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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.


Related in: MedlinePlus

Strategy of visualizing chromatin under force(A) Schematic representation of transgene insertions and fluorescence labeling of chromatin in live cells (Not drawn to scale) 26. Two yellow lines represent the long sequence of BAC. A stable cell line of DHFR D10 clone was used that had an insertion of 10 copies of the BAC into the same chromatin domain without any intervening CHO (Chinese Hamster Ovary) genomic DNA26. Here for visual simplicity only 2 copies of BAC (with DHFR gene and LacO) are drawn. (B) GFP tagged chromatin domains are shown inside the nucleus of a living CHO cell. Left image: nucleus is outlined by dashed lines; the brightfield image is overlaid with the fluorescent image. Right image: the magnified GFP-LacI image of the same cell; an arrow point to chromatin domains tagged with multiple GFP spots. (C) Schematic representation of the 3D Magnetic Twisting Cytometry (3D MTC). (D) A ferromagnetic bead was attached to the apical surface of the cells via integrins. A new strategy of altering stress angle but keeping stress amplitude constant was employed by which the bead was magnetized in Z direction and twisted in specified angles toward the X–Y plane by simultaneously modulating the amplitudes of the magnetic fields in X and Y directions. (E) A live CHO cell with a 4-μm ferromagnetic bead (a white arrow points to a solid black circle) with the GFP labeled chromatin (a yellow arrow points to the green spots). θ represents the angle of bead rotating direction with respect to the long axis of the cell (this notation applies to all cells in all figures). Scale bar, 5 μm. (F) Displacements of the center of the magnetic bead at the stress angle of θ=0º, 45º, or 90º in the same cell as in (E). Each displacement value is an average of data from 3 cycles. In all stress directions, the amplitudes of the sinusoidal magnetic fields (at 0.3 Hz) were modulated such that the peak stress amplitude remained constant at 15 Pa. The peak bead displacement was smaller along the long-axis of the cell than along the short axis. Note that due to a slight non-alignment between 0° stress angle (Y-axis) and the long axis of the cell in (D), thus 0° is in fact ~10°. Since the loading is cyclic and sinusoidal, a minus stress sign in (F) only represents the opposite direction of loading from the plus stress sign. (G) The dependence of cell stiffness (the ratio of the applied stress to the measured strain) on stress angles. Cell length to width ratio equals 2.84±0.377. Mean ± s.e.m.; n=30 cells, 21 independent experiments; *** P<0.001. (h) Schematic of two GFP spots of chromatin being deformed under stress. (i) Distance between two GFP spots (#1 and #2, corresponding to #2 and #3 GFP spots in Fig. 2d) in the same chromatin increases under a cyclic stress applied via integrins. Stress = 17.5 Pa at 0.3 Hz. Two white dashed lines are drawn only for visual aid.
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Figure 1: Strategy of visualizing chromatin under force(A) Schematic representation of transgene insertions and fluorescence labeling of chromatin in live cells (Not drawn to scale) 26. Two yellow lines represent the long sequence of BAC. A stable cell line of DHFR D10 clone was used that had an insertion of 10 copies of the BAC into the same chromatin domain without any intervening CHO (Chinese Hamster Ovary) genomic DNA26. Here for visual simplicity only 2 copies of BAC (with DHFR gene and LacO) are drawn. (B) GFP tagged chromatin domains are shown inside the nucleus of a living CHO cell. Left image: nucleus is outlined by dashed lines; the brightfield image is overlaid with the fluorescent image. Right image: the magnified GFP-LacI image of the same cell; an arrow point to chromatin domains tagged with multiple GFP spots. (C) Schematic representation of the 3D Magnetic Twisting Cytometry (3D MTC). (D) A ferromagnetic bead was attached to the apical surface of the cells via integrins. A new strategy of altering stress angle but keeping stress amplitude constant was employed by which the bead was magnetized in Z direction and twisted in specified angles toward the X–Y plane by simultaneously modulating the amplitudes of the magnetic fields in X and Y directions. (E) A live CHO cell with a 4-μm ferromagnetic bead (a white arrow points to a solid black circle) with the GFP labeled chromatin (a yellow arrow points to the green spots). θ represents the angle of bead rotating direction with respect to the long axis of the cell (this notation applies to all cells in all figures). Scale bar, 5 μm. (F) Displacements of the center of the magnetic bead at the stress angle of θ=0º, 45º, or 90º in the same cell as in (E). Each displacement value is an average of data from 3 cycles. In all stress directions, the amplitudes of the sinusoidal magnetic fields (at 0.3 Hz) were modulated such that the peak stress amplitude remained constant at 15 Pa. The peak bead displacement was smaller along the long-axis of the cell than along the short axis. Note that due to a slight non-alignment between 0° stress angle (Y-axis) and the long axis of the cell in (D), thus 0° is in fact ~10°. Since the loading is cyclic and sinusoidal, a minus stress sign in (F) only represents the opposite direction of loading from the plus stress sign. (G) The dependence of cell stiffness (the ratio of the applied stress to the measured strain) on stress angles. Cell length to width ratio equals 2.84±0.377. Mean ± s.e.m.; n=30 cells, 21 independent experiments; *** P<0.001. (h) Schematic of two GFP spots of chromatin being deformed under stress. (i) Distance between two GFP spots (#1 and #2, corresponding to #2 and #3 GFP spots in Fig. 2d) in the same chromatin increases under a cyclic stress applied via integrins. Stress = 17.5 Pa at 0.3 Hz. Two white dashed lines are drawn only for visual aid.

Mentions: We utilized a CHO DG44 (Chinese hamster ovarian) cell line containing a multi-copy insertion of a BAC with an ~180 kb mouse genomic insert containing the ~34 kb DHFR gene. The cell clone DHFR D10 stably expresses EGFP-dimer lac repressor (GFP-LacI), enabling visualization of the DHFR BAC, tagged with a 256mer lac operator repeat (~10 kb) (Fig. 1a)26. DHFR reduces dihydrofolate to tetrahydrofolate and is an essential enzyme for synthesizing thymidine. In order to allow for detection of local movements between chromatin regions, we used a cell clone that has multiple BAC copies in the same single chromatin without any intervening CHO genomic DNA26; the relative movement of multiple GFP spots (~2–7) therefore allows visualization of the relative motion of DHFR BAC chromatin26 (Fig. 1a, b, see Methods).


Transcription upregulation via force-induced direct stretching of chromatin
Strategy of visualizing chromatin under force(A) Schematic representation of transgene insertions and fluorescence labeling of chromatin in live cells (Not drawn to scale) 26. Two yellow lines represent the long sequence of BAC. A stable cell line of DHFR D10 clone was used that had an insertion of 10 copies of the BAC into the same chromatin domain without any intervening CHO (Chinese Hamster Ovary) genomic DNA26. Here for visual simplicity only 2 copies of BAC (with DHFR gene and LacO) are drawn. (B) GFP tagged chromatin domains are shown inside the nucleus of a living CHO cell. Left image: nucleus is outlined by dashed lines; the brightfield image is overlaid with the fluorescent image. Right image: the magnified GFP-LacI image of the same cell; an arrow point to chromatin domains tagged with multiple GFP spots. (C) Schematic representation of the 3D Magnetic Twisting Cytometry (3D MTC). (D) A ferromagnetic bead was attached to the apical surface of the cells via integrins. A new strategy of altering stress angle but keeping stress amplitude constant was employed by which the bead was magnetized in Z direction and twisted in specified angles toward the X–Y plane by simultaneously modulating the amplitudes of the magnetic fields in X and Y directions. (E) A live CHO cell with a 4-μm ferromagnetic bead (a white arrow points to a solid black circle) with the GFP labeled chromatin (a yellow arrow points to the green spots). θ represents the angle of bead rotating direction with respect to the long axis of the cell (this notation applies to all cells in all figures). Scale bar, 5 μm. (F) Displacements of the center of the magnetic bead at the stress angle of θ=0º, 45º, or 90º in the same cell as in (E). Each displacement value is an average of data from 3 cycles. In all stress directions, the amplitudes of the sinusoidal magnetic fields (at 0.3 Hz) were modulated such that the peak stress amplitude remained constant at 15 Pa. The peak bead displacement was smaller along the long-axis of the cell than along the short axis. Note that due to a slight non-alignment between 0° stress angle (Y-axis) and the long axis of the cell in (D), thus 0° is in fact ~10°. Since the loading is cyclic and sinusoidal, a minus stress sign in (F) only represents the opposite direction of loading from the plus stress sign. (G) The dependence of cell stiffness (the ratio of the applied stress to the measured strain) on stress angles. Cell length to width ratio equals 2.84±0.377. Mean ± s.e.m.; n=30 cells, 21 independent experiments; *** P<0.001. (h) Schematic of two GFP spots of chromatin being deformed under stress. (i) Distance between two GFP spots (#1 and #2, corresponding to #2 and #3 GFP spots in Fig. 2d) in the same chromatin increases under a cyclic stress applied via integrins. Stress = 17.5 Pa at 0.3 Hz. Two white dashed lines are drawn only for visual aid.
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Related In: Results  -  Collection

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Figure 1: Strategy of visualizing chromatin under force(A) Schematic representation of transgene insertions and fluorescence labeling of chromatin in live cells (Not drawn to scale) 26. Two yellow lines represent the long sequence of BAC. A stable cell line of DHFR D10 clone was used that had an insertion of 10 copies of the BAC into the same chromatin domain without any intervening CHO (Chinese Hamster Ovary) genomic DNA26. Here for visual simplicity only 2 copies of BAC (with DHFR gene and LacO) are drawn. (B) GFP tagged chromatin domains are shown inside the nucleus of a living CHO cell. Left image: nucleus is outlined by dashed lines; the brightfield image is overlaid with the fluorescent image. Right image: the magnified GFP-LacI image of the same cell; an arrow point to chromatin domains tagged with multiple GFP spots. (C) Schematic representation of the 3D Magnetic Twisting Cytometry (3D MTC). (D) A ferromagnetic bead was attached to the apical surface of the cells via integrins. A new strategy of altering stress angle but keeping stress amplitude constant was employed by which the bead was magnetized in Z direction and twisted in specified angles toward the X–Y plane by simultaneously modulating the amplitudes of the magnetic fields in X and Y directions. (E) A live CHO cell with a 4-μm ferromagnetic bead (a white arrow points to a solid black circle) with the GFP labeled chromatin (a yellow arrow points to the green spots). θ represents the angle of bead rotating direction with respect to the long axis of the cell (this notation applies to all cells in all figures). Scale bar, 5 μm. (F) Displacements of the center of the magnetic bead at the stress angle of θ=0º, 45º, or 90º in the same cell as in (E). Each displacement value is an average of data from 3 cycles. In all stress directions, the amplitudes of the sinusoidal magnetic fields (at 0.3 Hz) were modulated such that the peak stress amplitude remained constant at 15 Pa. The peak bead displacement was smaller along the long-axis of the cell than along the short axis. Note that due to a slight non-alignment between 0° stress angle (Y-axis) and the long axis of the cell in (D), thus 0° is in fact ~10°. Since the loading is cyclic and sinusoidal, a minus stress sign in (F) only represents the opposite direction of loading from the plus stress sign. (G) The dependence of cell stiffness (the ratio of the applied stress to the measured strain) on stress angles. Cell length to width ratio equals 2.84±0.377. Mean ± s.e.m.; n=30 cells, 21 independent experiments; *** P<0.001. (h) Schematic of two GFP spots of chromatin being deformed under stress. (i) Distance between two GFP spots (#1 and #2, corresponding to #2 and #3 GFP spots in Fig. 2d) in the same chromatin increases under a cyclic stress applied via integrins. Stress = 17.5 Pa at 0.3 Hz. Two white dashed lines are drawn only for visual aid.
Mentions: We utilized a CHO DG44 (Chinese hamster ovarian) cell line containing a multi-copy insertion of a BAC with an ~180 kb mouse genomic insert containing the ~34 kb DHFR gene. The cell clone DHFR D10 stably expresses EGFP-dimer lac repressor (GFP-LacI), enabling visualization of the DHFR BAC, tagged with a 256mer lac operator repeat (~10 kb) (Fig. 1a)26. DHFR reduces dihydrofolate to tetrahydrofolate and is an essential enzyme for synthesizing thymidine. In order to allow for detection of local movements between chromatin regions, we used a cell clone that has multiple BAC copies in the same single chromatin without any intervening CHO genomic DNA26; the relative movement of multiple GFP spots (~2–7) therefore allows visualization of the relative motion of DHFR BAC chromatin26 (Fig. 1a, b, see Methods).

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