Skidmore, J. M. | bioRxiv Science

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↗

bioRxiv2022

Chromatin remodeler Chd7 regulates photoreceptor development and outer segment length

Mutations in the chromatin remodeling factor CHD7 are the predominant cause of CHARGE syndrome, a congenital disorder that frequently includes ocular coloboma. Although CHD7 is known to be required for proper ocular morphogenesis, its role in retinal development has not been thoroughly investigated. Given that individuals with CHARGE syndrome can experience visual impairment even in the absence of coloboma, a better understanding of CHD7 function in the retina is needed. In this study, we characterized the expression pattern of Chd7 in the developing zebrafish and mouse retina and documented ocular and retinal phenotypes in Chd7 loss-of-function mutants. Zebrafish Chd7 was expressed throughout the retinal neuroepithelium when retinal progenitor cells were actively proliferating, and later in subsets of newly post-mitotic retinal cells. At stages of retinal development when most retinal cell types had terminally differentiated, Chd7 expression remained strong in the ganglion cell layer and in some cells in the inner nuclear layer. Intriguingly, strong expression of Chd7 was also observed in the outer nuclear layer where it was co-expressed with markers of post-mitotic cone and rod photoreceptors. Expression of mouse CHD7 displayed a similar pattern, including expression in the ganglion cells, subsets of inner nuclear layer cells, and in the distal outer nuclear layer as late as P15. Two different mutant chd7 zebrafish lines were characterized for ocular and retinal defects. These mutants displayed microphthalmia, reduced numbers of cone photoreceptors, and truncated rod and cone photoreceptor outer segments. Reduced cone photoreceptor number and abnormal outer segments were also observed in heterozygous Chd7 mutant mice. Taken together, our results in zebrafish and mouse reveal a conserved, previously undescribed role for Chd7 in retinal development and photoreceptor outer segment morphogenesis. Moreover, our work suggests an avenue of future investigation into the pathogenesis of visual system defects in CHARGE syndrome. HighlightsO_LIChd7 is expressed in both retinal progenitor cells and in differentiated retinal neurons, including post-mitotic rod and cone photoreceptors. C_LIO_LILoss of Chd7 results in a significant decrease in cone photoreceptors in both zebrafish and mouse. C_LIO_LICone and rod photoreceptor outer segments are truncated in chd7 mutants, suggesting a heretofore unappreciated role for Chd7 in outer segment morphogenesis. C_LI

developmental biology↗