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.

Many active genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that HSPA1B BAC transgenes and endogenous Hsp70 genes turn on 2–4 min after heat shock (HS), irrespective of their distance to speckles. However, both total HSPA1B mRNA counts and nascent transcript levels measured adjacent to the transgene are approximately twofold higher for speckle-associated alleles 15 min after HS. Nascent transcript level fold-increases for speckle-associated alleles are 12–56-fold and 3–7-fold higher 1–2 h after HS for HSPA1B transgenes and endogenous genes, respectively. Severalfold higher nascent transcript levels for several Hsp70 flanking genes also correlate with speckle association at 37°C. Live-cell imaging reveals that HSPA1B nascent transcript levels increase/decrease with speckle association/disassociation. Initial investigation reveals that increased nascent transcript levels accompanying speckle association correlate with reduced exosome RNA degradation and larger Ser2p CTD-modified RNA polymerase II foci. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through “gene expression amplification.”

Chromosome tagging using lac or tet operator repeats for in vivo visualization of chromosome dynamics has now become a standard methodology used in a range of organisms. One variation of this approach has been to build transgene arrays creating artificial chromosome blocks to study various aspects of chromatin structure, transcription, replication, or DNA repair. Previously, plasmid transgenes with or without subsequent gene amplification have been used to build these arrays. However, plasmid arrays typically show heterochromatic properties, while gene amplification typically results in chromosome instability of the amplified regions. To avoid these problems, we are now building transgene arrays from large genomic DNA inserts cloned in bacterial artificial chromosomes (BAC). These BAC transgenes show transcriptional levels within several fold of endogenous genes while also exhibiting targeting to specific nuclear compartments similar to the targeting of the endogenous genes. Here we describe Tn5 transposition and BAC recombineering methods used to retrofit BACs for their use in building BAC transgene arrays. This includes insertion of operator repeats and selectable markers into these BACs as well as targeted insertion or deletion of BAC sequences.

21Although the formation of RNA-protein bodies has been studied intensively, their 22 mobility and how their number and size are regulated are still poorly understood. Here, we show 23 significant increased mobility of nuclear speckles after transcriptional inhibition, including long-24 range directed motion of one speckle towards another speckle, terminated by speckle fusion, 25 over distances up to 4 um and with velocities between 0.2-1.5 µm/min. Frequently, 3 or even 4 26 speckles follow very similar paths, with new speckles appearing along the path followed by a 27 preceding speckle. Speckle movements and fusion events contribute to fewer but larger speckles 28 after transcriptional inhibition. These speckle movements are not actin-dependent, but occur 29 within chromatin-depleted channels enriched with small granules containing the speckle-marker 30 protein SON. Our observations suggest a mechanism for long-range, directed nuclear speckle 31 movements, contributing to overall regulation of nuclear speckle number and size as well as 32 overall nuclear organization. 33 enriched in RNA processing factors and polyadenylated RNAs 17, 18, 19 . Nuclei typically contain 57 20-40 irregular shaped nuclear speckles varying in size from ~0.5 µm to several µm 20 . 58Because "pure" liquid droplet bodies theoretically are predicted to merge into 59 progressively fewer and larger droplets, a major question regarding cell bodies in general is how 60 their number and size are regulated in the cell. Interestingly, with transcriptional inhibition 61 nuclear speckles become rounder and larger with a reduction in their overall number 21, 22 . Live-62 cell microscopy revealed that normally speckles were relatively stationary, but displayed 63 extension and dissociating particles. However, no peripheral dynamics were observed in the 64 absence of transcription 23 . These previous studies did not address directly whether nuclear 65 speckles also exhibit the general physical properties of RNA-protein bodies or how speckle 66 morphology changes dynamically in terms of speckle numbers, size and shape. 67Using the change in nuclear speckle morphology induced by transcriptional inhibition as 68 a model system, here we studied the mobility and liquid-like behaviors of nuclear speckles. 69Consistent with previous observations, we observed a general change in morphology after 70 transcriptional inhibition, with speckles becoming bigger and rounder. This was accompanied by 71 an increase in speckle mobility within the nucleus. 72 Surprisingly this increase in speckle mobility was not random. Instead we observed 73 repeated long-range, directional movement of multiple speckles over similar nuclear trajectories 74 or "tracks" which extended over micrometer distances. These movements ended with fusion of 75 the smaller, mobile speckles with larger, typically stationary speckles. These repeated fusion 76 events contributed to the overall decrease in nuclear speckle number and increased mean speckle 77 size as a function of time after transcrip...