Customize Consent Preferences

We use cookies to help you navigate efficiently and perform certain functions. You will find detailed information about all cookies under each consent category below.

The cookies that are categorized as "Necessary" are stored on your browser as they are essential for enabling the basic functionalities of the site. ... 

Always Active

Necessary cookies are required to enable the basic features of this site, such as providing secure log-in or adjusting your consent preferences. These cookies do not store any personally identifiable data.

Functional cookies help perform certain functionalities like sharing the content of the website on social media platforms, collecting feedback, and other third-party features.

Analytical cookies are used to understand how visitors interact with the website. These cookies help provide information on metrics such as the number of visitors, bounce rate, traffic source, etc.

Advertisement cookies are used to provide visitors with customized advertisements based on the pages you visited previously and to analyze the effectiveness of the ad campaigns.

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.

Go to