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Transcription upregulation via force-induced direct stretching of chromatin

View Article: PubMed Central - PubMed

ABSTRACT

Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green-fluorescent-protein (GFP) tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

No MeSH data available.


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The extent of chromatin stretching depends on stress directionsMean Squared Displacement (MSD) of individual GFP spots #1 (a), #2 (b), and #3 (c) in a representative cell (the same cell as in Fig. 1e), when the stress (15 Pa at 0.3 Hz) was applied at 0º, 45º, or 90º. The black dashed lines in a, b, and c were the no stress control. It is apparent that the MSD was largest when the stress was applied along the transverse direction relative to the long axis of the cell. Data from 7 cycles of displacements are averaged in MSD curves. (d) The fluorescent image of the three GFP spots in the same chromatin of the cell. (e, f) Chromatin stretching (both peak stretching amplitude in (e) and % stretching in (f)) depends on stress angles. The increase of distance between any two GFP spots (Δ Distance) as a function of the stress angle at a constant peak stress (15 Pa at 0.3 Hz) represents the extent of chromatin stretching. Note that % stretching represents “an apparent stretching of the chromatin”, i.e., the distance between two spots on the chromatin is increased; it does not suggest that the chromatin molecule itself is stretched or elongated. The peak compressing amplitude and % compressing were similar to those of stretching. Mean ± s.e.m; n=90 GFP spots from 30 cells of 21 separate experiments; *** P<0.001.
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Figure 2: The extent of chromatin stretching depends on stress directionsMean Squared Displacement (MSD) of individual GFP spots #1 (a), #2 (b), and #3 (c) in a representative cell (the same cell as in Fig. 1e), when the stress (15 Pa at 0.3 Hz) was applied at 0º, 45º, or 90º. The black dashed lines in a, b, and c were the no stress control. It is apparent that the MSD was largest when the stress was applied along the transverse direction relative to the long axis of the cell. Data from 7 cycles of displacements are averaged in MSD curves. (d) The fluorescent image of the three GFP spots in the same chromatin of the cell. (e, f) Chromatin stretching (both peak stretching amplitude in (e) and % stretching in (f)) depends on stress angles. The increase of distance between any two GFP spots (Δ Distance) as a function of the stress angle at a constant peak stress (15 Pa at 0.3 Hz) represents the extent of chromatin stretching. Note that % stretching represents “an apparent stretching of the chromatin”, i.e., the distance between two spots on the chromatin is increased; it does not suggest that the chromatin molecule itself is stretched or elongated. The peak compressing amplitude and % compressing were similar to those of stretching. Mean ± s.e.m; n=90 GFP spots from 30 cells of 21 separate experiments; *** P<0.001.

Mentions: First we measured movements of two GFP spots under a cyclic stress and found that the distance between them increased as a function of the stress cycle (Fig. 1h and i). Then we measured stress-induced displacements of individual GFP spots along the BAC insertions with a constant peak stress amplitude and with varying stress angles of 0°, 45°, and 90° (Supplementary Fig. 2a–c). All 3 GFP spots in the same chromatin were displaced synchronously with the bead displacement with phase lags (Supplementary Fig. 2a–c), indicating the viscoelastic features of the cytoplasm and the nucleus. To better visualize the dependence of GFP spot displacement on stress angles, we quantified Mean Squared Displacement (MSD) of each GFP spot as a function of stress angles. Each GFP spot exhibited highest MSDs when the stress was applied transverse cross the long axis (at 90°), followed by MSD amplitudes at 45° (diagonally) and at 0° (along the long axis) (Fig. 2a–d), showing strong dependence on stress angles. Since the increase of distance (Δ Distance) between any two GFP spots is a measure of the extent of chromatin stretching, we quantified Δ Distance as a function of stress angles. Interestingly, chromatin stretching at 90° is >3-fold larger than at 0° (Fig. 2e). Since cell stiffness at 90° was half of that at 0° (see Fig. 1g), these data suggest that for a given applied stress, chromatin stretching is inversely proportional to cell stiffness. Dividing Δ Distance by the original distance between GPF spots is a measure of % stretching; “apparent” chromatin stretching increased from 5% to ~18% when stress angles varied from 0° to 90° (Fig. 2f). Although the applied stress was applied along a particular direction on the cell surface, the resulting strains inside the nucleus could be complex. Using a published method13 to compute local chromatin strains from local chromatin GFP displacements, we found that both tensile strains and shear strains were stress-angle dependent, but tensile strains in the chromatin were ~2.5 times shear strains (Supplementary Fig. 3), suggesting that dilatational strains are the dominant mode of deformation in the chromatin. The differences between tensile strains and shear strains in the chromatin and their dependence on stress angles were supported by data from strain maps of the whole nucleus using histone 2B-green fluorescent protein (H2B-GFP) as a chromatin marker (Supplementary Figs. 4 and 5): peak bulk strain (both tensile and compressive strains) was ~4 times peak shear strain and both were stress-angle dependent, suggesting that a local surface stress of physiologic magnitudes could directly stretch chromatins and that BAC insertion did not cause any abnormal mechanical responses from the chromatin. Together, these results suggest that chromatin stretching is strongly dependent on how much the applied stress is propagated through the cell to extend the chromatin.


Transcription upregulation via force-induced direct stretching of chromatin
The extent of chromatin stretching depends on stress directionsMean Squared Displacement (MSD) of individual GFP spots #1 (a), #2 (b), and #3 (c) in a representative cell (the same cell as in Fig. 1e), when the stress (15 Pa at 0.3 Hz) was applied at 0º, 45º, or 90º. The black dashed lines in a, b, and c were the no stress control. It is apparent that the MSD was largest when the stress was applied along the transverse direction relative to the long axis of the cell. Data from 7 cycles of displacements are averaged in MSD curves. (d) The fluorescent image of the three GFP spots in the same chromatin of the cell. (e, f) Chromatin stretching (both peak stretching amplitude in (e) and % stretching in (f)) depends on stress angles. The increase of distance between any two GFP spots (Δ Distance) as a function of the stress angle at a constant peak stress (15 Pa at 0.3 Hz) represents the extent of chromatin stretching. Note that % stretching represents “an apparent stretching of the chromatin”, i.e., the distance between two spots on the chromatin is increased; it does not suggest that the chromatin molecule itself is stretched or elongated. The peak compressing amplitude and % compressing were similar to those of stretching. Mean ± s.e.m; n=90 GFP spots from 30 cells of 21 separate experiments; *** P<0.001.
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Figure 2: The extent of chromatin stretching depends on stress directionsMean Squared Displacement (MSD) of individual GFP spots #1 (a), #2 (b), and #3 (c) in a representative cell (the same cell as in Fig. 1e), when the stress (15 Pa at 0.3 Hz) was applied at 0º, 45º, or 90º. The black dashed lines in a, b, and c were the no stress control. It is apparent that the MSD was largest when the stress was applied along the transverse direction relative to the long axis of the cell. Data from 7 cycles of displacements are averaged in MSD curves. (d) The fluorescent image of the three GFP spots in the same chromatin of the cell. (e, f) Chromatin stretching (both peak stretching amplitude in (e) and % stretching in (f)) depends on stress angles. The increase of distance between any two GFP spots (Δ Distance) as a function of the stress angle at a constant peak stress (15 Pa at 0.3 Hz) represents the extent of chromatin stretching. Note that % stretching represents “an apparent stretching of the chromatin”, i.e., the distance between two spots on the chromatin is increased; it does not suggest that the chromatin molecule itself is stretched or elongated. The peak compressing amplitude and % compressing were similar to those of stretching. Mean ± s.e.m; n=90 GFP spots from 30 cells of 21 separate experiments; *** P<0.001.
Mentions: First we measured movements of two GFP spots under a cyclic stress and found that the distance between them increased as a function of the stress cycle (Fig. 1h and i). Then we measured stress-induced displacements of individual GFP spots along the BAC insertions with a constant peak stress amplitude and with varying stress angles of 0°, 45°, and 90° (Supplementary Fig. 2a–c). All 3 GFP spots in the same chromatin were displaced synchronously with the bead displacement with phase lags (Supplementary Fig. 2a–c), indicating the viscoelastic features of the cytoplasm and the nucleus. To better visualize the dependence of GFP spot displacement on stress angles, we quantified Mean Squared Displacement (MSD) of each GFP spot as a function of stress angles. Each GFP spot exhibited highest MSDs when the stress was applied transverse cross the long axis (at 90°), followed by MSD amplitudes at 45° (diagonally) and at 0° (along the long axis) (Fig. 2a–d), showing strong dependence on stress angles. Since the increase of distance (Δ Distance) between any two GFP spots is a measure of the extent of chromatin stretching, we quantified Δ Distance as a function of stress angles. Interestingly, chromatin stretching at 90° is >3-fold larger than at 0° (Fig. 2e). Since cell stiffness at 90° was half of that at 0° (see Fig. 1g), these data suggest that for a given applied stress, chromatin stretching is inversely proportional to cell stiffness. Dividing Δ Distance by the original distance between GPF spots is a measure of % stretching; “apparent” chromatin stretching increased from 5% to ~18% when stress angles varied from 0° to 90° (Fig. 2f). Although the applied stress was applied along a particular direction on the cell surface, the resulting strains inside the nucleus could be complex. Using a published method13 to compute local chromatin strains from local chromatin GFP displacements, we found that both tensile strains and shear strains were stress-angle dependent, but tensile strains in the chromatin were ~2.5 times shear strains (Supplementary Fig. 3), suggesting that dilatational strains are the dominant mode of deformation in the chromatin. The differences between tensile strains and shear strains in the chromatin and their dependence on stress angles were supported by data from strain maps of the whole nucleus using histone 2B-green fluorescent protein (H2B-GFP) as a chromatin marker (Supplementary Figs. 4 and 5): peak bulk strain (both tensile and compressive strains) was ~4 times peak shear strain and both were stress-angle dependent, suggesting that a local surface stress of physiologic magnitudes could directly stretch chromatins and that BAC insertion did not cause any abnormal mechanical responses from the chromatin. Together, these results suggest that chromatin stretching is strongly dependent on how much the applied stress is propagated through the cell to extend the chromatin.

View Article: PubMed Central - PubMed

ABSTRACT

Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green-fluorescent-protein (GFP) tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

No MeSH data available.


Related in: MedlinePlus