Integrin α4β1 controls G9a activity that regulates epigenetic changes and nuclear properties required for lymphocyte migration

The mechanical properties of the cell nucleus change to allow cells to migrate, but how chromatin modifications contribute to nuclear deformability has not been defined. Here, we demonstrate that a major factor in this process involves epigenetic changes that underpin nuclear structure. We investigated the link between cell adhesion and epigenetic changes in T-cells, and demonstrate that T-cell adhesion to VCAM1 via α4β1 integrin drives histone H3 methylation (H3K9me2/3) through the methyltransferase G9a. In this process, active G9a is recruited to the nuclear envelope and interacts with lamin B1 during T-cell adhesion through α4β1 integrin. G9a activity not only reorganises the chromatin structure in T-cells, but also affects the stiffness and viscoelastic properties of the nucleus. Moreover, we further demonstrated that these epigenetic changes were linked to lymphocyte movement, as depletion or inhibition of G9a blocks T-cell migration in both 2D and 3D environments. Thus, our results identify a novel mechanism in T-cells by which α4β1 integrin signaling drives specific chromatin modifications, which alter the physical properties of the nucleus and thereby enable T-cell migration.


Supplementary Materials and Methods
Immunofluorescence. Jurkat cells were cultured for 24 h on poly-Lysine, ICAM1 and VCAM1 coated coverslips. Cells were fixed in 4% formaldehyde (10 min), permeabilised with 0.5% Tx-100 PBS (5 min), blocked in 10% horse serum and incubated with appropriated primary antibodies for 1 h at RT. After several washes, samples with incubated with secondary antibodies. Samples were mounted in Dako and imaged with a Leica TCS SP5 confocal microscope. For H3K9me2/3 and H4K20me3 staining, after fixation samples were incubated in 100 mM sodium citrate preheated at 95ºC for 20 min prior cell permeabilisation.
after background subtraction. H3K9me2/3 and H4K20me3 foci counting analysis were performed manually using ImageJ.

RT-qPCR. Total RNAs were isolated by using RNAqueous-Micro Total RNA
Isolation Kit (Life Technologies), according to the manufacturer's instructions.
RNA were converted into cDNA by TaqMan Reverse Transcription Reagents (Life Technologies), qPCR was performed using the StepOnePlus System (Life technologies) and data were normalized to expression of two reference genes, ACTB and GAPDH. Gene expression is relative to that of the control samples. Values shown are the mean ± standard deviation of three independent experiments. Primer sequences are shown as Supplemental table 1.

Production of lentivirus
Cell migration assay. We coated 96-well plates with poly-Lysine (5 μg/ml) or VCAM1 (2.5 μg/ml). Jurkat cells pretreated or not with chaetocin (0.5 μM) or Farmingdale, NY). After preclearing with protein G-Agarose beads (Pierce), supernatants were incubated with antibodies followed by coupling to protein G-or protein A-Sepharose. Proteins were resolved by SDS-PAGE. For total lysates, proteins were extracted using SDS-loading buffer and sonication before boilling. Proteins were resolved in 12.5% polyacrylamide gels. Gels were transferred to nitrocellulose membranes then blocked in 5% low fat milk in TBS-Tween (0.5%) for 1 hour at room temperature. Membranes were incubated with primary antibodies in 5% low fat milk in TBS-tween (0.5%) at the appropriate dilution at 4°C overnight. Membranes were washed in TBS-Tween (0.5%) and incubated with appropriated IRDye secondary antibodies (Li-cor Biosciences; Lincoln NE) in 5% low fat milk in TBS-Tween (0.5%) for 1 hour at room temperature. Protein signal was analysed by Odyssey (Li-cor).

Osmotic stress analysis. Jurkat cells were cultured on coated plates for 24
h. Then, medium was replaced with hypotonic (normal medium diluted 1:5 with water) or hypertonic (medium with 0.4 M NaCl) medium and incubated for 5 min before fixation with 4% formaldehyde (10 min). Cells were permeabilized with 0.5% Tx-100 PBS (5 min