Forces Directly Induce Chromatin Deformation And Gene Expression

Mechanical forces play critical roles in regulating normal physiological functions such as breath,hearing and sports as well as tumorigenesis,metastasis and self-renewal of malignant,stem cell-like tumor-repopulating cells.However,how forces control and regulate gene expression and cellular functions remains largely elusive.A local dynamic force via cytoskeleton to nuclear results in direct dissociation of associated complexes in Cajal bodies;mechano signal can indirect control gene expression by activate cytoplasmic proteins translocation.It still remains unclear if force can directly influence chromatin.Mechanical signal is rapid and propagates fast and efficient.To study how force impacts chromatin structure,we must quantify chromatin deformation while applying the force on it.Chromatin is in the nucleus of the cell and are composed of DNA and its associated proteins.It is difficult to label a chromatin domain by multiple fluorescence spots and quantify its displacements.Chromatin visualization and real-time tracking are two major challenges in this project.Three-dimensional magnetic twisting cytometry(3D-MTC)can apply force in any direction on the living cells,stimulated emission depletion(STED)nanoscopy can rapid capture living cells structure high-resolution images.We interfaced the three-dimensional magnetic twisting cytometry with the STED nanoscopy to apply cyclic local forces at different directions on living cells and real-time track cellular structure deformation.We used green fluorescent protein(GFP)-tagged bacterial artificial chromosome(BAC)inserted dihydrofolate reductase(DHFR,a housekeeping gene)to visualize chromatin and measure deformation of the same chromatin domain in living CHO cells.The chromatin moved synchronous with the sine force applied by 3D-MTC,quantified chromtin displacement suggested it was directly stretching by the mechano force,chromatin stretching depended on force directions and force amplitudes.Quantified DHFR transcription using FISH(fluorescence in situ hybridization)found transcription started as soon as the force was applied and partial transcripts were detected as early as 15 seconds after force application,DHFR upregulation depended on force directions,amplitudes and duration times.Our findings suggested that local forces applied via integrins rapidly propagate along the tensed actin cytoskeleton to the LINC(linker of nucleoskeleton and cytoskeleton)complex and to the chromatin to directly stretch chromatin and upregulate DHFR transcription.