Session 2B: Connecting the Chromatin Remodeler CHD7 in the Regulation of Autism and CHARGE Syndrome
Abstract
A strong hypothesis is emerging in connection with the CHD7 protein: dysfunction of this protein might play a key role in CHARGE syndrome, autism, and several cardiovascular diseases. As there is no cellular or animal model system to study the function of CHD7, the goal of this investigation was to create the disease in-a-dish by the use of CRISPR/Cas9 to edit the CHD7 gene in human aortic endothelial cells (hAECs). Accordingly, we generated lentivirus particles encoding CRISPR/Cas9-CHD7 sgRNAs in an all-in-one vector, and transduced hAECs with viral particles. We selected clones that were puromycin resistant. These cells were passaged for 5-6 times in puromycin containing media. DNAs were prepared from these clones, and CRISPR/Cas9 mediated CHD7 mutations were confirmed by Sanger DNA sequencing. Decreased expression of CHD7 was confirmed by Western blot analyses. We showed that haploinsufficiency of CHD7 mediate decreased expression VEGFR2/FLK1, but increased expression of p53 and p21 cell cycle inhibitors, thereby inducing apoptosis of these cells. These findings indicate that CHD7 protein regulates the expression of VEGFR2/FLK1 in cardiovascular cells, and therefore its downregulation is likely to affect several different cell types, including the fate of neuronal cells that depend on VEGFR2 signaling. We propose that altered CHD7 and VEGFR2/FLK1 function is the key to CHARGE, a subset of autism, and cardiovascular diseases.
Session 2B: Connecting the Chromatin Remodeler CHD7 in the Regulation of Autism and CHARGE Syndrome
Lecture Hall
A strong hypothesis is emerging in connection with the CHD7 protein: dysfunction of this protein might play a key role in CHARGE syndrome, autism, and several cardiovascular diseases. As there is no cellular or animal model system to study the function of CHD7, the goal of this investigation was to create the disease in-a-dish by the use of CRISPR/Cas9 to edit the CHD7 gene in human aortic endothelial cells (hAECs). Accordingly, we generated lentivirus particles encoding CRISPR/Cas9-CHD7 sgRNAs in an all-in-one vector, and transduced hAECs with viral particles. We selected clones that were puromycin resistant. These cells were passaged for 5-6 times in puromycin containing media. DNAs were prepared from these clones, and CRISPR/Cas9 mediated CHD7 mutations were confirmed by Sanger DNA sequencing. Decreased expression of CHD7 was confirmed by Western blot analyses. We showed that haploinsufficiency of CHD7 mediate decreased expression VEGFR2/FLK1, but increased expression of p53 and p21 cell cycle inhibitors, thereby inducing apoptosis of these cells. These findings indicate that CHD7 protein regulates the expression of VEGFR2/FLK1 in cardiovascular cells, and therefore its downregulation is likely to affect several different cell types, including the fate of neuronal cells that depend on VEGFR2 signaling. We propose that altered CHD7 and VEGFR2/FLK1 function is the key to CHARGE, a subset of autism, and cardiovascular diseases.