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.

Summary Association and disassociation of gene loci with respect to specific nuclear compartments accompany changes in gene expression, yet little is known concerning the mechanisms by which this occurs or its functional consequences. Previously we showed that tethering acidic activators to a peripheral chromosome site led to movement of the chromosome site away from the nuclear periphery, but the physiological relevance of this movement was unclear [1]. Nuclear speckles, or interchromatin granule clusters, are enriched in factors involved in RNA processing [2], and the association of a subset of active genes at their periphery suggests speckles may play a role in gene expression [3, 4]. Here we show an actin dependent association of Hsp70 transgenes with nuclear speckles after heat shock. We visualized Hsp70 transgenes moving curvilinearly towards nuclear speckles over ~0.5–6 μm distances at velocities of 1–2 μm min−1. Chromatin stretching in the direction of movement demonstrates a force generating mechanism. Transcription in nearly all cases increased noticeably only after initial contact with a nuclear speckle. Moreover, blocking new Hsp70 transgene/speckle association by actin depolymerization prevented significant heat-shock induced transcriptional activation in transgenes not associated with speckles, although robust transcriptional activation was observed for Hsp70 transgenes associated with nuclear speckles. Our results demonstrate the existence of a still to be revealed machinery for moving chromatin in a direct path over long distances towards nuclear speckles in response to transcriptional activation; moreover this speckle association enhances the heat-shock activation of these Hsp70 transgenes.

Diverse antibody repertoires are generated through remote genomic interactions involving immunoglobulin variable (V H ), diversity (D H ) and joining (J H ) gene segments. How such interactions are orchestrated remains unknown. Here we develop a strategy to track V H -D H J H motion in B-lymphocytes. We find that V H and D H J H segments are trapped in configurations that allow only local motion, such that spatially proximal segments remain in proximity, while spatially remote segments remain remote. Within a subset of cells, however, abrupt changes in V H -D H J H motion are observed, plausibly caused by temporal alterations in chromatin configurations. Comparison of experimental and simulated data suggests that constrained motion is imposed by a network of cross-linked chromatin chains characteristic of a gel phase, yet poised near the sol phase, a solution of independent chromatin chains. These results suggest that chromosome organization near the sol-gel phase transition dictates the timing of genomic interactions to orchestrate gene expression and somatic recombination.

Multiple cis-elements surrounding the β-globin gene locus combine to target this locus to the nuclear periphery through at least two different epigenetic marks.

Although the formation of RNA-protein bodies has been studied intensively, their mobility and how their number and size are regulated are still poorly understood. Here, we show significantly increased mobility of nuclear speckles after transcriptional inhibition, including long-range directed motion of one speckle towards another speckle, terminated by speckle fusion, over distances up to 4 µm and with velocities between 0.2 µm/min and 1.5 µm/min. Frequently, three or even four speckles follow very similar paths, with new speckles appearing along the path followed by a preceding speckle. Speckle movements and fusion events contribute to fewer, but larger, speckles after transcriptional inhibition. These speckle movements are not actin dependent, but occur within chromatin-depleted channels enriched with small granules containing the speckle marker protein SON. Similar longrange speckle movements and fusion events were observed after heat shock or heavy metal stress, and during late G2 and early prophase. Our observations suggest a mechanism for long-range, directional nuclear speckle movements, contributing to overall regulation of nuclear speckle number and size as well as overall nuclear organization. This article has an associated First Person interview with the first author of the paper.