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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.

Chromatin is the complex assembly of nucleic acids and proteins that makes up the physiological form of the eukaryotic genome. The nucleosome is the fundamental repeating unit of chromatin, and is composed of ∼147 bp of DNA wrapped around a histone octamer formed by two copies of each core histone: H2A, H2B, H3 and H4. Prior to nucleosome assembly, and during histone eviction, histones are typically assembled into soluble H2A/H2B dimers and H3/H4 dimers and tetramers. A multitude of factors interact with soluble histone dimers and tetramers, including chaperones, importins, histone-modifying enzymes and chromatin-remodeling enzymes. It is still unclear how many of these proteins recognize soluble histones; therefore, there is a need for new structural tools to study non-nucleosomal histones. Here, a single-chain, tailless Xenopus H2A/H2B dimer was created by directly fusing the C-terminus of H2B to the N-terminus of H2A. It is shown that this construct (termed scH2BH2A) is readily expressed in bacteria and can be purified under non-denaturing conditions. A 1.31 Å resolution crystal structure of scH2BH2A shows that it adopts a conformation that is nearly identical to that of nucleosomal H2A/H2B. This new tool is likely to facilitate future structural studies of many H2A/H2B-interacting proteins.

Xenopus laevis development is marked by accelerated cell division solely supported by the proteins maternally deposited in the egg. Oocytes mature to eggs with concomitant transcriptional silencing. The unique maternal chromatin state contributing to this silencing and subsequent zygotic activation is likely established by histone posttranslational modifications and histone variants. Therefore, tools for understanding the nature and function of maternal and embryonic histones are essential to deciphering mechanisms of regulation of development, chromatin assembly, and transcription. Here we describe protocols for isolating pronuclear sperm chromatin from Xenopus egg extracts and hydroxyapatite-based histone purification from this chromatin. The histones purified through this method can be directly assembled into chromatin through in vitro assembly reactions, providing a unique opportunity to biochemically dissect the effect of histone variants, histone modifications, and other factors in chromatin replication and assembly. We also describe how to isolate chromatin from staged embryos and analyze the proteins to reveal dynamic developmental histone modifications. Finally, we present protocols to measure chromatin assembly in extracts, including supercoiling and micrococcal nuclease assays. Using these approaches, analysis of maternal and zygotic histone posttranslational modifications concomitant with cell-cycle and developmental transitions can be tested., (© 2019 Cold Spring Harbor Laboratory Press.)

Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear. Using an NMR-based approach, here we demonstrate that the Xenopus laevis Npm tail domain controls the binding of histones at its largest acidic stretch (A2) via direct competition with both the C-terminal basic stretch and basic nuclear localization signal. NMR and small-angle X-ray scattering (SAXS) structural analyses allowed us to construct models of both the tail domain and the pentameric complex. Functional analyses demonstrate that these competitive intramolecular interactions negatively regulate Npm histone chaperone activity in vitro. Together these data establish a potentially generalizable mechanism of histone chaperone regulation via dynamic and specific intramolecular shielding of histone interaction sites.

Chromatin is the complex of eukaryotic DNA and proteins required for the efficient compaction of the nearly 2-meter-long human genome into a roughly 10-micron-diameter cell nucleus. The fundamental repeating unit of chromatin is the nucleosome: 147bp of DNA wrapped about an octamer of histone proteins. Nucleosomes are stable enough to organize the genome yet must be dynamically displaced and reassembled to allow access to the underlying DNA for transcription, replication, and DNA damage repair. Histone chaperones are a non-catalytic group of proteins that are central to the processes of nucleosome assembly and disassembly and thus the fluidity of the ever-changing chromatin landscape. Histone chaperones are responsible for binding the highly basic histone proteins, shielding them from non-specific interactions, facilitating their deposition onto DNA, and aiding in their eviction from DNA. Although most histone chaperones perform these common functions, recent structural studies of many different histone chaperones reveal that there are few commonalities in their folds. Importantly, sequence-based predictions show that histone chaperones are highly enriched in intrinsically disordered regions (IDRs) and acidic stretches. In this review, we focus on the molecular mechanisms underpinning histone binding, selectivity, and regulation of these highly dynamic protein regions. We highlight new evidence suggesting that IDRs are often critical for histone chaperone function and play key roles in chromatin assembly and disassembly pathways., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Protein arginine methyltransferase 5 (PRMT5) complexed with MEP50/WDR77 catalyzes arginine methylation on histones and other proteins. PRMT5-MEP50 activity is elevated in cancer cells and its expression is highly correlated with poor prognosis in many human tumors. We demonstrate that PRMT5-MEP50 is essential for transcriptional regulation promoting cancer cell invasive phenotypes in lung adenocarcinoma, lung squamous cell carcinoma and breast carcinoma cancer cells. RNA-Seq transcriptome analysis demonstrated that PRMT5 and MEP50 are required to maintain expression of metastasis and Epithelial-to-mesenchymal transition (EMT) markers and to potentiate an epigenetic mechanism of the TGFβ response. We show that PRMT5-MEP50 activity both positively and negatively regulates expression of a wide range of genes. Exogenous TGFβ promotes EMT in a unique pathway of PRMT5-MEP50 catalyzed histone mono- and dimethylation of chromatin at key metastasis suppressor and EMT genes, defining a new mechanism regulating cancer invasivity. PRMT5 methylation of histone H3R2me1 induced transcriptional activation by recruitment of WDR5 and concomitant H3K4 methylation at targeted genes. In parallel, PRMT5 methylation of histone H4R3me2s suppressed transcription at distinct genomic loci. Our decoding of histone methylarginine at key genes supports a critical role for complementary PRMT5-MEP50 transcriptional activation and repression in cancer invasion pathways and in response to TGFβ stimulation and therefore orients future chemotherapeutic opportunities., Competing Interests: The authors declare no conflict of interest.

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1-20) and H4(1-20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Histone proteins carry information contained in post-translational modifications. Eukaryotic cells utilize this histone code to regulate the usage of the underlying DNA. In the maturing oocytes and eggs of the frog Xenopus laevis, histones are synthesized in bulk in preparation for deposition during the rapid early developmental cell cycles. During this key developmental time frame, embryonic pluripotent chromatin is established. In the egg, non-chromatin-bound histones are complexed with storage chaperone proteins, including nucleoplasmin. Here we describe the identification and characterization of a complex of the protein arginine methyltransferase 5 (Prmt5) and the methylosome protein 50 (Mep50) isolated from Xenopus eggs that specifically methylates predeposition histones H2A/H2A.X-F and H4 and the histone chaperone nucleoplasmin on a conserved motif (GRGXK). We demonstrate that nucleoplasmin (Npm), an exceedingly abundant maternally deposited protein, is a potent substrate for Prmt5-Mep50 and is monomethylated and symmetrically dimethylated at Arg-187. Furthermore, Npm modulates Prmt5-Mep50 activity directed toward histones, consistent with a regulatory role for Npm in vivo. We show that H2A and nucleoplasmin methylation appears late in oogenesis and is most abundant in the laid egg. We hypothesize that these very abundant arginine methylations are constrained to pre-mid blastula transition events in the embryo and therefore may be involved in the global transcriptional repression found in this developmental time frame.

Histones are the major protein components of chromatin, the physiological form of the genome in all eukaryotic cells. Chromatin is the substrate of information-directed biological processes, such as gene regulation and transcription, replication, and mitosis. A long-standing experimental model system to study many of these processes is the extract made from the eggs of the anuran Xenopus laevis. Since work in recent years has solidified the importance of post-translational modification of histones in directing biological processes, the study of histones in a biochemically dissectible model such as Xenopus is crucial for the understanding of their biological significance. Here we present a rationale and methods for isolating and studying histones and chromatin in different Xenopus egg and oocyte extracts. In particular, we present protocols for the preparation of: cell-free egg and oocyte extract; nucleoplasmic extract ("NPE"); biochemical purification of maternally-deposited, stored histones in the oocyte and the egg; assembly of pronuclei in egg extract and the isolation of pronuclear chromatin and histones; and an extract chromatin assembly assay. We also demonstrate aspects of the variability of the system to be mindful of when working with extract and the importance of proper laboratory temperature in preparing quality extracts. We expect that these methods will be of use in promoting further understanding of embryonic chromatin in a unique experimental system.

Histone H2A.X is an H2A variant present in multicellular organisms that is specifically phosphorylated on the serine in the C-terminal consensus sequence, canonically "SQEY," in response to DNA damage. We have recently shown the significance of phosphorylation of the penultimate tyrosine for maintenance and processing of the DNA damage response in mammalian cells. Here, we report the identification of distinct H2A.X variants in the eggs and early embryos of the frog Xenopus laevis that contain a C-terminal SQEF, among other changes; we have denoted these proteins as "H2A.X-F." H2A.X-F is present only in late-staged oocytes, eggs, and premidblastula transition embryos and is not present in somatic cells. Similar unannotated isoforms were identified in other rapidly developing aquatic species, such as Xenopus tropicalis, goldfish, and zebrafish, and in Arabidopsis and chickpea. Furthermore, we demonstrate by mass spectrometry and phospho-specific antibodies that H2A.X-F is phosphorylated in the absence of exogenous DNA damage, in both actively dividing, unperturbed embryos and cell-free egg extract in the absence and presence of DNA damage and S-phase checkpoint conditions. We propose that this isoform may be involved in modulating the cellular response to the rapid early cell cycles in externally developing species.

Histone proteins contain epigenetic information that is encoded both in the relative abundance of core histones and variants and particularly in the post-translational modification of these proteins. We determined the presence of such variants and covalent modifications in seven tissue types of the anuran Xenopus laevis, including oocyte, egg, sperm, early embryo equivalent (pronuclei incubated in egg extract), S3 neurula cells, A6 kidney cells, and erythrocytes. We first developed a new robust method for isolating the stored, predeposition histones from oocytes and eggs via chromatography on heparin-Sepharose, whereas we isolated chromatinized histones via conventional acid extraction. We identified two previously unknown H1 isoforms (H1fx and H1B.Sp) present on sperm chromatin. We immunoblotted this global collection of histones with many specific post-translational modification antibodies, including antibodies against methylated histone H3 on Lys(4), Lys(9), Lys(27), Lys(79), Arg(2), Arg(17), and Arg(26); methylated histone H4 on Lys(20); methylated H2A and H4 on Arg(3); acetylated H4 on Lys(5), Lys(8), Lys(12), and Lys(16) and H3 on Lys(9) and Lys(14); and phosphorylated H3 on Ser(10) and H2A/H4 on Ser(1). Furthermore, we subjected a subset of these histones to two-dimensional gel analysis and subsequent immunoblotting and mass spectrometry to determine the global remodeling of histone modifications that occurs as development proceeds. Overall, our observations suggest that each metazoan cell type may have a unique histone modification signature correlated with its differentiation status.

Epigenetic information is hypothesized to be encoded in histone variants and post-translational modifications. Varied cell- and locus-specific combinations of these epigenetic marks are likely contributors to regulation of chromatin-templated transactions, including transcription, replication, recombination, and repair. Therefore, the relative abundance of histone modifications in a given cell type is a potential index of cell fate and specificity. Here, we utilize mass spectrometry techniques to characterize the relative abundance index of cell type-specific modifications on histones H3 and H4 in distinct cell types from the frog Xenopus laevis, including the sperm, the stored predeposition histones in the egg, the early embryo equivalent pronuclei, cultured somatic cells, and erythrocytes. We used collisionally associated dissociation to identify the modifications present on histone H3 in a variety of cell types, resolving 26 distinctly modified H3 peptides. We employed the electron transfer dissociation fragmentation technique in a "middle-down" approach on the H4 N-terminal tail to explore the overlap of post-translational modifications. We observed 66 discrete isoforms of the H4 1-23 fragment in four different cell types. Isolation of the stored, predeposition histone H4 from the frog egg also revealed a more varied pattern of modifications than the previously known diacetylation on Lys(5) and Lys(12). The developmental transitions of modifications on H3 and H4 were strikingly varied, implying a strong correlation of the histone code with cell type and fate. Our results are consistent with a histone code index for each cell type and uncover potential cross-talk between modifications on a single tail.

DNA double-stranded breaks present a serious challenge for eukaryotic cells. The inability to repair breaks leads to genomic instability, carcinogenesis and cell death. During the double-strand break response, mammalian chromatin undergoes reorganization demarcated by H2A.X Ser 139 phosphorylation (gamma-H2A.X). However, the regulation of gamma-H2A.X phosphorylation and its precise role in chromatin remodelling during the repair process remain unclear. Here we report a new regulatory mechanism mediated by WSTF (Williams-Beuren syndrome transcription factor, also known as BAZ1B)-a component of the WICH complex (WSTF-ISWI ATP-dependent chromatin-remodelling complex). We show that WSTF has intrinsic tyrosine kinase activity by means of a domain that shares no sequence homology to any known kinase fold. We show that WSTF phosphorylates Tyr 142 of H2A.X, and that WSTF activity has an important role in regulating several events that are critical for the DNA damage response. Our work demonstrates a new mechanism that regulates the DNA damage response and expands our knowledge of domains that contain intrinsic tyrosine kinase activity.

Histone proteins are the major protein components of chromatin, the physiologically relevant form of the genome (or epigenome) in all eukaryotic cells. Chromatin is the substrate of many biological processes, such as gene regulation and transcription, replication, mitosis and apoptosis. Since histones are extensively post-translationally modified, the identification of these covalent marks on canonical and variant histones is crucial for the understanding of their biological significance. Many different biochemical techniques have been developed to purify and separate histone proteins. Here, we present standard protocols for acid extraction and salt extraction of histones from chromatin; separation of extracted histones by reversed-phase HPLC; analysis of histones and their specific post-translational modification profiles by acid urea (AU) gel electrophoresis and the additional separation of non-canonical histone variants by triton AU(TAU) and 2D TAU electrophoresis; and immunoblotting of isolated histone proteins with modification-specific antibodies.