HSP70 Transgene Directed Motion to Nuclear Speckles Facilitates Heat Shock Activation

Abstract

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.

Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis

“…7). This observation is in contrast to the dynamic changes in nuclear organization observed under stress response conditions (13,30,31), in which loci move in less time than is required for a cell cycle. The nuclear topology did not return to its prestress configuration until after release from a mitotic block, however (30).…”

“…Specific genes, generally those involved in a rapid cellular response to a stimulus such as stress, can dynamically change positions (13), and these changes can alter CT positioning (30,31); however, interphase chromatin demonstrates constrained diffusion for the most part, consistent with the Brownian motion of an object tethered in space (32,33). During mitosis, the relationship between gene expression and organization is lost on dissolution of the nucleus (34).…”

“…The combination of genomic sequences and genome-wide expression studies have revealed another form of nonrandom gene distribution, termed tandem gene arrays (TGAs), which represent contiguous stretches of gene loci that display differential regulation during cellular development and/or function (4,5). Importantly, gene clusters and TGAs also demonstrate expression-dependent nuclear localization patterns (6-9); therefore, gene loci are both deterministically arrayed along chromosomes and positioned within the nucleus (10)(11)(12)(13)(14)(15).…”

3D orbital tracking for super-resolving the dynamics of gene expression

“…However, there is not yet consensus whether intrinsic factors such as the position, local mobility or an active molecular mechanism at a genetic locus could be the determinants of the observed heterogeneity [3][4][5][6]. We show that the high-speed 3D orbital tracking techniques can resolve at the single cell level multiple, distinct regions of mRNA synthesis within a labeled transgene array.…”

Design (and) principles of nuclear dynamics in Stockholm

“…Andrew Belmont (University of Illinois at Urbana-Champaign, USA) reviewed his group's recently published and unpublished work showing long-range, apparently active movement of Hsp70 transgenes to nuclear speckles and the strong enhancement of heat-shock induced transcriptional activation seen after contact of Hsp70 transgenes with nuclear speckles. 20 The Hsp70 DNA locus was detected using the lacOLacI system, 21 the HSP70 mRNA by MS2 tagging, and the nuclear speckles using a GFP-fusion protein marker, thereby allowing the detection of active and non-active Hsp70 in living cells. To determine how prevalent nuclear speckle association is for endogenous genes, they developed a new genomic method, termed TSA-Seq, for mapping cytological proximity to nuclear compartments.…”