Molecular Control of Genome Architecture

Abstract

Heterochromatin (HC) tethering at the nuclear lamina (NL) is observed in almost every cell, and mutations in tethering proteins lead to a variety of diseases called laminopathies that have been the subject of intense study. Nonetheless, the importance of HC tethering for genome regulation and/or other cellular functions is not fully understood. Unlike most cells, murine rod photoreceptors have an inverted organization, in which euchromatin (EC) is localized to the nuclear periphery, and HC is untethered. Using rods as a model system devoid of any tethering proteins, two players were identified as crucial in HC tethering. The lamin B receptor (Lbr) is sufficient for HC tethering and is expressed during development, but Lbr levels decline upon rod photoreceptor differentiation. A second tether is the intermediate filament lamin A (LA), which is not normally expressed in murine rods. In my first published data chapter, I found that LA ectopically upregulates and reorganizes the genome during rod degeneration.

Using ATAC-seq to examine genome accessibility, we showed that LA and Lbr tethering proteins both lead to marked increases in genome accessibility at regions associated with stress responsive genes. However scRNA-seq on LA- and Lbr-expressing rods revealed relatively minor stress-responsive gene transcription. Together, our data reveal that HC tethers have a global effect on genome accessibility and suggest that HC tethering primes the photoreceptor genome to respond to stress.

In my next data chapter, I performed experiments designed to understand why LA upregulates during degeneration. Along with Jasmine Levesque , we found that in the rd1 model of retinitis pigmentosa, LA upregulation correlates with markers of DNA damage. Moreover, functional manipulations suggest that LA upregulation promotes rod photoreceptor survival. Together, these data imply that LA may upregulate as an adaptive response to DNA damage.

Next, to better understand the logic of HC tethering, I performed experiments designed to elucidate the tethering mechanism. I performed a structure/function study of LA-dependent HC tethering. More than 400 different LMNA gene mutations are associated with laminopathic disease. Studies have previously linked mutations to reduction of HC, abnormal nuclear morphology and DNA damage, but most experiments have been performed in conventionally organized cell types - many of which express wild-type LA/C and Lbr. It has therefore been difficult to determine whether genome disorganization is directly caused by specific LA mutations, or is a consequence of other pathological mechanisms. I expressed a panel of mutant LA constructs in rod photoreceptors to determine how mutations affect tethering competence.

I identified protein domains necessary for HC tethering within the LA C-terminus and IG-fold domain. Moreover, progeroid and non-progeroid laminopathic disease mutations exhibited a complete loss of tethering competence. However, defarnesylating these laminopathic mutants partially restored HC tethering. My results suggest that the LA C-terminus is necessary and sufficient for HC tethering, in part, due to the requirement for C-terminal processing.

In my final data chapter, since LA does not have an obvious chromatin binding domain, we evaluated the hypothesis that partner proteins act as a bridge between chromatin and LA. Using rod photoreceptors as a model for tethering sufficiency, I ectopically expressed a set of LA- interacting proteins. However, my results suggest that these partner proteins could not act in trans to restore tethering to versions of LA that were tethering-incompetent. Together with proteomic experiments profiling LA and molecular modelling, our results suggest that LA does not depend on known partner proteins to interact with HC, but interacts directly with nucleosomes. Our findings might impact the field of human disease genomics and provide insight into mechanisms regulating genome organization.