Lamin A upregulation reorganizes the genome during rod photoreceptor degeneration

Two proteins have previously been shown to be necessary and sufficient to tether heterochromatin at the nuclear envelope. The lamin B receptor (Lbr) is expressed during development, but downregulates upon rod differentiation. A second tether is the intermediate filament lamin A (LA), which is not normally expressed in murine rods.To determine how heterochromatin tethering affects the genome, we used in vivo electroporation to misexpress LA or Lbr in mature rods in the absence of degeneration, resulting in the restoration of conventional nuclear architecture. Using scRNA-seq, we show that reorganizing the nucleus via LA/Lbr misexpression has only minor effects on rod gene expression. Next, using ATAC-seq, we show that LA and Lbr similarly increase genome accessibility. Novel ATAC-seq peaks tended to distal to genes, but some peaks were associated with stress-responsive genes. Together, our data reveal that heterochromatin tethers have a global effect on genome accessibility, and suggest that LA –dependent genome reconfiguration might contribute to the stress response of rod photoreceptors. Control, lamin A, or Lbr expression plasmids were transfected into retinal progenitor cells in vivo at P0. After ~8 weeks, retinas were harvested and dissociated using Accutase. Cells were sorted via flow cytometry using reporter gene expression and Dapi exclusion to gate viable, transfected cells. For ATAC-seq experiments, we utilized a rod-specific reporter plasmid (pRho2.2-DsRed) to mark rods specifically. ATAC-seq data were generated from FACs sorted rods following Buenrostro et al. (PMID: 25559105). Briefly, 50 000 flow sorted cells were lysed in cold lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Lysed nuclei were tagmented using 6.5 µl of TDE1 transposase from the Nextera DNA Flex Library kit (Illumina). Samples were purified using Zymo-Spin IC columns (Zymo), and libraries constructed according to the Nextera workflow. Libraries were cleaned up using the AMPure XP kit (Beckman Coulter). PE 150 sequencing was performed using the NextSeq 500 platform to a read-depth of 25-35 million reads per sample. Data were processed using the Galaxy platform. Fastq files were processed with Fastq Groomer, Trimmomatic, and mapped to the mm10 genome using Bowtie2. We used Macs2 to call narrowpeaks.