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. 2018 Nov 26;16(1):142.
doi: 10.1186/s12915-018-0608-2.

Migration through a small pore disrupts inactive chromatin organization in neutrophil-like cells

Elsie C Jacobson  1 , Jo K Perry  1 , David S Long  2   3 , Ada L Olins  4 , Donald E Olins  4 , Bryon E Wright  2 , Mark H Vickers  1 , Justin M O'Sullivan  5
Affiliations

Migration through a small pore disrupts inactive chromatin organization in neutrophil-like cells

Elsie C Jacobson et al. BMC Biol. .

Abstract

Background: Mammalian cells are flexible and can rapidly change shape when they contract, adhere, or migrate. The nucleus must be stiff enough to withstand cytoskeletal forces, but flexible enough to remodel as the cell changes shape. This is particularly important for cells migrating through confined spaces, where the nuclear shape must change in order to fit through a constriction. This occurs many times in the life cycle of a neutrophil, which must protect its chromatin from damage and disruption associated with migration. Here we characterized the effects of constricted migration in neutrophil-like cells.

Results: Total RNA sequencing identified that migration of neutrophil-like cells through 5- or 14-μm pores was associated with changes in the transcript levels of inflammation and chemotaxis-related genes when compared to unmigrated cells. Differentially expressed transcripts specific to migration with constriction were enriched for groups of genes associated with cytoskeletal remodeling. Hi-C was used to capture the genome organization in control and migrated cells. Limited switching was observed between the active (A) and inactive (B) compartments after migration. However, global depletion of short-range contacts was observed following migration with constriction compared to migration without constriction. Regions with disrupted contacts, TADs, and compartments were enriched for inactive chromatin.

Conclusion: Short-range genome organization is preferentially altered in inactive chromatin, possibly protecting transcriptionally active contacts from the disruptive effects of migration with constriction. This is consistent with current hypotheses implicating heterochromatin as the mechanoresponsive form of chromatin. Further investigation concerning the contribution of heterochromatin to stiffness, flexibility, and protection of nuclear function will be important for understanding cell migration in relation to human health and disease.

Keywords: Chromatin conformation; Epigenetics; Heterochromatin; Hi-C; Immune; Mechanotransduction; Migration; Neutrophil; Nuclear remodeling; Transcription.

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Figures

Fig. 1
Fig. 1
Neutrophil-like cells (7 μm diameter) were migrated through two sizes of porous membrane. a A classical Boyden chamber was modified to allow a continuous flow of media (0.2 mL/min) through the lower well. Migrated cells were processed every 30 min. Three experimental conditions were used: b cells that were not migrated, c cells that migrated through pores larger than themselves (14 μm diameter), and d cells that migrated through pores smaller than themselves (5 μm diameter)
Fig. 2
Fig. 2
Gene expression changes. a Venn diagram of significantly differentially expressed genes (DESeq2, FDR < 0.05) between 14-μm and 5-μm pores, unmigrated and 5-μm pores, and unmigrated and 14-μm pores. Eighty percent of the genes differentially expressed after migration through 14-μm pores also change after migration through 5-μm pores. b The 420 genes that changed in both conditions had highly correlated (R2 = 0.92) log2 fold changes (logFC). c While 1621 additional genes change expression after migration through 5 μm, only 178 of these were significantly different between the two pore sizes. The 1443 that were not significantly different had highly correlated changes compared to unmigrated cells (R2 = 0.82). d The relatively small subset of 199 genes that were uniquely different after migration through 5-μm pores. We believe these genes are associated with the effects of remodeling and had low correlation (R2 = 0.36) between 5- and 14-μm pore migration
Fig. 3
Fig. 3
Compartment switching following migration with or without constriction. a Compartment structure is mostly conserved, with a small proportion of regions in different compartments in cells migrated with or without constriction. 100 kb bins were considered switched if they had the opposite PC1 sign and low correlation (R < 0.6) between the two conditions. b Bootstrapping found that switching from compartment B occurred more frequently than switching from compartment A. c Compartment switching was not associated with gene expression changes. The log2 fold change of differentially expressed genes (5-μm vs 14-μm pores) did not correlate with compartment or switching status. d TADs identified in migration with constriction were considered conserved if they overlapped < 80% with TADs identified in migration without constriction. Conserved TADs were more likely to be found in compartment A than non-conserved TADs.
Fig. 4
Fig. 4
Short-range contacts were depleted after migration with constriction. a The whole genome contact matrix at 100 kb resolution was normalized with iterative correction and eigenvector decomposition (ICE), and distance-dependent contact probability plotted on a log-log scale. There was no significant difference between the distributions of unmigrated cells, and cells migrated without constriction (14-μm pores) (KS test, p = 0.14), while cells migrated with constriction (5-μm pores) have a significantly different distribution from both unmigrated and migrated without constriction (KS test 5.74 × 10− 9 and 3.11 × 10− 13 respectively). The inset highlights the rapid decay of contact frequency between 100 kb and 500 kb in migration with constriction. b Differential intrachromosomal contacts between migration with and without constriction were called at 100 kb resolution, tiling across the chromosome in 40-kb bins. Long-range (> 1 Mb) differential contacts were equally likely to be lost or gained, while short-range (100 kb–1 Mb) contacts were significantly more likely to be lost after migration with constriction. Regions involved in a contact were assigned to compartment A or B. c Distribution of disrupted contacts. Bin sizes are uneven. i Number of significant contacts (FDR < 0.05) in compartment A and B in cells migrated without constriction. ii Number of significantly decreased contacts (FDR < 0.1) in compartments A and B between migration with and without constriction. iii Bootstrapping of significant contacts found a strong enrichment of decreased short-range (< 1 Mb) contacts in compartment B. Error bars represent 99% CI of the expected proportion of contacts in compartment A or B based on 10,000 bootstraps. d 100-kb bins were defined as transcriptionally active if they contained one of more expressed gene. i Stable A compartment regions were most likely to be active, and stable B compartment genes were least likely to be active. ii A two sample test for equality of proportions found that disrupted contacts in either compartment A or B were less transcriptionally active than total contacts in the same compartment. Error bars show the 95% confidence interval of the difference in proportion of disrupted and total contacts that contain at least one expressed gene
Fig. 5
Fig. 5
An example region of chromosome 17(q24.1-q25.1) showing genome organization features following migration with or without constriction. a Significant contacts (FDR < 0.05, 100 kb resolution) in unmigrated cells, and cells migrated with or without constriction. Line thickness indicates significance. b Hi-C heatmap PC1 values, indicating compartments A (positive, black) and B (negative, grey) in the three conditions. c Correlation values show how similar the contact patterns are between two conditions. Comparisons between all three conditions are shown here, with a dotted line at R = 0.6 indicating the cutoff used to call compartment switching. d Domain (TAD) locations for all three conditions are shown here. The boundaries of TADs that are not conserved between migration with and without constriction are indicated with dashed grey lines. e The positions of expressed genes, and log mean gene expression of the two migration conditions. One gene in this region was differentially expressed (red) between migration with and without constriction; however, it was in a region with stable compartments, TADs, and contacts. f Hi-C heatmaps of the ICE normalized contact frequency at 128 kb resolution, visualized in HiGlass [100]. Lighter colour indicates higher contact frequency between regions in i unmigrated cells, ii cells migrated without constriction, and iii cells migrated with constriction
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