LIM-domain transcription complexes interact with ring-finger ubiquitin ligases and thereby impact islet β-cell function

Abstract

Edited by Joel M. Gottesfeld Diabetes is characterized by a loss of ␤-cell mass, and a greater understanding of the transcriptional mechanisms governing ␤-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ␤-cell function, but few proteins are known to interact with this complex in ␤-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)-and MSbased experiments with mouse ␤-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ␤-cell function. We observed that Rnf20 and Rnf40 depletion reduces ␤-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ␤-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.

Egr2-guided histone H2B monoubiquitination is required for peripheral nervous system myelination

“…In line with previous reports on cellular systems ( 12 , 14 , 16 , 21 ), we found evidence for a role of H2Bub1 in determining cell-specific gene expression and differentiation. In the absence of Rnf40 and H2Bub1, we observed only a mild axon sorting defect.…”

LDB1-mediated transcriptional complexes are sensitive to islet stress

“…To correlate downregulation of Ldb1 mRNA with target genes also impacted by cytokine treatments (e.g., MafA ), we performed an siRNA Ldb1 knockdown ( siLdb1 ) in dispersed primary mouse islet cells without cytokines, as described in our past studies. 23 , 33 SiLdb1 transfection resulted in an approximately 80% reduction of Ldb1 mRNA, consistent with levels of Ldb1 loss upon cytokine cocktail treatment ( Figure 3D , I). Loss of Ldb1 resulted in reductions in Ins1, MafA and Pdx1 , known targets of LDB1:ISL1 transcriptional complexes ( Figure 3I ).…”

The transcriptional co-regulator LDB1 is required for brown adipose function

“…Western blotting was performed as described previously [ 20 , 24 ]. Polyvinylidene difluoride (PVDF) membranes were probed using the following primary antibodies: rabbit α-LDB1 (1:1000, generously provided by Dr. Paul Love, NIH), rabbit α-UCP1 (1:1000, Cell Signaling #14670S (D9D6X)), mouse a-Actin (1:1000, Abcam ab3280), rabbit α-pAKT S473 (1:1000, Cell Signaling #9271S), rabbit Pan AKT (1:3000, Cell Signaling #4691S), rabbit α-phospho-p44/42 MAPK (T202/4204) (1:1000, Cell Signaling #4370S), and rabbit α-p44/42 MAPK (T202/4204) (1:1000, Cell Signaling #4695S).…”

“…Quantitative PCR was performed on the ChIP DNA using an SYBR Green PCR master mix (Bio-Rad). Enriched ChIP DNA was normalized to Albumin promoter DNA enrichment and calculated relative to IgG, set as one-fold (ΔΔCT) [ 11 , 20 , 24 ]. ChIP experiments were performed with independent chromatin preparations.…”