Citation
- Authors: Beucher A. et al.
- Year: 2022
- Journal: Nat Cell Biol
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293FT
Method
CRISPRi and CRISPR–SAM lentiviral particles were produced as described56. 293FT cells were seeded at 75,000 cells per cm2 in T75 flasks and, 24 h later, transfected with CRISPR and packaging plasmids pMDLg/pRRE, pRSV-Rev and pMD2.G (12251, 12253 and 12259; Addgene) with PEIpro (Polyplus-transfection) in antibiotic-free media using a 1:1 ratio of total µg DNA to µl PEIpro. The medium was replaced with 9 ml fresh 293FT antibiotic-free media 18 h post-transfection and lentiviral particles were collected 72 h post-transfection.
Abstract
The biological purpose of long non-coding RNAs (lncRNAs) is poorly understood. Haploinsufficient mutations in HNF1A homeobox A (HNF1A), encoding a homeodomain transcription factor, cause diabetes mellitus. Here, we examine HASTER, the promoter of an lncRNA antisense to HNF1A. Using mouse and human models, we show that HASTER maintains cell-specific physiological HNF1A concentrations through positive and negative feedback loops. Pancreatic β cells from Haster mutant mice consequently showed variegated HNF1A silencing or overexpression, resulting in hyperglycaemia. HASTER-dependent negative feedback was essential to prevent HNF1A binding to inappropriate genomic regions. We demonstrate that the HASTER promoter DNA, rather than the lncRNA, modulates HNF1A promoter-enhancer interactions in cis and thereby regulates HNF1A transcription. Our studies expose a cis-regulatory element that is unlike classic enhancers or silencers, it stabilizes the transcription of its target gene and ensures the fidelity of a cell-specific transcription factor program. They also show that disruption of a mammalian lncRNA promoter can cause diabetes mellitus.