Fig 2: Ronin and Hcf-1 Control Target Genes during Early Cardiac Development by Modulating H3K4 Trimethylation(A) ChIP-seq using H3K4me3-specific antibodies and GO analysis of genes with significantly lower H3K4me3 status than expected (Z score > 2) in Nkx2.5::Cre-driven Ronin knockout embryos at E11.5 show significant enrichment of categories related to cardiac function. Depicted are the top categories with fold enrichment scores of >8.(B) Boxplot (median and interquantile values) of ChIP-seq signal differences comparing H3K4me3 levels between Nkx2.5-knockout and control hearts at E11.5 (knockout minus control) at promoter regions (TSS to +500 bp) of all H3K4me3-marked genes, downregulated Ronin target genes, or downregulated genes that are not bound by Ronin/Hcf-1. The H3K4me3 signal of Ronin-regulated target genes is significantly reduced (p < 0.0002 by t test). TSS, transcriptional start site.(C) The effect of Ronin loss on the spatial distribution of H3K4me3 marks in the vicinity of relevant promoters, reported as the average difference in H3K4me3 signal between knockout and control, according to the positions of Ronin target genes located ± 1 kb from the TSS. Black line, all H3K4me3-marked genes; blue line, downregulated Ronin target genes; red line, all downregulated genes that are not bound by Ronin. Note reduction in H3K4me3 upstream of the TSS as well as 200–400 kb downstream of the TSS.(D) Examples of ChIP-seq signals at selected Ronin/Hcf-1 target genes show that the H3K4me3 marks at Ronin target genes.(E) ChIP-qPCR using an H3K4me3-specific antibody demonstrates that the H3K4me3 histone mark is greatly diminished in the promoter region of Ronin target genes in hearts isolated at E11.5 of Nkx2.5::Cre and at P0 of αMHC::Cre animals after knockout of Ronin. The PCR signal after ChIP is shown relative to the signal of the corresponding input sample (∗p < 0.05 by t test, n = 3 per group). Mean ± SEM values are shown.See also Table S3.

Fig 3: Two serine residues, S448/S451, are critical for O-GlcNAcylation of NRF1. (A) Detection of O-GlcNAcylation of NRF1 protein. Nuclear extracts of 293T cells expressing NRF1-3×FLAG were prepared and then pulled down with an anti-FLAG antibody. Immunoprecipitated samples were subjected to immunoblot analysis with antibodies against O-GlcNAc peptides and the FLAG tag. (B) Motifs in NRF1, which mediate interaction with Fbw7 (left) and β-TrCP (right). Serine residues that were replaced with alanine in subsequent experiments are underlined. The numbers beneath the underlines denote positions of the serine residues in the amino acid sequence of the NRF1 protein. (C) Summary of serine-to-alanine substitutions in the two motifs of NRF1. (D and E) Nuclear accumulation of NRF1 and its mutant molecules in response to enhanced cellular O-GlcNAcylation induced by high-glucose condition (D) or PugNAc treatment (E). 293T cells expressing wild-type or mutant NRF1-3×FLAG (WT, A-SS, S-AA, and A-AA) were cultured in low- or high-glucose culture medium (D) or with or without 100 μM PugNAc (E) for 24 h. Nuclear extracts were prepared and subjected to immunoblot analysis with antibodies against the FLAG tag and lamin B. (F) Effects of OGA expression on the protein levels of NRF1 and its mutant molecules. 293T cells expressing wild-type or mutant NRF1-3×FLAG were transfected with an empty or a Myc-OGA expression vector. At 24 h after transfection, the cells were cultured in high-glucose medium for another 24 h and harvested. Nuclear extracts were prepared and subjected to immunoblot analysis with antibodies against the FLAG tag, Myc tag, and lamin B. (G) Identification of critical serine residues for O-GlcNAcylation of the NRF1 protein. Nuclear extracts of 293T cells expressing the WT or S-AA mutant of NRF1-3×FLAG were prepared and pulled down with an anti-FLAG antibody. Immunoprecipitated samples were subjected to immunoblot analysis with antibodies against O-GlcNAc peptides and the FLAG tag. (H) Effects of PugNAc on protein interactions with NRF1. 293T cells that were stably transfected with an empty vector or an expression vector of the WT or S-AA mutant of NRF1-3×FLAG were pretreated with 100 μM PugNAc or left untreated for 24 h before nuclear extracts were prepared for immunoprecipitation with an anti-FLAG antibody. Immunoprecipitated proteins were subjected to immunoblot analysis with antibodies against β-TrCP, OGT, HCF-1, and the FLAG tag. (I) Effects of PugNAc on ubiquitination of NRF1. 293F cells that were stably transfected with an empty vector or an expression vector of NRF1-3×FLAG were pretreated with or without 100 μM PugNAc for 24 h and incubated with 10 μM MG132 for another 4 h before whole-cell extracts were prepared for immunoprecipitation with an anti-FLAG antibody. Immunoprecipitated proteins were subjected to immunoblot analysis with antibodies against NRF1 and the FLAG tag.

Fig 4: Ronin Cooperates with Hcf-1 to Regulate Expression of Metabolic Genes during Cardiac Development(A) Venn diagram of ChIP-seq data representing embryonic hearts collected at E11.5. Most of the genes targeted by Ronin are also bound by Hcf-1 and are H3K4me3 positive.(B) Consensus sequences of the Ronin binding motif identified after ChIP-seq analysis are almost identical in embryonic heart (top) and embryonic stem cells (bottom).(C) Examples of genes bound by both Ronin and Hcf-1 showing that signals are enriched immediately upstream of transcriptional start sites (arrow).(D) PANTHER analysis classifying GO categories of all Ronin/Hcf-1 target genes assigns the vast majority of Ronin-bound genes to metabolic processes.See also Table S1.

Fig 5: EBNA1 interacts with HCF1 in an OriP-dependent manner. (A) IP with EBNA1 or control IgG from MutuI cells were probed with antibody to OCT2, HCF1, or EBNA1 (αOCT2, αHCF1, or αEBNA1). (B) IP with OCT2 or control IgG from MutuI cells were probed with EBNA1 or HCF1 antibody. (C) EBNA1 or control IgG from DG75 or MutuI cells was assayed with antibody to HCF1. (D) FLAG-tagged EBNA1 (f-EBNA1) was expressed in 293T or HeLa cells with or without OriP plasmid and assayed by FLAG-IP and probed with antibody to HCF1. (E) Western blot of total cell lysates from DG75, MutuI, LCL, HeLa, or HEK 293T cells probed with antibody to HCF1, EBNA1, or actin as indicated in the figure.