Article Figures & Data
Figures
- Figure 1. hnRNP E1 binds polycytosine-rich DNA tracts.
(A) Oligonucleotides and deoxyoligonucleotides used in RNA electrophoretic mobility shift assay (REMSA)/electrophoretic mobility shift assay (EMSA). (B) Immunofluorescence of hnRNP E1 and DAPI staining showing localization of hnRNP E1. (C) ChIP-PCR products from A549 DNA showing enrichment of hnRNP E1 at promoter regions of c-MYC, h-Ras, and EGFR genes. (D) hnRNP E1 protein and its KH domains. (D) Top. SDS–PAGE gel showing GST fusion proteins of hnRNP E1; KH1, KH2, and KH3; and hnRNP D. Bottom. The three KH domains of hnRNP E1. (E, F, G, H, I, J, K, L) Autoradiograms of representative REMSA/EMSA assays. (E) REMSA showing hnRNP E1 binding to BAT RNA. (F, G, H, I, J) EMSA showing hnRNP E1 binding to c-MYC C (F), c-MYC G (G), TEL DNA C (H), TEL DNA G (I), and c-MYC C, BCL2, EGFR, HIF1-α, PDGF, and VEGF (J). (K) EMSA showing binding of hnRNP E1, KH1, KH2, and KH3 to BAT RNA. (L) EMSA showing binding of hnRNP E1, KH1, KH2, and KH3 to TEL DNA C. (M) Quantification of REMSA and EMSA assay.
- Figure 2. Genome-wide DNA binding by hnRNP E1 and its cellular function.
(A) Per cent of regions in the mouse (NMuMG) genome with at least 50% of “C” nucleotide. (B) Per cent (and number) of promoter proximal regions containing high-C sequences. (C) Two poly-C representative sequences from Table S1. (D) Autoradiographs of electrophoretic mobility shift assay showing differential binding of telomeric oligodeoxynucleotides containing one to five repeats of “CCCTAA” to GST-hnRNP E1 protein. (E) PAGE gel showing binding of telomeric oligodeoxynucleotides containing one to five repeats of “CCCTAA” upon loading on GST-hnRNP E1 immobilized on agarose beads. (F) Model showing i-motif and G4 structures. (G) Autoradiograph of urea-PAGE and PAGE (pH 7.5 and 5.5) gels showing fractionation pattern of various oligonucleotides. (H) Autoradiograph of a PAGE gel (run at pH 7.5) showing binding of hnRNP E1 to c-MYC, TEL DNA C and hRas2 deoxynucleotides incubated at pH 5.5. (I) Immunofluorescence of NMuMG control and E1KD cells showing i-motif foci. (J) Immunofluorescence of A549 control and E1KD showing i-motif foci, hnRNP E1 foci, and their colocalization. (K) Immunofluorescence of A549 control and E1KD cells showing G4 foci.
- Figure S1. Representative graphs showing quantification of hnRNP E1 binding to BAT RNA and poly-C–rich DNA tracts.
(A) BAT RNA. (B) TEL DNA C. (C) c-MYC. (D) BCL2. (E) HIF1-a. (F) PDGF. (G) VEGF. (H) EGFR.
- Figure S2. Representative graphs showing quantification of binding by hnRNP E1, KH1, KH2 and KH3 to BAT RNA.
(A) Binding of hnRNP E1 to BAT RNA. (B) Binding of KH1, KH2, and KH3 to BAT RNA.
- Figure S3. Autoradiographs showing protein–nucleic acid interactions.
(A) Competition of TEL DNA C and BAT RNA with TEL DNA C for binding to hnRNP E1. (B) Binding of hnRNP E1, KH1, KH2, and KH3 to TEL RNA C. (C) Electrophoretic mobility shift assay showing hnRNP E1 binding to TEL DNA C and TEL RNA C. (D) Competition of TEL DNA C and TEL RNA C with TEL DNA C for binding to hnRNP E1. (E) Binding of hnRNP E1 to TEL DNA C, and competition of TEL DNA C and TEL DNA G with TEL DNA C for binding to hnRNP E1.
- Figure S4. Binding of hnRNP E1 to telomeric DNA.
(A, B) Agarose gel showing fractionation of ChIP-PCR products generated by telomere primers (tel1 and tel2) from mouse cell line NMuMG. M, DNA molecular weight markers; Control: no antibody control, FLAG: FLAG antibody used to pull-down FLAG-tagged hnRNP E1, Trf2:Trf2 antibody used to pull-down Trf2. (C) Autoradiogram of electrophoretic mobility shift assay showing hnRNP E1 binding to mutants of TEL DNA C that contain mutations in four or five first “C”s of CCC repeats present in a 33 base deoxyoligonucleotide with five repeats of “CCCTAA.” (D) Immunofluorescence showing i-motifs in a single nucleus of A549 and E1KD cells. Fig 2J shows multiple nuclei.
- Figure 3. Role of hnRNP E1 in genome integrity.
(A, B) Immunofluorescence showing γ-H2AX activation in E1KD cells. (C) Western blot showing γ-H2AX activation in E1KD cells. (D) Immunofluorescence showing increase in and colocalization of γ-H2AX and G4 foci in E1KD cells. (E) ChIP-qPCR analysis showing γ-H2AX localization at c-MYC promoter of scrambled (Ascr) and hnRNP E1 knockdown (E1KD) cells.
- Figure 4. E1KD phenotype acts synergistically with HU/UV and promotes mutation.
(A, B) Cell cycle distribution of A549 control and E1KD cells exposed to UV. (C) Blot showing CPD incorporation in wild-type and E1KD cells. (D, E) Western blots showing effects of UV on checkpoint proteins in A549 control and E1KD (D) cells, and checkpoint proteins in NMuMG control and E1KD cells (E). (F) Western blot showing proliferating cell nuclear antigen monoubiquitination, Usp1, and Usp7 in A549 control and E1KD cells recovering from UV exposure (h, hours). (G, H) Western blots showing γ-H2AX activation (G) and proliferating cell nuclear antigen monoubiquitination along with Usp1 and Usp7 (H) in A549 control and E1KD cells treated with HU. (I) Histogram of clonogenic assay with A549 control and E1KD cells exposed to HU. (J) Mutation frequency in A549 control and E1KD cells.
- Figure 5. Model showing the role of hnRNP E1 in genome integrity.
Upon DNA damage, hnRNP E1 bound at polycytosine tracts activates damage signaling and repair with the loading and assistance of RPA (R). Proliferating cell nuclear antigen (PCNA) is monoubiquitinated, replaces DNA polymerase δ with TLS polymerases, which are often error-free, and finally PCNA is deubiquitinated. When DNA damage occurs in the absence of hnRNP E1, only RPA functions at polycytosine tracts albeit slowly, PCNA monoubiquitination is enhanced and lingers, error-prone TLS polymerases replace DNA polymerase δ, and signaling and repair are attenuated.
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