Holding, M. L. | Search bioRxiv

bioRxiv2022

Regulation of cellular LDL uptake by PROX1 and CHD7

An elevated level of low-density lipoprotein (LDL) in the bloodstream is a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). The low-density lipoprotein receptor (LDLR) is a critical regulator of circulating LDL, and increasing LDLR activity is an effective therapeutic approach to reduce circulating LDL cholesterol levels. In this study, we characterize PROX1 and CHD7, two genes we previously identified in a genome-scale CRISPR screen as positive regulators of LDL uptake in HuH7 cells. We found that although disruption of either PROX1 or CHD7 significantly reduced LDL uptake, only PROX1 disruption significantly reduced the cellular levels of LDLR mRNA and surface-displayed LDLR protein. Consistent with a direct role for PROX1 in LDLR gene regulation, we also observed in publicly available data sets the presence of two liver-specific PROX1 binding sites near the LDLR locus, one of which colocalized with biochemical hallmarks of enhancer activity in hepatic tissue. Both PROX1 LDLR binding sites contained predicted PROX1 transcription factor binding motifs and colocalized with binding sites for HNF4, a known interactor for PROX1 and regulator of hepatic lipid metabolism and LDL uptake. In contrast to PROX1, no CHD7 binding sites were detected near the LDLR locus. Together, our results support a model in which both PROX1 and CHD7 promote cellular LDL uptake through distinct mechanisms, with PROX1 directly promoting LDLR gene expression and CHD7 functioning through an LDLR-independent pathway.

molecular biology↗

bioRxiv2021

Trio-binned genomes of the woodrats Neotoma bryanti and N. lepida reveal novel gene islands and rapid copy number evolution of xenobiotic metabolizing cytochrome p450 genes

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third-generation sequencing and novel analytical toolsets. Woodrats of the genus Neotoma provide a unique opportunity to study how vertebrate herbivores respond to climate change, as two sister species (N. bryanti and N. lepida) independently achieved a major dietary feat - switching to the novel and toxic food source creosote bush (Larrea tridentata) - in the aftermath of a natural warming event. To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti x N. lepida F1 hybrid, resulting in phased, chromosome-level, highly complete, haploid genome assemblies for each species from one individual. Using these new assemblies, we explored the genomic architecture of three cytochrome p450 subfamilies (2A, 2B, and 3A) that play key roles in the metabolism of naturally occurring toxic dietary compounds. We found that woodrats show expansions of all three p450 gene families, including the evolution of multiple novel gene islands within the 2B and 3A subfamilies. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago. Turnover and novelty in detoxification gene islands in herbivores is widespread within distinct p450 subfamilies, and may have provided the crucial substrate for dietary adaptation during environmental change.

genomics↗