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

doi:10.1186/s13072-020-00366-4

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.

Highlights

Ubiquitin-like with plant homeodomain (PHD) and RING finger domains 1 (UHRF1, UniProtKB Q96T88, or Np95 in M. musculus, UniProtKB Q8VDF2) is an E3 ubiquitin ligase that bindsVaughan et al Epigenetics & Chromatin (2020) 13:44 toward multiple sites of mono-ubiquitination on histone H3 (UniProtKB, P68431) [8, 13, 14] and PCNA-associated factor (PAF15, UniProtKB Q15004) [15], which are bound by the ubiquitin-interacting motif (UIM) of deoxyribonucleic acid (DNA)-methyltransferase 1 (DNMT1, UniProtKB P26358) [16,17,18]
Saturation in peptide hybridization was evident by an inability to discriminate the UHRF1 tandem Tudor domain (TTD) interaction between LIG1K126me0 and LIG1K126me2, a binding preference that was previously determined by both fluorescence polarization (FP) [21] and isothermal titration calorimetry (ITC) [22]
We observed that LIG1K126me2 interacted primarily with Tudor domains (PHD finger protein 20 (PHF20, UniProtKB Q9BVI0) and UHRF1) and chromodomains (testis-specific chromodomain protein Y 1 (CDY1, UniProtKB Q9Y6F8), chromodomain Y-like protein isoform 2 (CDYL1b, UniProtKB Q9Y2322), chromobox protein homolog 3 (CBX3, UniProtKB Q13185), and chromodomain Y-like protein 2 (CDYL2, UniProtKB Q8N8U2)) that bound H3K9me2 (Fig. 1a, right)

Summary

Introduction

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1, UniProtKB Q96T88, or Np95 in M. musculus, UniProtKB Q8VDF2) is an E3 ubiquitin ligase that bindsVaughan et al Epigenetics & Chromatin (2020) 13:44 toward multiple sites of mono-ubiquitination on histone H3 (UniProtKB, P68431) [8, 13, 14] and PCNA-associated factor (PAF15, UniProtKB Q15004) [15], which are bound by the ubiquitin-interacting motif (UIM) of DNA (cytosine-5)-methyltransferase 1 (DNMT1, UniProtKB P26358) [16,17,18]. The UHRF1 tandem Tudor domain (TTD) binds lysine 9 di- and tri-methylation on histone H3 (H3K9me2/me3) and all three methylation states of lysine 126 on LIG1 (LIG1K126) [21] The contribution of this non-histone methyllysine-driven interaction to UHRF1-dependent DNA methylation maintenance is unclear. In mouse embryonic stem cells (mESCs), deletion of LIG1 had no effect on DNA methylation measured by mass spectrometry of bulk 5mC, luminometric methylation assay, and reduced representation bisulfite sequencing (RRBS) [21]. In this same study, DNA methylation analysis revealed a defect in mESCs in which 32 or 66 residues surrounding lysine 126 on LIG1 (the reported site of LIG1 methylation) were deleted. The contribution of this methyllysine-driven interaction to cancer cell DNA methylation maintenance through UHRF1 has not been considered

Methods

Results

Conclusion