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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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A model for direct force impact on gene activationA local surface force via integrins is propagated though the myosin-II tensed actin cytoskeleton to the LINC (via SUN1 and SUN2) complex, to nuclear lamins, and then is transferred to the flanking chromatin through BAF and HP1 proteins and other molecules. The flanking chromatin transfers the force to deform and to stretch the chromatin segment containing the DHFR gene at the nuclear interior, facilitating binding of the RNA Polymerase II and transcription factors to upregulate DHFR transcription. Note that underneath each nuclear protein is its gene name. Not drawn to scale.
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Figure 6: A model for direct force impact on gene activationA local surface force via integrins is propagated though the myosin-II tensed actin cytoskeleton to the LINC (via SUN1 and SUN2) complex, to nuclear lamins, and then is transferred to the flanking chromatin through BAF and HP1 proteins and other molecules. The flanking chromatin transfers the force to deform and to stretch the chromatin segment containing the DHFR gene at the nuclear interior, facilitating binding of the RNA Polymerase II and transcription factors to upregulate DHFR transcription. Note that underneath each nuclear protein is its gene name. Not drawn to scale.

Mentions: We speculate that transient chromatin stretching of ~20–100 nm can open sufficient space to decondense the chromatin for increased accessibility of the transcriptional machinery transcription, leading to an increased rate of DHFR transcriptional initiation. It is noted that DHFR transcripts after completion of transcription will move/diffuse away from the GFP spots (BAC transgene) and therefore what we measure 1 hr after stress application could be an underestimate of the actual number of stress-induced new transcripts. However, assuming a constant probability of transport of newly synthesized messages away from the transcription site, at steady-state the size of the accumulated transcript pool adjacent to the transgenes should be proportional to the rate of transcription. We acknowledge that we have only examined a chromatin-stretch dependent DHFR transgene upregulation in the current study; however, previous work has shown that these DHFR BAC transgenes exhibit ~50% the expression levels of the endogenous DHFR gene33, suggesting that the transgene is physiologically relevant. It remains to be seen if other genes can also be upregulated by direct chromatin stretching and if our current findings can be extended to other cell types, but it is remarkable that a house-keeping gene like DHFR can respond to direct chromatin stretching, suggesting the potential generality of gene activation by force-induced direct chromatin stretching. It is likely that the DHFR transgene may be already active at baseline and molecules are ready to be recruited (e.g., RNA Poly II is active and ready to be recruited (Supplementary Fig. 8)); or the transgene site presumably has a partially open configuration and is located in the nuclear interior (Supplementary Fig. 15). It is possible that some endogenous genes that are not active at all may not be able to be activated by an applied force alone. This may be the reason that when an external force (either a stretching force or a shear force) is applied, only some genes are activated while other genes are not. However, we have found that DHFR transcription could still be elevated by force after serum deprivation, which is known to condense the chromatin and decrease DHFR gene expression26, suggesting that the applied force is potent enough to decondense the chromatin to elevate DHFR transcription in serum-deprived CHO cells. One possible interpretation of our current findings is that the DHFR gene, due to the BAC insertion, might be uniquely sensitive to this increase in transcription driven by mechanical forces. To test this possibility, we applied forces to these CHO cells and assayed a known mechanosensitive endogenous gene (egr-1, early growth response gene 1) expression34 under identical culture and mechanical conditions as the DHFR gene. We found that egr-1 gene is upregulated by >14-fold at 17.5 Pa at 0.3 Hz for 1 hr (Supplementary Fig. 16), much more than DHFR (~2-fold) under the same culture and stress conditions, suggestion that the DHFR gene is not particularly sensitive to the increase in transcription driven by mechanical forces. Since we have not been able to insert multiple fluorescent tags to the same chromatin that are near the egr-1 gene because it is a non-trivial task, we cannot say definitively that this gene upregulation is chromatin-stretch dependent. However, since the H2B-GFP mapping of the BAC CHO cells shows that the magnitudes of peak tensile strains of H2B are similar to what is observed with the BAC GFP spots under similar stress conditions, these findings suggest that egr-1 upregulation by the magnetic bead stress might be at least partially due to local stretching of the chromatin segment that contains egr-1 gene. It would be important to note that DHFR is used here as a model system to demonstrate that mechanical coupling of forces from the outside of the cell to the inside of the nucleus has the potential to directly activate gene expression. There is an advantage of this type of experimental designs, because it is unlikely that there would exist a biochemical signaling pathway to activate DHFR expression in the presence of force. Thus using DHFR is another argument supporting our conclusion that the observed increased expression of DHFR in response to stress is due to mechanical force transduction. We envision that known mechanically-responsive genes may have evolved mechanisms such that they are much more sensitive to force than other genes. The increased response of egr-1 to force suggests that this might be the case. In the future it would be interesting to determine if our conclusion that the extent of chromatin stretching is critical in determining the level of DHFR transcription applies to many other genes that are being actively transcribed. It is also of interest that the loading in this study is symmetric and thus transient compression is also induced in the chromatin GFP spots; it is not clear why these stretching and compressing impacts do not cancel each other. One possibility is on and off rates and kinetics of molecular binding are different such that transient compressing does not cancel transient stretching effects. The exact mechanisms need to be explored in the future. Nevertheless, our current findings, together with the reports on the established physical linkage from the ECM to the nucleus and the role of tensed actin bundles in long-distance force propagation28, support the model of a direct force impact on flanking chromatin to induce DHFR gene transcription at the nuclear interior (Fig. 6).


Transcription upregulation via force-induced direct stretching of chromatin
A model for direct force impact on gene activationA local surface force via integrins is propagated though the myosin-II tensed actin cytoskeleton to the LINC (via SUN1 and SUN2) complex, to nuclear lamins, and then is transferred to the flanking chromatin through BAF and HP1 proteins and other molecules. The flanking chromatin transfers the force to deform and to stretch the chromatin segment containing the DHFR gene at the nuclear interior, facilitating binding of the RNA Polymerase II and transcription factors to upregulate DHFR transcription. Note that underneath each nuclear protein is its gene name. Not drawn to scale.
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Related In: Results  -  Collection

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Figure 6: A model for direct force impact on gene activationA local surface force via integrins is propagated though the myosin-II tensed actin cytoskeleton to the LINC (via SUN1 and SUN2) complex, to nuclear lamins, and then is transferred to the flanking chromatin through BAF and HP1 proteins and other molecules. The flanking chromatin transfers the force to deform and to stretch the chromatin segment containing the DHFR gene at the nuclear interior, facilitating binding of the RNA Polymerase II and transcription factors to upregulate DHFR transcription. Note that underneath each nuclear protein is its gene name. Not drawn to scale.
Mentions: We speculate that transient chromatin stretching of ~20–100 nm can open sufficient space to decondense the chromatin for increased accessibility of the transcriptional machinery transcription, leading to an increased rate of DHFR transcriptional initiation. It is noted that DHFR transcripts after completion of transcription will move/diffuse away from the GFP spots (BAC transgene) and therefore what we measure 1 hr after stress application could be an underestimate of the actual number of stress-induced new transcripts. However, assuming a constant probability of transport of newly synthesized messages away from the transcription site, at steady-state the size of the accumulated transcript pool adjacent to the transgenes should be proportional to the rate of transcription. We acknowledge that we have only examined a chromatin-stretch dependent DHFR transgene upregulation in the current study; however, previous work has shown that these DHFR BAC transgenes exhibit ~50% the expression levels of the endogenous DHFR gene33, suggesting that the transgene is physiologically relevant. It remains to be seen if other genes can also be upregulated by direct chromatin stretching and if our current findings can be extended to other cell types, but it is remarkable that a house-keeping gene like DHFR can respond to direct chromatin stretching, suggesting the potential generality of gene activation by force-induced direct chromatin stretching. It is likely that the DHFR transgene may be already active at baseline and molecules are ready to be recruited (e.g., RNA Poly II is active and ready to be recruited (Supplementary Fig. 8)); or the transgene site presumably has a partially open configuration and is located in the nuclear interior (Supplementary Fig. 15). It is possible that some endogenous genes that are not active at all may not be able to be activated by an applied force alone. This may be the reason that when an external force (either a stretching force or a shear force) is applied, only some genes are activated while other genes are not. However, we have found that DHFR transcription could still be elevated by force after serum deprivation, which is known to condense the chromatin and decrease DHFR gene expression26, suggesting that the applied force is potent enough to decondense the chromatin to elevate DHFR transcription in serum-deprived CHO cells. One possible interpretation of our current findings is that the DHFR gene, due to the BAC insertion, might be uniquely sensitive to this increase in transcription driven by mechanical forces. To test this possibility, we applied forces to these CHO cells and assayed a known mechanosensitive endogenous gene (egr-1, early growth response gene 1) expression34 under identical culture and mechanical conditions as the DHFR gene. We found that egr-1 gene is upregulated by >14-fold at 17.5 Pa at 0.3 Hz for 1 hr (Supplementary Fig. 16), much more than DHFR (~2-fold) under the same culture and stress conditions, suggestion that the DHFR gene is not particularly sensitive to the increase in transcription driven by mechanical forces. Since we have not been able to insert multiple fluorescent tags to the same chromatin that are near the egr-1 gene because it is a non-trivial task, we cannot say definitively that this gene upregulation is chromatin-stretch dependent. However, since the H2B-GFP mapping of the BAC CHO cells shows that the magnitudes of peak tensile strains of H2B are similar to what is observed with the BAC GFP spots under similar stress conditions, these findings suggest that egr-1 upregulation by the magnetic bead stress might be at least partially due to local stretching of the chromatin segment that contains egr-1 gene. It would be important to note that DHFR is used here as a model system to demonstrate that mechanical coupling of forces from the outside of the cell to the inside of the nucleus has the potential to directly activate gene expression. There is an advantage of this type of experimental designs, because it is unlikely that there would exist a biochemical signaling pathway to activate DHFR expression in the presence of force. Thus using DHFR is another argument supporting our conclusion that the observed increased expression of DHFR in response to stress is due to mechanical force transduction. We envision that known mechanically-responsive genes may have evolved mechanisms such that they are much more sensitive to force than other genes. The increased response of egr-1 to force suggests that this might be the case. In the future it would be interesting to determine if our conclusion that the extent of chromatin stretching is critical in determining the level of DHFR transcription applies to many other genes that are being actively transcribed. It is also of interest that the loading in this study is symmetric and thus transient compression is also induced in the chromatin GFP spots; it is not clear why these stretching and compressing impacts do not cancel each other. One possibility is on and off rates and kinetics of molecular binding are different such that transient compressing does not cancel transient stretching effects. The exact mechanisms need to be explored in the future. Nevertheless, our current findings, together with the reports on the established physical linkage from the ECM to the nucleus and the role of tensed actin bundles in long-distance force propagation28, support the model of a direct force impact on flanking chromatin to induce DHFR gene transcription at the nuclear interior (Fig. 6).

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