Chromatin condensation regulates endothelial cell adaptation to shear stress.

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS.

Quantifying site-specific chromatin mechanics and DNA damage response.

DNA double-strand breaks pose a direct threat to genomic stability. Studies of DNA damage and chromatin dynamics have yielded opposing results that support either increased or decreased chromatin motion after damage. In this study, we independently measure the dynamics of transcriptionally active or repressed chromatin regions using particle tracking microrheology. We find that the baseline motion of transcriptionally repressed regions of chromatin are significantly less mobile than transcriptionally active chromatin, which is statistically similar to the bulk motion of chromatin within the nucleus. Site specific DNA damage using KillerRed tags induced in loci within repressed chromatin causes an increased motion, while loci within transcriptionally active regions remains unchanged at similar time scales. We also observe a time-dependent response associated with a further increase in chromatin decondensation. Global induction of damage with bleocin displays similar trends of chromatin decondensation and increased mobility only at 53BP1-labeled damage sites but not at non-damaged sites, indicating that chromatin dynamics are tightly regulated locally after damage. These results shed light on the evolution of the local and global DNA damage response associated with chromatin remodeling and dynamics, with direct implications for their role in repair.

SIRT6 facilitates directional telomere movement upon oxidative damage.

Oxidative damage to telomeres leads to telomere attrition and genomic instability, resulting in poor cell viability. Telomere dynamics contribute to the maintenance of telomere integrity; however, whether oxidative damage induces telomere movement and how telomere mobility is regulated remain poorly understood. Here, we show that oxidative damage at telomeres triggers directional telomere movement. The presence of the human Sir2 homolog, Sirtuin 6 (SIRT6) is required for oxidative damage-induced telomeric movement. SIRT6 knock out (KO) cells show neither damage-induced telomere movement nor chromatin decondensation at damaged telomeres; both are observed in wild type (WT) cells. A deacetylation mutant of SIRT6 increases damage-induced telomeric movement in SIRT6 KO cells as well as WT SIRT6. SIRT6 recruits the chromatin-remodeling protein SNF2H to damaged telomeres, which appears to promote chromatin decondensation independent of its deacetylase activity. Together, our results suggest that SIRT6 plays a role in the regulation of telomere movement upon oxidative damage, shedding new light onto the function of SIRT6 in telomere maintenance.

SSRP1 Cooperates with PARP and XRCC1 to Facilitate Single-Strand DNA Break Repair by Chromatin Priming.

DNA single-strand breaks (SSB) are the most common form of DNA damage, requiring repair processes that to initiate must overcome chromatin barriers. The FACT complex comprised of the SSRP1 and SPT16 proteins is important for maintaining chromatin integrity, with SSRP1 acting as an histone H2A/H2B chaperone in chromatin disassembly during DNA transcription, replication, and repair. In this study, we show that SSRP1, but not SPT16, is critical for cell survival after ionizing radiation or methyl methanesulfonate-induced single-strand DNA damage. SSRP1 is recruited to SSB in a PARP-dependent manner and retained at DNA damage sites by N-terminal interactions with the DNA repair protein XRCC1. Mutational analyses showed how SSRP1 function is essential for chromatin decondensation and histone H2B exchange at sites of DNA strand breaks, which are both critical to prime chromatin for efficient SSB repair and cell survival. By establishing how SSRP1 facilitates SSB repair, our findings provide a mechanistic rationale to target SSRP1 as a general approach to selectively attack cancer cells. Cancer Res; 77(10); 2674-85. ©2017 AACR.

Early Passage Dependence of Mesenchymal Stem Cell Mechanics Influences Cellular Invasion and Migration.

The cellular structures and mechanical properties of human mesenchymal stem cells (hMSCs) vary significantly during culture and with differentiation. Previously, studies to measure mechanics have provided divergent results using different quantitative parameters and mechanical models of deformation. Here, we examine hMSCs prepared for clinical use and subject them to mechanical testing conducive to the relevant deformability associated with clinical injection procedures. Micropipette aspiration of hMSCs shows deformation as a viscoelastic fluid, with little variation from cell to cell within a population. After two passages, hMSCs deform as viscoelastic solids. Further, for clinical applicability during stem cell migration in vivo, we investigated the ability of hMSCs to invade into micropillar arrays of increasing confinement from 12 to 8 µm spacing between adjacent micropillars. We find that hMSC samples with reduced deformability and cells that are more solid-like with passage are more easily able to enter the micropillar arrays. Increased cell fluidity is an advantage for injection procedures and optimization of cell selection based on mechanical properties may enhance efficacy of injected hMSC populations. However, the ability to invade and migrate within tight interstitial spaces appears to be increased with a more solidified cytoskeleton, likely from increased force generation and contractility. Thus, there may be a balance between optimal injection survival and in situ tissue invasion.

Active cytoskeletal force and chromatin condensation independently modulate intranuclear network fluctuations.

Chromatin remodeling, including the movement of genes and regulatory factors, precedes or accompanies stimulated changes in gene expression. Here we quantify chromatin fluctuations in primary human cells using particle-tracking microrheology and determine the physical mechanisms which influence chromatin reorganization. We find that intranuclear movements are enhanced beyond thermal motion by active force generation from cytoskeletal motor activity propagated through the LINC complex; intranuclear movements are also dependent on the viscoelasticity of the DNA-protein polymer network. Chromatin movements were dramatically altered by modulation of chromatin condensation state, which we independently verified using fluorescence lifetime imaging microscopy (FLIM). These findings suggest that chromatin condensation and cytoskeletal force generation play distinct functional roles in regulating intranuclear movements, and these effects are decoupled as measured by particle tracking. We further utilize this approach in identifying the nuclear responsiveness of primary human endothelial cells to vascular endothelial growth factor (VEGF): early in the response chromatin movements increase and are dominated by cytoskeletal force, which transitions at later times to a chromatin decondensation event. Given the hierarchical genome organization in primary cells, our work generally suggests an important role for force generation and chromatin mechanics in altered gene expression kinetics.