A Binary Arginine Methylation Switch on Histone H3 Arginine 2 Regulates Its Interaction with WDR5.

Histone H3 arginine 2 (H3R2) is post-translationally modified in three different states by "writers" of the protein arginine methyltransferase (PRMT) family. H3R2 methylarginine isoforms include PRMT5-catalyzed monomethylation (me1) and symmetric dimethylation (me2s) and PRMT6-catalyzed me1 and asymmetric dimethylation (me2a). WD-40 repeat-containing protein 5 (WDR5) is an epigenetic "reader" protein that interacts with H3R2. Previous studies suggested that H3R2me2s specified a high-affinity interaction with WDR5. However, our prior biological data prompted the hypothesis that WDR5 may also interact with H3R2me1. Here, using highly accurate quantitative binding analysis combined with high-resolution crystal structures of WDR5 in complex with unmodified (me0) and me1/me2s l-arginine amino acids and in complex with the H3R2me1 peptide, we provide a rigorous biochemical study and address long-standing discrepancies of this important biological interaction. Despite modest structural differences at the binding interface, our study supports an interaction model regulated by a binary arginine methylation switch: H3R2me2a prevents interaction with WDR5, whereas H3R2me0, -me1, and -me2s are equally permissive.

Cellular consequences of arginine methylation.

Arginine methylation is a ubiquitous post-translational modification. Three predominant types of arginine-guanidino methylation occur in Eukarya: mono (Rme1/MMA), symmetric (Rme2s/SDMA), and asymmetric (Rme2a/ADMA). Arginine methylation frequently occurs at sites of protein-protein and protein-nucleic acid interactions, providing specificity for binding partners and stabilization of important biological interactions in diverse cellular processes. Each methylarginine isoform-catalyzed by members of the protein arginine methyltransferase family, Type I (PRMT1-4,6,8) and Type II (PRMT5,9)-has unique downstream consequences. Methylarginines are found in ordered domains, domains of low complexity, and in intrinsically disordered regions of proteins-the latter two of which are intimately connected with biological liquid-liquid phase separation. This review highlights discoveries illuminating how arginine methylation affects genome integrity, gene transcription, mRNA splicing and mRNP biology, protein translation and stability, and phase separation. As more proteins and processes are found to be regulated by arginine methylation, its importance for understanding cellular physiology will continue to grow.

Glutamylation of Npm2 and Nap1 acidic disordered regions increases DNA mimicry and histone chaperone efficiency.

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

Glutamylation of Npm2 and Nap1 acidic disordered regions increases DNA charge mimicry to enhance chaperone efficiency.

Histone chaperones-structurally diverse, non-catalytic proteins enriched with acidic intrinsically disordered regions (IDRs)-protect histones from spurious nucleic acid interactions and guide their deposition into and out of nucleosomes. Despite their conservation and ubiquity, the function of the chaperone acidic IDRs remains unclear. Here, we show that the Xenopus laevis Npm2 and Nap1 acidic IDRs are substrates for TTLL4 (Tubulin Tyrosine Ligase Like 4)-catalyzed post-translational glutamate-glutamylation. We demonstrate that, to bind, stabilize, and deposit histones into nucleosomes, chaperone acidic IDRs function as DNA mimetics. Our biochemical, computational, and biophysical studies reveal that glutamylation of these chaperone polyelectrolyte acidic stretches functions to enhance DNA electrostatic mimicry, promoting the binding and stabilization of H2A/H2B heterodimers and facilitating nucleosome assembly. This discovery provides insights into both the previously unclear function of the acidic IDRs and the regulatory role of post-translational modifications in chromatin dynamics.

Independent transcriptomic and proteomic regulation by type I and II protein arginine methyltransferases.

Protein arginine methyltransferases (PRMTs) catalyze the post-translational monomethylation (Rme1), asymmetric (Rme2a), or symmetric (Rme2s) dimethylation of arginine. To determine the cellular consequences of type I (Rme2a) and II (Rme2s) PRMTs, we developed and integrated multiple approaches. First, we determined total cellular dimethylarginine levels, revealing that Rme2s was ∼3% of total Rme2 and that this percentage was dependent upon cell type and PRMT inhibition status. Second, we quantitatively characterized in vitro substrates of the major enzymes and expanded upon PRMT substrate recognition motifs. We also compiled our data with publicly available methylarginine-modified residues into a comprehensive database. Third, we inhibited type I and II PRMTs and performed proteomic and transcriptomic analyses to reveal their phenotypic consequences. These experiments revealed both overlapping and independent PRMT substrates and cellular functions. Overall, this study expands upon PRMT substrate diversity, the arginine methylome, and the complex interplay of type I and II PRMTs.

Rinf Regulates Pluripotency Network Genes and Tet Enzymes in Embryonic Stem Cells.

The Retinoid inducible nuclear factor (Rinf), also known as CXXC5, is a nuclear protein, but its functions in the context of the chromatin are poorly defined. We find that in mouse embryonic stem cells (mESCs), Rinf binds to the chromatin and is enriched at promoters and enhancers of Tet1, Tet2, and pluripotency genes. The Rinf-bound regions show significant overlapping occupancy of pluripotency factors Nanog, Oct4, and Sox2, as well as Tet1 and Tet2. We found that Rinf forms a complex with Nanog, Oct4, Tet1, and Tet2 and facilitates their proper recruitment to regulatory regions of pluripotency and Tet genes in ESCs to positively regulate their transcription. Rinf deficiency in ESCs reduces expression of Rinf target genes, including several pluripotency factors and Tet enzymes, and causes aberrant differentiation. Together, our findings establish Rinf as a regulator of the pluripotency network genes and Tet enzymes in ESCs.