IMPORTIN-4, the primary nuclear import receptor of core histones H3 and H4, binds the H3-H4 dimer and histone chaperone ASF1 prior to nuclear import. However, how H3-H3-ASF1 is recognized for transport cannot be explained by available crystal structures of IMPORTIN-4-histone tail peptide complexes. Our 3.5-Å IMPORTIN-4-H3-H4-ASF1 cryoelectron microscopy structure reveals the full nuclear import complex and shows a binding mode different from suggested by previous structures. The N-terminal half of IMPORTIN-4 clamps the globular H3-H4 domain and H3 αN helix, while its C-terminal half binds the H3 N-terminal tail weakly; tail contribution to binding energy is negligible. ASF1 binds H3-H4 without contacting IMPORTIN-4. Together, ASF1 and IMPORTIN-4 shield nucleosomal H3-H4 surfaces to chaperone and import it into the nucleus where RanGTP binds IMPORTIN-4, causing large conformational changes to release H3-H4-ASF1. This work explains how full-length H3-H4 binds IMPORTIN-4 in the cytoplasm and how it is released in the nucleus.

During mammalian spermatogenesis, the paternal genome is extensively remodelled via replacement of histones with protamines forming the highly compact mature sperm nucleus. Compaction occurs in post-meiotic spermatids and is accompanied by extensive double strand break (DSB) formation. We investigate the epigenomic and genomic context of mouse spermatid DSBs, identifying primary sequence motifs, secondary DNA structures and chromatin contexts associated with this damage. Consistent with previously published results we find spermatid DSBs positively associated with short tandem repeats and LINE elements. We further show spermatid DSBs preferentially occur in association with (CA)n, (NA)n and (RY)n repeats, in predicted Z-DNA, are not associated with G-quadruplexes, are preferentially found in regions of low histone mark coverage and engage the remodelling/NHEJ factor BRD4. Locations incurring DSBs in spermatids also show distinct epigenetic profiles throughout later developmental stages: regions retaining histones in mature sperm, regions susceptible to oxidative damage in mature sperm, and fragile two-cell like embryonic stem cell regions bound by ZSCAN4 all co-localise with spermatid DSBs and with each other. Our results point to a common 'vulnerability code' unifying several types of DNA damage occurring on the paternal genome during reproduction, potentially underpinned by torsional changes during sperm chromatin remodelling.

The body plan along the anteroposterior axis and regional identities are specified by the spatiotemporal expression of Hox genes. Multistep controls are required for their unique expression patterns; however, the molecular mechanisms behind the tight control of Hox genes are not fully understood. In this study, we demonstrated that the Lin28a/let-7 pathway is critical for axial elongation. Lin28a-/- mice exhibited axial shortening with mild skeletal transformations of vertebrae, which were consistent with results in mice with tail bud-specific mutants of Lin28a. The accumulation of let-7 in Lin28a-/- mice resulted in the reduction of PRC1 occupancy at the Hox cluster loci by targeting Cbx2. Consistently, Lin28a loss in embryonic stem-like cells led to aberrant induction of posterior Hox genes, which was rescued by the knockdown of let-7. These results suggest that the Lin28/let-7 pathway is involved in the modulation of the 'Hox code' via Polycomb regulation during axial patterning.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections of mammalian germline cells. A large proportion of ERVs lose their open reading frames (ORFs), while others retain them and become exapted by the host species. However, it remains unclear what proportion of ERVs possess ORFs (ERV-ORFs), become transcribed, and serve as candidates for co-opted genes.

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation.

It is widely accepted that transcriptional regulation of eukaryotic genes is intimately coupled to covalent modifications of the underlying chromatin template, and in certain cases the functional consequences of these modifications have been characterized. Here we present evidence that gene activation in the silent heterochromatin of the yeast Saccharomyces cerevisiae can occur in the context of little, if any, covalent histone modification. Using a SIR-regulated heat shock-inducible transgene, hsp82-2001, and a natural drug-inducible subtelomeric gene, YFR057w, as models we demonstrate that substantial transcriptional induction (>200-fold) can occur in the context of restricted histone loss and negligible levels of H3K4 trimethylation, H3K36 trimethylation and H3K79 dimethylation, modifications commonly linked to transcription initiation and elongation. Heterochromatic gene activation can also occur with minimal H3 and H4 lysine acetylation and without replacement of H2A with the transcription-linked variant H2A.Z. Importantly, absence of histone modification does not stem from reduced transcriptional output, since hsp82-ΔTATA, a euchromatic promoter mutant lacking a TATA box and with threefold lower induced transcription than heterochromatic hsp82-2001, is strongly hyperacetylated in response to heat shock. Consistent with negligible H3K79 dimethylation, dot1Δ cells lacking H3K79 methylase activity show unimpeded occupancy of RNA polymerase II within activated heterochromatic promoter and coding regions. Our results indicate that large increases in transcription can be observed in the virtual absence of histone modifications often thought necessary for gene activation.

Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD) histone pre-mRNA processing requires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.

Histone proteins and the nucleosomal organization of chromatin are near-universal eukaroytic features, with the exception of dinoflagellates. Previous studies have suggested that histones do not play a major role in the packaging of dinoflagellate genomes, although several genomic and transcriptomic surveys have detected a full set of core histone genes. Here, transcriptomic and genomic sequence data from multiple dinoflagellate lineages are analyzed, and the diversity of histone proteins and their variants characterized, with particular focus on their potential post-translational modifications and the conservation of the histone code. In addition, the set of putative epigenetic mark readers and writers, chromatin remodelers and histone chaperones are examined. Dinoflagellates clearly express the most derived set of histones among all autonomous eukaryote nuclei, consistent with a combination of relaxation of sequence constraints imposed by the histone code and the presence of numerous specialized histone variants. The histone code itself appears to have diverged significantly in some of its components, yet others are conserved, implying conservation of the associated biochemical processes. Specifically, and with major implications for the function of histones in dinoflagellates, the results presented here strongly suggest that transcription through nucleosomal arrays happens in dinoflagellates. Finally, the plausible roles of histones in dinoflagellate nuclei are discussed.

Arginine methylation broadly occurs in histones and has been linked to transcriptional regulation, cell cycle regulation and DNA repair. While numerous proteins (histone code effectors) that specifically recognize or read the methylated lysine residues in core histones have been identified, little is known for effectors specific for methylated arginines in histones. In this study, we attempted to identify effector(s) recognizing asymmetrically methylated R17 and R26 in H3, which are catalyzed by CARM1/PRMT4, through an unbiased biochemical approach. Although we have yet to identify such effector using this approach, we find that these modifications function cooperatively with histone acetylation to inhibit the binding of the nucleosome remodeling and deacetylase complex (NuRD) and TIF1 family corepressors to H3 tail in vitro. In support of this finding, we show that overexpression of CARM1 in 293 T cells leads to reduced association of NuRD with chromatin, whereas knockdown of CARM1 in HeLa cells leads to increased association of NuRD with chromatin and decreased level of histone acetylation. Furthermore, in the Carm1-/- MEF cells there is an increased association of NuRD and TIF1β with chromatin and a global decrease in histone acetylation. By chromatin immunoprecipitation assay, we show that overexpression of CARM1 results in reduced association of NuRD complex and TIF1β with an episomal reporter and that CARM1 is required in MEF cells for LPS-induced dissociation of NuRD from a NF-κb target gene. Taking together, our study provides evidence for a role of CARM1-mediated arginine methylation in regulation of histone acetylation and transcription: facilitating transcription by discharging corepressors from chromatin.

Nucleosome, the fundamental unit of chromatin, is formed by wrapping nearly 147bp of DNA around an octamer of histone proteins. This histone core has many variants that are different from each other by their biochemical compositions as well as biological functions. Although the deposition of histone variants onto chromatin has been implicated in many important biological processes, such as transcription and replication, the mechanisms of how they are deposited on target sites are still obscure.

The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases is associated with various malignancies, including aggressive acute leukemia with NUP98-NSD1 translocation. While NSD proteins represent attractive drug targets, their catalytic SET domains exist in autoinhibited conformation, presenting notable challenges for inhibitor development. Here, we employed a fragment-based screening strategy followed by chemical optimization, which resulted in the development of the first-in-class irreversible small-molecule inhibitors of the nuclear receptor-binding SET domain protein 1 (NSD1) SET domain. The crystal structure of NSD1 in complex with covalently bound ligand reveals a conformational change in the autoinhibitory loop of the SET domain and formation of a channel-like pocket suitable for targeting with small molecules. Our covalent lead-compound BT5-demonstrates on-target activity in NUP98-NSD1 leukemia cells, including inhibition of histone H3 lysine 36 dimethylation and downregulation of target genes, and impaired colony formation in an NUP98-NSD1 patient sample. This study will facilitate the development of the next generation of potent and selective inhibitors of the NSD histone methyltransferases.

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.

The histone code is an epigenetic regulatory system thought to play a crucial role in cellular events such as development, differentiation and in the maintenance of pluripotency. In order to gain an insight into the role variant histones may play during mammalian development; we studied gene expression of histone variants and remodelling enzymes in mouse embryonic stem (ES) cells and during mouse preimplantation development. Using quantitative reverse-transcription PCR (qRT-PCR) we document the gene expression pattern of 12 histone variants and 2 of their associated remodelling enzymes in undifferentiated ES cells and during preimplantation embryo development. All histone variants were detected in undifferentiated ES cells, with H2AZ showing the highest expression levels of all the histone variants tested. The results also show that H2A variant levels tend to increase later in embryo development whilst H3 variant levels are elevated in early preimplantation stages. In addition, the expression of SWI/SNF, a remodeler protein involved in specifically remodelling H2A-H2B dimers, mirrors the expression of H2B and H2A variants, and the H3-H4 specific chaperone CAF-1 expression mirrors H3 variant expression. These results provide a foundation for further studies on the functions of histone variants during development, differentiation and in pluripotency.

Yin Yang 1 (YY1) is a member of polycomb protein family involved in epigenetic modifications and transcriptional controls. We have shown that YY1 acts as positive regulator of tumor growth and angiogenesis by interfering with the VEGFA network. Yet, the link between polycomb chromatin complex and hypoxia regulation of VEGFA is still poorly understood. Here, we establish that hypoxia impairs YY1 binding to VEGFA mRNA 3'UTR (p<0.001) in bone malignancy. Moreover, RNA immunoprecipitation reveals the formation of triplex nuclear complexes among YY1, VEGFA DNA, mRNA, and unreached about 200 fold primiRNA 200b and 200c via Dicer protein. In this complex, YY1 is necessary to maintain the steady-state level of VEGFA expression while its silencing increases VEGFA mRNA half-life at 4 h and impairs the maturation of miRNA 200b/c. Hypoxia promotes histone modification through ubiquitination both of YY1 and Dicer proteins. Hypoxia-mediated down-regulation of YY1 and Dicer changes post-transcriptional VEGFA regulation by resulting in the accumulation of primiRNA200b/c in comparison to mature miRNAs (p<0.001). Given the regulatory functions of VEGFA on cellular activities to promote neoangiogenesis, we conclude that YY1 acts as novel critical interface between epigenetic code and miRNAs machinery under chronic hypoxia in malignancy.

MLL3 and MLL4 are two closely related members of the SET1/MLL family of histone H3K4 methyltransferases and are responsible for monomethylating histone H3K4 on enhancers, which are essential in regulating cell-type-specific gene expression. Mutations of MLL3 or MLL4 have been reported in different types of cancer. Recently, the PHD domains of MLL3/4 have been reported to recruit the MLL3/4 complexes to their target genes by binding to histone H4 during the NT2/D1 stem cell differentiation. Here we show that an extended PHD domain (ePHD6) involving the sixth PHD domain and its preceding zinc finger in MLL3 and MLL4 specifically recognizes an H4H18-containing histone H4 fragment and that modifications of residues surrounding H4H18 modulate H4 binding to MLL3/4. Our in vitro methyltransferase assays and cellular experiments further reveal that the interaction between ePHD6 of MLL3/4 and histone H4 is required for their nucleosomal methylation activity and MLL4-mediated neuronal differentiation of NT2/D1 cells.

Serine ADP-ribosylation (Ser-ADPr) is a recently discovered protein modification that is catalyzed by PARP1 and PARP2 when in complex with the eponymous histone PARylation factor 1 (HPF1). In addition to numerous other targets, core histone tails are primary acceptors of Ser-ADPr in the DNA damage response. Here, we show that specific canonical histone marks interfere with Ser-ADPr of neighboring residues and vice versa. Most notably, acetylation, but not methylation of H3K9, is mutually exclusive with ADPr of H3S10 in vitro and in vivo. We also broaden the O-linked ADPr spectrum by providing evidence for tyrosine ADPr on HPF1 and other proteins. Finally, we facilitate wider investigations into the interplay of histone marks with Ser-ADPr by introducing a simple approach for profiling posttranslationally modified peptides. Our findings implicate Ser-ADPr as a dynamic addition to the complex interplay of modifications that shape the histone code.

Chromosomal duplication requires de novo assembly of nucleosomes from newly synthesized histones, and the process involves a dynamic network of interactions between histones and histone chaperones. sNASP and ASF1 are two major histone H3-H4 chaperones found in distinct and common complexes, yet how sNASP binds H3-H4 in the presence and absence of ASF1 remains unclear. Here we show that, in the presence of ASF1, sNASP principally recognizes a partially unfolded Nα region of histone H3, and in the absence of ASF1, an additional sNASP binding site becomes available in the core domain of the H3-H4 complex. Our study also implicates a critical role of the C-terminal tail of H4 in the transfer of H3-H4 between sNASP and ASF1 and the coiled-coil domain of sNASP in nucleosome assembly. These findings provide mechanistic insights into coordinated histone binding and transfer by histone chaperones.

N-terminal tails of H2A, H2B, H3 and H4 histone families are subjected to posttranslational modifications that take part in transcriptional regulation mechanisms, such as transcription factor binding and gene expression. Regulation mechanisms under control of histone modification are important but remain largely unclear, despite of emerging datasets for comprehensive analysis of histone modification. In this paper, we focus on what we call genetic harmonious units (GHUs), which are co-occurring patterns among transcription factor binding, gene expression and histone modification. We present the first genome-wide approach that captures GHUs by combining ChIP-chip with microarray datasets from Saccharomyces cerevisiae. Our approach employs noise-robust soft clustering to select patterns which share the same preferences in transcription factor-binding, histone modification and gene expression, which are all currently implied to be closely correlated. The detected patterns are a well-studied acetylation of lysine 16 of H4 in glucose depletion as well as co-acetylation of five lysine residues of H3 with H4 Lys12 and H2A Lys7 responsible for ribosome biogenesis. Furthermore, our method further suggested the recognition of acetylated H4 Lys16 being crucial to histone acetyltransferase ESA1, whose essential role is still under controversy, from a microarray dataset on ESA1 and its bypass suppressor mutants. These results demonstrate that our approach allows us to provide clearer principles behind gene regulation mechanisms under histone modifications and detect GHUs further by applying to other microarray and ChIP-chip datasets. The source code of our method, which was implemented in MATLAB (http://www.mathworks.com/), is available from the supporting page for this paper: http://www.bic.kyoto-u.ac.jp/pathway/natsume/hm_detector.htm.

Pioneer transcription factors have the ability to access DNA in compacted chromatin1. Multiple transcription factors can bind together to a regulatory element in a cooperative way, and cooperation between the pioneer transcription factors OCT4 (also known as POU5F1) and SOX2 is important for pluripotency and reprogramming2-4. However, the molecular mechanisms by which pioneer transcription factors function and cooperate on chromatin remain unclear. Here we present cryo-electron microscopy structures of human OCT4 bound to a nucleosome containing human LIN28B or nMATN1 DNA sequences, both of which bear multiple binding sites for OCT4. Our structural and biochemistry data reveal that binding of OCT4 induces changes to the nucleosome structure, repositions the nucleosomal DNA and facilitates cooperative binding of additional OCT4 and of SOX2 to their internal binding sites. The flexible activation domain of OCT4 contacts the N-terminal tail of histone H4, altering its conformation and thus promoting chromatin decompaction. Moreover, the DNA-binding domain of OCT4 engages with the N-terminal tail of histone H3, and post-translational modifications at H3K27 modulate DNA positioning and affect transcription factor cooperativity. Thus, our findings suggest that the epigenetic landscape could regulate OCT4 activity to ensure proper cell programming.