Salahudeen, A. A. | Search bioRxiv

bioRxiv2019

HAT1 drives a gene-metabolite circuit that links nutrient metabolism to histone production

The energetic costs of duplicating chromatin along with DNA replication are large and therefore likely depend on nutrient sensing checkpoints and metabolic inputs. By studying chromatin modifiers regulated by epithelial growth factor, we identify histone acetyltransferase 1 (HAT1) as an induced gene that enhances cell proliferation by coordinating histone production with glucose metabolism. In addition to its canonical role as a cytoplasmic free histone H4 acetyltransferase, a HAT1-containing complex binds specifically at promoters of H4 genes. HAT1 stimulated acetate delivery and consumption at H4 promoters to drive S-phase H4 transcription. This required the presence of a histone H4-specific promoter element in the region of HAT1 chromatin binding. These data describe a feed-forward circuit whereby HAT1-dependent capture of acetyl-groups drives further H4 production to support growth-factor dependent proliferation. These findings also extend to human disease and animal models, as high HAT1 levels associate with poor outcomes across multiple cancer types.

molecular biology↗

bioRxiv2019

Highly Efficient Repair of the ΔF508 Mutation in Airway Stem Cells of Cystic Fibrosis Patients with Functional Rescue of the Differentiated Epithelia

Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl- channel. CF results in multiorgan dysfunction and ultimately mortality from respiratory sequelae. Although pharmacologic approaches have demonstrated efficacy in reducing symptoms and respiratory decline, a curative treatment modality remains elusive. Gene therapy, a promising curative strategy, has been limited due to poor correction efficiencies both in vitro and in vivo. Here, we use Cas9 and adeno-associated virus 6 (AAV6) to correct the {Delta}F508 mutation (found in [~]70% of CF alleles and [~]90% of CF patients in North America) in upper airway basal stem cells (UABCs) obtained from CF and non-CF patients undergoing functional endoscopic sinus surgery (FESS). In UABCs from homozygous ({Delta}F508/{Delta}F508) and compound heterozygous ({Delta}F508/Other) CF patients, we achieved 28 {+/-} 5 % and 42 {+/-} 15% correction, respectively. In homozygous human bronchial epithelial cells (HBECs), we achieved 41{+/-} 4 % correction. Upon differentiation in air-liquid interface (ALI), cultures of corrected CF cells displayed partial restoration of CFTRinh-172 sensitive Cl- currents relative to non-CF controls: 31{+/-} 5 % in UABCs and 51 {+/-} 3 % in HBECs (both from subjects homozygous for {Delta}F508 CFTR). Finally, gene edited cells embedded successfully and retained expression of cytokeratin 5 (KRT5), a basal cell marker, on a FDA-approved porcine small intestinal submucosal (pSIS) membrane previously shown to improve re-mucosalization after FESS. In summary, we present an efficient, feeder-free, selection-free and clinically compatible approach to generate cell-based therapies for CF from autologous airway stem cells. This approach represents a first step towards developing patient-specific autologous airway stem cell transplant as a curative treatment for CF.

bioengineering↗