Mechanisms and functional significance of nuclear compartmentalization

The mammalian cell nucleus is a complex yet highly organized structure. There is significant amount of knowledge about the organization of nucleus; however, the influence of such structural complexity of the nucleus in gene regulation is poorly understood. For better understanding of the effects of nuclear repositioning of a gene locus on gene expression, I investigated the physiological significance and mechanism of association of a gene locus with a nuclear sub-compartment. In chapter 1, I showed an actin dependent association of HSP70 transgenes with nuclear speckles after heat shock. I visualized HSP70 transgenes moving curvilinearly towards nuclear speckles over ~0.5-6 μm distances at velocities of 1-2 μm min-1. Observation of chromatin stretching in the direction of movement demonstrated a force generating mechanism. Transcription in nearly all cases increased noticeably only after initial contact with a nuclear speckle. The time lag between initial speckle contact and increased transcription was inversely proportional to the speckle size at first contact with the HSP70 transgene and after initial contact with small speckles an increase in speckle size typically preceded increased transcription. 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. In Chapter 2, we used autonomous targeting of BAC transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs), including the 6 kb PTR1, target the ~100 kb beta-globin gene cluster and LCR region to the nuclear periphery and confer increased H3K9me3 modification, as assayed both by immunofluorescence and chromatin immunoprecipitation. PTRs within HBB BACs bias a competition between peripheral versus pericentric heterochromatin (PCH) targeting towards peripheral targeting. Targeting to both heterochromatin compartments is dependent on H3K9 trimethylation. More generally, PTRs confer targeting from the PCH to the periphery, from the interior to the PCH, or have no targeting activity depending on the flanking DNA context, with peripheral targeting correlating with higher, domain-wide H3K9m3 levels. A combination of FISH, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over several hundred kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent Lamin Associated Domain. Our results demonstrate multiple cis elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting