Molecular investigation of the tandem Tudor domain and plant homeodomain histone binding domains of the epigenetic regulator UHRF2

Abstract

Ubiquitin‐like containing PHD and ring finger (UHRF)1 and UHRF2 are multidomain epigenetic proteins that play a critical role in bridging crosstalk between histone modifications and DNA methylation. Both proteins contain two histone reader domains, called tandem Tudor domain (TTD) and plant homeodomain (PHD), which read the modification status on histone H3 to regulate DNA methylation and gene expression. To shed light on the mechanism of histone binding by UHRF2, we have undergone a detailed molecular investigation with the TTD, PHD and TTD‐PHD domains and compared the binding activity to its UHRF1 counterpart. We found that unlike UHRF1 where the PHD is the primary binding contributor, the TTD of UHRF2 has modestly higher affinity toward the H3 tail, while the PHD has a weaker binding interaction. We also demonstrated that like UHRF1, the aromatic amino acids within the TTD are important for binding to H3K9me3 and a conserved aspartic acid within the PHD forms an ionic interaction with R2 of H3. However, while the aromatic amino acids in the TTD of UHRF1 contribute to selectivity, the analogous residues in UHRF2 contribute to both selectivity and affinity. We also discovered that the PHD of UHRF2 contains a distinct asparagine in the H3R2 binding pocket that lowers the binding affinity of the PHD by reducing a potential electrostatic interaction with the H3 tail. Furthermore, we demonstrate the PHD and TTD of UHRF2 cooperate to interact with the H3 tail and that dual domain engagement with the H3 tail relies on specific amino acids. Lastly, our data indicate that the unique stretch region in the TTD of UHRF2 can decrease the melting temperature of the TTD‐PHD and represents a disordered region. Thus, these subtle but important mechanistic differences are potential avenues for selectively targeting the histone binding interactions of UHRF1 and UHRF2 with small molecules.

Impaired Repopulating Ability of Uhrf2−/− Hematopoietic Progenitor Cells in Mice

“…We next analyzed enrichment of histone H3 trimethylated at lysine 9 (H3K9me3) in the region encompassing 5 kb upstream and downstream of gene body regions in LSK cells using CUT&Tag, because it has been reported that the tandem Tudor domain (TTD) of Uhrf2 interacts with H3K9me3 [ 2 , 20 , 21 , 22 ]. CUT&Tag showed that the increases in H3K9me3 was more frequent, but not universal, in Uhrf2 −/− LSK cells compared to Uhrf2 +/+ LSK cells ( Figure 5 ).…”

“…CUT&Tag showed a general enrichment of H3K9me3 in HSPCs of Uhrf2 −/− mice compared to those of Uhrf2 +/+ mice. Because the TTD domain of UHRF2 protein, with activity of a ubiquitin ligase, recognizes H3K9me3 [ 2 , 20 , 21 , 22 ], the alteration of H3K9me3 status in Uhrf2 −/− LSK cells may be associated with deletion of UHRF2 protein containing the TTD domain. However, we could not find a factor leading to HSPC proliferation that is regulated by H3K9me3.…”

Advancing thermostability of the key photorespiratory enzyme glycerate 3-kinase by structure-based recombination

“…For determining T m , differential scanning fluorimetry was employed using SYPRO Orange (Huynh and Partch, 2015). For these assays, fluorescence measurements were collected from 30 μl reactions in white, 0.2 ml PCR tubes with optical flat caps (Bio-Rad Laboratories, Hercules, California, USA) containing 12.5 mM Na-phosphate (pH 7.5), 15x SYPRO Orange (Sigma, Sigma-Aldrich, St. Louis, MO, USA), and 15 μg of His6- tagged recombinant enzyme (Ginnard et al ., 2022). Starting at 20 °C, samples were warmed to 95 °C at 1 °C per min with readings collected every 15 seconds using a 7500 Fast Real-Time PCR System equipped with a ROX filter (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA USA).…”

The UHRF protein family in epigenetics, development, and carcinogenesis

“…With respect to UHRF2, the TTD and PHD domains recognize H3K9me3 and H3R2me0, respectively, although the binding mode appears to be slightly different from binding modes of UHRF1. 32 ) Recent studies also showed that the SRA domain of UHRF2 recognizes 5hmC. 33 , 34 ) Finally, the RING domain of UHRF2 has multiple substrates for ubiquitylation including PCNP, cyclin D1, cyclin E1, and X-ray repair cross complementing 1 (XRCC1).…”