Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1.

Long noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for the progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligonucleotide pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study and now provide a detailed analysis on a how high-glucose milieu downregulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other coregulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1), were enriched at the Tug1 promoter under high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high-glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1 and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.

In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation.

RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5'-3' proximity and unexplored subcellular localizations of poly(A)+ RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.