Figure 1
From: Hnf1b renal expression directed by a distal enhancer responsive to Pax8
Identification of a conserved non-coding sequence as candidate for Hnf1b enhancer regulated by Pax8. (a) Localization of two conserved non-coding sequence (CNS1 and CNS2, red bars) in M. musculus and X. tropicalis. CNS1 is localized in the intergenic region between Hnf1b and the upstream gene Heatr6. CNS2 is localized in the 4th intron of Hnf1b. Arrows indicate the transcription initiation of Hnf1b. Protein coding exons are represented by blue bars, exonic sequences corresponding to 5ʹ and 3ʹ UTRs are in yellow. (b) Nucleotide sequence comparison of CNS1 in different species. The table indicates the % of conserved nucleotides. (c) Nucleotide sequence alignment of CNS1 in X. tropicalis, H. sapiens and M. musculus. Conserved nucleotides are in red. Underlined are conserved putative binding sites of TFs known to be expressed in the developing kidney according to (https//sckidney.flatironinstitute.org)79, GUDMAP (https://www.gudmap.org)80,81 or Xenbase (https://www.xenbase.org/entry/). The blue box highlights the conserved Pax8 putative binding site. Sequences targeted by the gRNAa and gRNAb used for CRISPR editing in Fig. 6 are shown in green. (d) Pax8 binding site sequence identified in CNS1 and Pax8 binding consensus sequence as defined by Ref.31 shown as sequence logo. For each position, the size of the characters represents the relative frequency of the corresponding position. The lower sequence is the Pax8 binding sequence in CNS1. Nucleotides mutated in the Pax8-BS mut oligonucleotide used for Electrophoretic Mobility Shift Assay (EMSA), and in plasmids CNS1 mut-Luc and CNS1 mut-eGFP are labelled in red. (e) EMSA. Biotin-labelled oligonucleotide probes were incubated with nuclear extracts from MDCK as indicated: Pax8-BS, Pax8-BS mut and Control TPO (Pax8 binding site identified in the thyroperoxidase promoter, used as a positive control). An arrow marks the Pax8 specific complexes. Specificity of the Pax8 complex was demonstrated by competition with 10- and 100-fold molar excesses of unlabelled Pax8-BS oligonucleotide (compare lanes 5 and 7) as well as incubation with 1 μg of Pax8 monoclonal antibodies (Pax8 AB) leading to a decrease in the Pax8 complex signal (compare lanes 5 with 9). The figure is representative of 3 independent experiments. Original uncropped autoradiography of the whole membrane is presented in Supplementary Fig. S5.
