UC San Diego

The nuclear envelope (NE) is associated with two major protein complexes: nuclear pore complexes (NPCs) and the nuclear lamina. NPCs are ~120 MDa structures that perforate the NE and mediate nucleocytoplasmic transport, and the nuclear lamina is a network of type V intermediate filaments that forms a meshwork lining the nucleoplasmic side of the inner nuclear membrane (INM) and provides structure to the nucleus. Aside from these canonical functions of NPCs and the nuclear lamina, it has been discovered that these complexes and their subcomponents have additional roles, such as those in chromatin organization and gene regulation. This dissertation aims to expand the known functions of proteins within these complexes, and focuses on two NPC components, Nup98 and Nup96 (Nup98/96), and the lamin isoform, Lamin B1 (LmnB1). Chapter 2 suggests a novel function for Nup98/96 in maintaining the integrity of the nuclear lamina. Small interfering RNA (siRNA) mediated knockdown (KD) of Nup98/96 enlarges the lamina meshwork across the nuclear surface and increases the frequency of transient nuclear envelope rupture. This weakening of the nuclear lamina was further determined to be a consequence of reduced LmnB1 expression, and Chapter 3 shows that LmnB1 is post-transcriptionally regulated through its 3’ untranslated region upon Nup98/96 KD. However, further analysis indicated that the siRNA oligonucleotides designed against Nup98/96 also target LmnB1 for degradation. These siRNAs mimicked microRNAs, hsa-miR-218 and hsa-miR-636, through sequence similarities within their seed regions, allowing these siRNAs to directly target LmnB1. Therefore, Nup98/96 does not regulate the nuclear lamina through LmnB1, and the ability of siRNAs to mimic miRNAs should be carefully considered when designing and utilizing siRNAs in future studies. Chapter 4 describes the effects of LmnB1 overexpression (OE) on chromatin organization and senescence induction. LmnB1 OE induces the formation of heterochromatic DNA foci within the nucleoplasm, coinciding with a reduction of heterochromatin at the nuclear periphery. This leads to changes in gene expression, which may be a consequence of altered chromatin accessibility or changes in histone modifications. The release of heterochromatin from the nuclear periphery is not a consequence of reducing other NE proteins, such as Lamin A or lamin B receptor, that are important for tethering heterochromatin at the nuclear periphery. This suggests that LmnB1 OE might increase the thickness of the nuclear lamina and disrupt the binding of heterochromatin tethers at the nuclear periphery. Finally, although the induced heterochromatin foci are reminiscent of the DNA organization described in senescent cells, LmnB1 OE slows cell proliferation but does not induce senescence. Overall, this dissertation demonstrates the importance of tightly regulating LmnB1 expression to maintain nuclear integrity and chromatin organization. Several human diseases show misregulation of LmnB1, and an interesting observation is the increased expression of LmnB1 observed in various types of cancer, some of which also correlate with higher tumor grade and poor prognosis. This suggests that LmnB1 OE might promote tumorigenesis, which may be mediated through changes in chromatin organization and gene expression, as shown in Chapter 4. However, given the reduction in cell proliferation that was also observed upon LmnB1 OE, further mechanistic studies will be required to reconcile whether LmnB1 OE may be pro-tumorigenic or tumor suppressive.