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. 2020 Oct 23;13(1):44.
doi: 10.1186/s13072-020-00366-4.

The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

Robert M Vaughan  1 , Ariana Kupai  1 , Caroline A Foley  2 , Cari A Sagum  3 , Bailey M Tibben  1 , Hope E Eden  1 , Rochelle L Tiedemann  1 , Christine A Berryhill  1 , Varun Patel  1 , Kevin M Shaw  1 , Krzysztof Krajewski  4 , Brian D Strahl  4 , Mark T Bedford  3 , Stephen V Frye  2 , Bradley M Dickson  1 , Scott B Rothbart  5
Affiliations

The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells

Robert M Vaughan et al. Epigenetics Chromatin. .

Abstract

The chromatin-binding E3 ubiquitin ligase ubiquitin-like with PHD and RING finger domains 1 (UHRF1) contributes to the maintenance of aberrant DNA methylation patterning in cancer cells through multivalent histone and DNA recognition. The tandem Tudor domain (TTD) of UHRF1 is well-characterized as a reader of lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and, more recently, lysine 126 di- and tri-methylation on DNA ligase 1 (LIG1K126me2/me3). However, the functional significance and selectivity of these interactions remain unclear. In this study, we used protein domain microarrays to search for additional readers of LIG1K126me2, the preferred methyl state bound by the UHRF1 TTD. We show that the UHRF1 TTD binds LIG1K126me2 with high affinity and selectivity compared to other known methyllysine readers. Notably, and unlike H3K9me2/me3, the UHRF1 plant homeodomain (PHD) and its N-terminal linker (L2) do not contribute to multivalent LIG1K126me2 recognition along with the TTD. To test the functional significance of this interaction, we designed a LIG1K126me2 cell-penetrating peptide (CPP). Consistent with LIG1 knockdown, uptake of the CPP had no significant effect on the propagation of DNA methylation patterning across the genomes of bulk populations from high-resolution analysis of several cancer cell lines. Further, we did not detect significant changes in DNA methylation patterning from bulk cell populations after chemical or genetic disruption of lysine methyltransferase activity associated with LIG1K126me2 and H3K9me2. Collectively, these studies identify UHRF1 as a selective reader of LIG1K126me2 in vitro and further implicate the histone and non-histone methyllysine reader activity of the UHRF1 TTD as a dispensable domain function for cancer cell DNA methylation maintenance.

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Conflict of interest statement

B.D.S and M.T.B. are co-founders of Epicypher, Inc. The other authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
LIG1K126me2 is read by a high-affinity interaction through the UHRF1 TTD. a Protein reader domain microarrays consisting of 308 GST-tagged domains (see Additional file 1: Fig. S1), each printed in duplicate, were probed by either anti-GST antibody, or Cy3-labeled LIG1(118–130)K126me2, H3(1–20)K9me2, or LIG1(118–130)K126me0 (left). Reader arrays were quantified using ArrayNinja software. Data were normalized to the brightest signal for each peptide (right). Error bars represent the range from duplicate spots. Full reader array datasets are available in Additional file 2: Table S1. b The indicated GST-tagged reader domains were hybridized to histone peptide microarrays at 1 µM, followed by fluorescent detection and quantified by ArrayNinja. Results for interactions with H3(1–20)K9me2 and LIG1(118–130)K126me2 are shown. Error bars represent standard error of the mean of six printed spots. Full histone peptide array data are available in Additional file 3: Table S2. c Fluorescence polarization binding assays between the indicated GST-tagged reader domains and either FAM-LIG1(118–130)K126me2 (left) or H3(1–20)K9me2-FAM (right). Error bars represent the 95% confidence interval from triplicate measurements
Fig. 2
Fig. 2
Unlike binding to H3K9me2, the UHRF1 PHD and its N-terminal linker do not modulate LIG1K126me2 recognition through the TTD. a Structural models of UHRF1 TTD bound to LIG1K126me3 (PDB:5YYA), UHRF1 PBR (PDB:6B9M), H3K9me3 (PDB:2L3R), and UHRF1 TTD–PHD bound to H3K9me3 (PDB:3ASK) with the linker between TTD and PHD shown in pink. b, c FP binding assays between the indicated GST-tagged b UHRF1 domains or c TTD–PHD mutants reader domains and FAM-LIG1(118–130)K126me2, H3(1–20)K9me2-FAM, or FAM-H3(1–20)K9me2. Error bars represent standard error of the mean from triplicate measurements
Fig. 3
Fig. 3
A LIG1K126me2 cell-penetrating peptide has no significant effects on HeLa cell DNA methylation. a Fluorescence polarization binding assays between full-length UHRF1 and FAM-labeled LIG1(118–130)K126me2 peptides with (red) and without (blue) a cell-penetrating peptide (CPP) sequence, -polyethylene glycol-kkkrkv. b Fluorescence microscopy of HeLa cells after 5-h incubation with control solvent (water) or with FAM-LIG1K126me2-CPP. c Chloroalkane penetration assay (CAPA) for chloroalkane-tagged (ct) LIG1K126me2-CPP in HeLa cells (CP50), concentration where 50% of maximal penetration was observed; error bars, the standard error from the independent experiments—three independent curve fits from three independent experiments). d Infinium MethylationEPIC BeadChip analysis of HeLa cells (beta values: 0, unmethylated; 1, methylated) after 7 days of incubation with control solvent or LIG1K126me2-CPP peptide at 20 µM. Scatter plots with density for all probes (left) and those that had beta values > 0.8 in control cells (right). e Distribution of beta-value differences (∆β) between control and LIG1K126me2-CPP-treated cells for probes in panel (d) that were > 0.8 in control cells (n)
Fig. 4
Fig. 4
LIG1 depletion does not affect DNA methylation maintenance in HCT116. a Western blots confirming LIG1 or UHRF1 knockdown in HCT116 cells. b, c Infinium MethylationEPIC BeadChip analysis of HCT116 cells (beta values: 0, unmethylated; 1, methylated) 12 days after incorporation of shRNAs targeting UHRF1 (b) or LIG1 (c) relative to the control cell line (shLuc, shRNA targeting luciferase). Scatter plots with density for all cytosine probes (left) and those that had beta values > 0.8 in control cells (middle) are shown. Distribution of beta-value differences (∆β) between shLuc and target knockdown for probes that were > 0.8 in control cells (n) is shown (right)
Fig. 5
Fig. 5
DNA methylation maintenance is stable despite global disruptions to LIG1K126- and H3K9-associated lysine methylation signaling. a In vitro methylation of recombinant full-length LIG1 by the catalytic domain of G9a (EHMT2). 3H-SAM incorporation was monitored by autoradiography and Coomassie staining is used as a loading control. b Western blots of HCT116 cells treated with either DMSO control or the G9a inhibitor UNC0638 (1 µM) for 48 h. cbb, Coomassie brilliant blue stain of membrane. c Infinium MethylationEPIC BeadChip analysis of HCT116 cells from b. Data are presented as in Fig. 4b, c. d Western blots of HCT116 cells 12 days after incorporation of lentivirus expressing wild-type H3.3 (WT) or H3.3 K9M. e Infinium MethylationEPIC BeadChip analysis of HCT116 cells from d. Data are presented as in Fig. 4b, c
Fig. 6
Fig. 6
Molecular interaction analysis and theoretical modeling do not support a UHRF1–LIG1 interaction in cells. a Western blots for the indicated proteins following chromatin fractionation of HCT116 cells in the absence (−, empty vector control) or presence (+) of a LIG1 transgene. Total, whole cell extracts; chr, chromatin fraction; sol, soluble fraction. b IP of endogenous UHRF1 from HCT116 cells followed by western blot for LIG1 and UHRF1. c A competitive binding model for UHRF1 interactions with methylated forms of H3 and LIG1. Shaded regions show the range of approximate ratios for the methylated (purple) or unmethylated (green) binding partners of UHRF1
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