resources from INFN, CINECA, and Scope at the University of Naples.

Author information

A.P. and M.N. designed the project. M.B., A.M.C., and S.B. performed the polymer modeling analysis. S.Q.X. performed the wet-lab experiments and image analysis. I.d.S. and E.T.T. performed the bioinformatics analyses. D.R. provided conceptual advice and co-mentored the work of S.Q.X. with A.P. M.R.B. developed the image analysis pipeline. A.P., M.N., M.B., S.Q.X., A.M.C. and S.B. wrote the paper, and all authors revised the manuscript.

Correspondence to Mario Nicodemi or Ana Pombo.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Schematic representation of the extended HoxB locus and classification of gene promoter states or CTCF occupancy sites.

(a) An illustration of the 1Mb genomic region centered on the HoxB gene cluster (chr11:95685813-96650449; mouse genome mm9). (b) Classification of genes within the HoxB locus according to the state of transcriptional activation of their promoters: promoters classified as Active are shown in green, Poised promoters in red and Silent promoter in grey. Gip was the only gene classified as Silent. (c) Classification of genes based only on the presence of RNAPII-S5p at promoters (blue). (d) Distribution of CTCF binding sites (orange).

Supplementary Figure 2 The four stable phases of the homotypic interaction model obtained varying the affinities of binders.

The system phase diagram identifies the stable architectural classes of the model, which correspond to its thermodynamics phases, defined by binding affinity.

Supplementary Figure 3 Contact matrices across the HoxB locus simulated using the SBS model.

(a) Contact matrices summarize chromatin contacts obtained using the SBS model, which considers homotypic interactions between Active and/or Poised genes. The four larger panels illustrate the predicted full contact probability matrices in the four thermodynamics states of the model. The four smaller panels represent the model-predicted contact frequency matrix restricted to the probes considered in the FISH experiments. (b) Contact matrix results using the SBS model in the cases in which: left, a 50:50 mixture of polymers is considered, composed of polymers in phases 2 and 3; or, right, all promoters genes marked by RNAPII-S5p can bind with each other (right). (c) The contact matrix predicted by an SBS model based only on contacts that depend on contacts between CTCF binding sites.

Supplementary Figure 4 Chromatin occupancy maps across the Skap1 gene in mouse ES cells.

To identify a control region for FISH at similar distance to Active or Poised (Polycomb-repressed) windows, we identified a region within the coding region of the Skap1 gene. Although the Skap1 promoter is classified at Polycomb-repressed and occupied by RNAPII-S5p, its long coding region (>200kb) is devoid of specific enrichment for the promoter marks studied. The control region chosen (grey horizontal bar) is highly enriched for CTCF occupancy and is expected to interact extensively in the CTCF-only models but not the promoter state models. Orange vertical lines: CTCF binding sites.

Supplementary Figure 5 Strategy for semi-automated distance measurement in cryoFISH images of genomic loci within the HoxB locus.

(a) Summary of the semi-automated approach developed here to locate FISH signals in nuclear profiles from cryosections and to measure their inter-locus distances. (b) Table listing the number of nuclear profiles analysed for the ten pairs of probes tested, the frequency of probe detection and average locus inter-distances from FISH and from the SBS model for the equivalent genomic regions, considering the ensemble of homotypic polymers modelled in phase 4.

Supplementary Figure 6 Distance distributions of genomic regions within the HoxB locus.

This figure presents the histograms of inter-locus physical distances between different pairs of probes within the HoxB locus that were not shown in Fig. 4. The distance distributions of heterotypic pairs of loci are more spread than the distributions of distances of pairs belonging to the same class (homotypic pairs; Fig. 4). Around 70% of distances are >250 nm for heterotypic pairs. Number of distance measurements from left to right, top to bottom are: 78, 76, 92, 139, 100 and 128.

Supplementary Figure 7 Comparison of inter-locus distances from cryoFISH imaging of the HoxB locus and the SBS homotypic (phase 4) interaction model.

Similar distance distributions were obtained experimentally by cryoFISH from a population of mouse ES cells and by polymer physics using an ensemble of SBS polymers corresponding to the homotypic interaction case, although free fitting parameters were not used to optimise the match. Most (typically ~70%) of heterotypic distances and distances to control probe are much more broader (>150nm). A 50:50 mixture of polymers in phases 2 and 3 is also represented for comparison. Number of distance measurements were Hoxb13-Snf8 (phase 4, 76; cryoFISH, 149), Snf8-Control (64; 92), Hoxb1-Snx11 (64; 128), Hoxb1-Control (46; 78), Hoxb13-Snx11 (97; 139), and Hoxb13-Control (67; 102).

Supplementary Figure 8 Testing different modelling parameters of the SBS model: an analysis of correlations with cryoFISH results.

(a) Scheme of two different conditions of polymer modelling considered in our study, which differ by the binding multiplicity of the polymer beads. In the case of multiplicity equal to 1 (right), the polymer architectures produced can have different compared with the high multiplicity case (left). (b) Table listing the Pearson’s correlation coefficients between the contact matrix obtained experimentally from single cell imaging of genomic positioning across the extended HoxB locus and the matrices calculated from ensembles of folded polymers obtained by different variants of the SBS model. The model with multiplicity 6 correlates better in comparison with cryoFISH experimental results than the one with multiplicity 1. The case corresponding to ‘phase 4’ is preferred to the ‘mix phase 2-3’ case (i.e., a 50-50 mixture of phase 2 and 3; panel d) as seen by comparing the specific values of their contact matrices with those obtained by FISH (panel g). (c) The full contact probability matrix for the case with multiplicity 6 shows the formation of both local clustering and long-range contacts. (d) Comparison of the mini-contact matrices obtained with the SBS model using multiplicity 6 in phase 4, mix phase 2-3 and phase 1, relative to the five polymer positions corresponding to the five FISH probes. (e) The contact probability matrix for the case with multiplicity 1 lacks longer-range contacts, namely between the two active (green) loci containing Snx11 and Snf8. Note that these mini-matrices were produced with more signal resolution than in previous figures, to allow more sensitivity in the calculations of correlations between matrices. (f) The mini-contact matrix in the model with multiplicity 1. (g) The contact matrix in our cryoFISH experiments for the five FISH probes calculated also with more signal resolution, to allow sensitive calculations of correlations with SBS matrices.

Supplementary Figure 9 Molecular Dynamics confirms Monte Carlo results.

The contact matrix derived in phase 4 by full scale Molecular Dynamics computer simulations (a) is 97% similar to the one derived by Monte Carlo methods (b), when using similar values of parameters (here ER =EG =5.2 kBT; FENE constants: K=30 kBT/σ2, R0 =1.6σ).

Supplementary Figure 10 Molecular Dynamics results are robust to changes in the force-field parameters.

As predicted by Statistical Mechanics, the structure of the contact matrix in phase 4 is robust to changes in the force-field parameters of our Molecular Dynamics computer simulations.(a) The parameters are those reported in Suppl. Fig. 9.(b) ER =EG =6.0 kBT; FENE parameters: K=30kBT/σ2, R0 =1.6σ.(c) ER=EG =5.2 kBT; K=35kBT/σ2, R0 =1.6σ.(d) ER=EG =5.2 kBT; K=25kBT/σ2, R0 =1.6σ.(e) ER=EG =5.2 kBT; K=30kBT/σ2, R0 =1.7σ.(f) ER=EG=5.2 kBT; K=30kBT/σ2, R0 =1.4σ.

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Barbieri, M., Xie, S., Torlai Triglia, E. et al. Active and poised promoter states drive folding of the extended HoxB locus in mouse embryonic stem cells. Nat Struct Mol Biol 24, 515–524 (2017) doi:10.1038/nsmb.3402

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