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Complex organisms can rapidly induce select genes in response to diverse environmental cues. This regulation occurs in the context of large genomes condensed by histone proteins into chromatin. The sensing of pathogens by macrophages engages conserved signalling pathways and transcription factors to coordinate the induction of inflammatory genes1–3. Enriched integration of histone H3.3, the ancestral histone H3 variant, is a general feature of dynamically regulated chromatin and transcription4–7. However, how chromatin is regulated at induced genes, and what features of H3.3 might enable rapid and high-level transcription, are unknown. The amino terminus of H3.3 contains a unique serine residue (Ser31) that is absent in ‘canonical’ H3.1 and H3.2. Here we show that this residue, H3.3S31, is phosphorylated (H3.3S31ph) in a stimulation-dependent manner along rapidly induced genes in mouse macrophages. This selective mark of stimulation-responsive genes directly engages the histone methyltransferase SETD2, a component of the active transcription machinery, and ‘ejects’ the elongation corepressor ZMYND118,9. We propose that features of H3.3 at stimulation-induced genes, including H3.3S31ph, provide preferential access to the transcription apparatus. Our results indicate dedicated mechanisms that enable rapid transcription involving the histone variant H3.3, its phosphorylation, and both the recruitment and the ejection of chromatin regulators. The histone variant H3.3 is phosphorylated at Ser31 in induced genes, and this selective mark stimulates the histone methyltransferase SETD2 and ejects the ZMYND11 repressor, thus revealing a role for histone phosphorylation in amplifying de novo transcription.

Background SWI/SNF (BAF) chromatin remodeling complexes regulate lineage-specific enhancer activity by promoting accessibility for diverse DNA-binding factors and chromatin regulators. Additionally, they are known to modulate the function of the epigenome through regulation of histone post-translational modifications and nucleosome composition, although the way SWI/SNF complexes govern the epigenome remains poorly understood. Here, we investigate the function of ARID1A, a subunit of certain mammalian SWI/SNF chromatin remodeling complexes associated with malignancies and benign diseases originating from the uterine endometrium. Results Through genome-wide analysis of human endometriotic epithelial cells, we show that more than half of ARID1A binding sites are marked by the variant histone H3.3, including active regulatory elements such as super-enhancers. ARID1A knockdown leads to H3.3 depletion and gain of canonical H3.1/3.2 at ARID1A-bound active regulatory elements, and a concomitant redistribution of H3.3 toward genic elements. ARID1A interactions with the repressive chromatin remodeler CHD4 (NuRD) are associated with H3.3, and ARID1A is required for CHD4 recruitment to H3.3. ZMYND8 interacts with CHD4 to suppress a subset of ARID1A, CHD4, and ZMYND8 co-bound, H3.3+ H4K16ac+ super-enhancers near genes governing extracellular matrix, motility, adhesion, and epithelial-to-mesenchymal transition. Moreover, these gene expression alterations are observed in human endometriomas. Conclusions These studies demonstrate that ARID1A-containing BAF complexes are required for maintenance of the histone variant H3.3 at active regulatory elements, such as super-enhancers, and this function is required for the physiologically relevant activities of alternative chromatin remodelers.

The Su(var)3–9, enhancer of zeste, and trithorax (SET) and really interesting new gene (RING) finger–associated (SRA) protein domain is conserved across bacteria and eukaryota and coordinates extrahelical or “flipped” DNA bases. A functional SRA domain is required for ubiquitin-like with PHD and RING finger domains 1 (UHRF1) E3 ubiquitin ligase activity toward histone H3, a mechanism for recruiting the DNA methylation maintenance enzyme DNA methyltransferase 1 (DNMT1). The SRA domain supports UHRF1 oncogenic activity in colon cancer cells, highlighting that UHRF1 SRA antagonism could be a cancer therapeutic strategy. Here we used molecular dynamics simulations, DNA binding assays, in vitro ubiquitination reactions, and DNA methylation analysis to identify the SRA finger loop as a regulator of UHRF1 ubiquitin targeting and DNA methylation maintenance. A chimeric UHRF1 (finger swap) with diminished E3 ligase activity toward nucleosomal histones, despite tighter binding to unmodified or asymmetric or symmetrically methylated DNA, uncouples DNA affinity from regulation of E3 ligase activity. Our model suggests that SRA domains sample DNA bases through flipping in the presence or absence of a cytosine modification and that specific interactions of the SRA finger loop with DNA are required for downstream host protein function. Our findings provide insight into allosteric regulation of UHRF1 E3 ligase activity, suggesting that UHRF1's SRA finger loop regulates its conformation and function.

International audience; Landmark discoveries made nearly two decades ago identified known transcriptional regulators as histone lysine methyltransferases. Since then, the field of lysine methylation signaling has been dominated by studies of how this small chemical posttranslational modification regulates gene expression and other chromatin-based processes. However, recent advances in mass-spectrometry-based proteomics have revealed that histones are just a subset of the thousands of eukaryotic proteins marked by lysine methylation. As the writers, erasers, and readers of histone lysine methylation are emerging as a promising therapeutic target class for cancer and other diseases, a key challenge for the field is to define the full spectrum of activities for these proteins. Here we summarize recent discoveries implicating non-histone lysine methylation as a major regulator of diverse cellular processes. We further discuss recent technological innovations that are enabling the expanded study of lysine methylation signaling. Collectively, these findings are shaping our understanding of the fundamental mechanisms of non-histone protein regulation through this dynamic and multi-functional posttranslational modification.

Purpose:The successful clinical translation of compounds that target specific oncogenic transcription factors will require an understanding of the mechanism of target suppression to optimize the dose and schedule of administration. We have previously shown trabectedin reverses the gene signature of the EWS-FLI1 transcription factor. In this report, we establish the mechanism of suppression and use it to justify the reevaluation of this drug in the clinic in patients with Ewing sarcoma.Experimental Design: We demonstrate a novel epigenetic mechanism of trabectedin using biochemical fractionation and chromatin immunoprecipitation sequencing. We link the effect to drug schedule and EWS-FLI1 downstream target expression using confocal microscopy, qPCR, Western blot analysis, and cell viability assays. Finally, we quantitate target suppression within the three-dimensional architecture of the tumor in vivo using 18F-FLT imaging.Results:Trabectedin evicts the SWI/SNF chromatin-remodeling complex from chromatin and redistributes EWS-FLI1 in the nucleus leading to a marked increase in H3K27me3 and H3K9me3 at EWS-FLI1 target genes. These effects only occur at high concentrations of trabectedin leading to suppression of EWS-FLI1 target genes and a loss of cell viability. In vivo, low-dose irinotecan is required to improve the magnitude, penetrance, and duration of target suppression in the three-dimensional architecture of the tumor leading to differentiation of the Ewing sarcoma xenograft into benign mesenchymal tissue.Conclusions:These data provide the justification to evaluate trabectedin in the clinic on a short infusion schedule in combination with low-dose irinotecan with 18F-FLT PET imaging in patients with Ewing sarcoma.

Mutations in thePHIP/BRWD2chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations inPHIPexpression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease., This study shows that the cell cycle E3 ubiquitin ligase APC/C is a regulator of several chromatin regulatory proteins, including the multivalent epigenetic reader and writer UHRF1. Perturbing UHRF1 ubiquitylation and degradation alters cell cycle and DNA methylation patterning, pointing to a key role for cell cycle degradation in shaping chromatin environments.

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.

Tazemetostat is the first epigenetic therapy to gain FDA approval in a solid tumor. This lysine methyltransferase inhibitor targets EZH2, the enzymatic subunit of the PRC2 transcriptional silencing complex. Tumors with mutations in subunits of the SWI/SNF chromatin remodeling complex, inclusive of most epithelioid sarcomas, are sensitive to EZH2 inhibition.

Significance DNA methylation and histone posttranslational modifications are key epigenetic marks that contribute to the fine-tuned regulation of gene expression and other chromatin-templated biological processes. Here, we build artificial chromatin templates and reveal key chromatin structural features and epigenetic marks that coordinately regulate the binding and enzymatic activity of the DNA methylation regulator UHRF1. Studying activities of epigenetic regulators in the context of defined chromatin templates, particularly for multidomain histone and DNA binding proteins such as UHRF1, is critical for understanding molecular mechanisms of epigenetic crosstalk and mechanics regulating epigenetic signaling, and for determining how epigenetic dysregulation contributes to human disease., Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.

UHRF1 is a histone- and DNA-binding E3 ubiquitin ligase that functions with DNMT1 to maintain mammalian DNA methylation. UHRF1 facilitates DNMT1 recruitment to replicating chromatin through a coordinated mechanism involving histone and DNA recognition and histone ubiquitination. UHRF2 shares structural homology with UHRF1, but surprisingly lacks functional redundancy to facilitate DNA methylation maintenance. Molecular mechanisms uncoupling UHRF2 from DNA methylation maintenance are poorly defined. Through comprehensive and comparative biochemical analysis of recombinant human UHRF1 and UHRF2 reader and writer activities, we reveal conserved modes of histone PTM recognition but divergent DNA binding properties. While UHRF1 and UHRF2 diverge in their affinities toward hemi-methylated DNA, we surprisingly show that both hemi-methylated and hemi-hydroxymethylated DNA oligonucleotides stimulate UHRF2 ubiquitin ligase activity toward histone H3 peptide substrates. This is the first example of an E3 ligase allosterically regulated by DNA hydroxymethylation. However, UHRF2 is not a productive histone E3 ligase toward purified mononucleosomes, suggesting UHRF2 has an intra-domain architecture distinct from UHRF1 that is conformationally constrained when bound to chromatin. Collectively, our studies reveal that uncoupling of UHRF2 from the DNA methylation maintenance program is linked to differences in the molecular readout of chromatin signatures that connect UHRF1 to ubiquitination of histone H3.

Bromodomains (BD) are conserved reader modules that bind acetylated lysine residues on histones. Although much has been learned regarding the in vitro properties of these domains, less is known about their function within chromatin complexes. SWI/SNF chromatin-remodeling complexes modulate transcription and contribute to DNA damage repair. Mutations in SWI/SNF subunits have been implicated in many cancers. Here we demonstrate that the BD of Caenorhabditis elegans SMARCA4/BRG1, a core SWI/SNF subunit, recognizes acetylated lysine 14 of histone H3 (H3K14ac), similar to its Homo sapiens ortholog. We identify the interactions of SMARCA4 with the acetylated histone peptide from a 1.29 Å-resolution crystal structure of the CeSMARCA4 BD–H3K14ac complex. Significantly, most of the SMARCA4 BD residues in contact with the histone peptide are conserved with other proteins containing family VIII bromodomains. Based on the premise that binding specificity is conserved among bromodomain orthologs, we propose that loop residues outside of the binding pocket position contact residues to recognize the H3K14ac sequence. CRISPR-Cas9-mediated mutations in the SMARCA4 BD that abolish H3K14ac binding in vitro had little or no effect on C. elegans viability or physiological function in vivo. However, combining SMARCA4 BD mutations with knockdown of the SWI/SNF accessory subunit PBRM-1 resulted in severe developmental defects in animals. In conclusion, we demonstrated an essential function for the SWI/SNF bromodomain in vivo and detected potential redundancy in epigenetic readers in regulating chromatin remodeling. These findings have implications for the development of small-molecule BD inhibitors to treat cancers and other diseases.

Summary: DNA methylation plays crucial roles in chromatin structure and gene expression. Aberrant DNA methylation patterns, including global hypomethylation and regional hypermethylation, are associated with cancer and implicated in oncogenic events. How DNA methylation is regulated in developmental and cellular processes and dysregulated in cancer is poorly understood. Here, we show that PRMT6, a protein arginine methyltransferase responsible for asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a), negatively regulates DNA methylation and that PRMT6 upregulation contributes to global DNA hypomethylation in cancer. Mechanistically, PRMT6 overexpression impairs chromatin association of UHRF1, an accessory factor of DNMT1, resulting in passive DNA demethylation. The effect is likely due to elevated H3R2me2a, which inhibits the interaction between UHRF1 and histone H3. Our work identifies a mechanistic link between protein arginine methylation and DNA methylation, which is disrupted in cancer. : Veland et al. find that PRMT6, an arginine methyltransferase responsible for histone H3 arginine 2 (H3R2) methylation, negatively regulates maintenance DNA methylation by impairing UHRF1 recruitment to chromatin. The authors also find that PRMT6 upregulation contributes to global DNA hypomethylation in cancer cells. Keywords: PRMT6, arginine methylation, UHRF1, DNMT1, DNA methylation, cancer

Histone methyltransferase MLL4 is centrally involved in transcriptional regulation and is often mutated in human diseases, including cancer and developmental disorders. MLL4 contains a catalytic SET domain that mono-methylates histone H3K4 and seven PHD fingers of unclear function. Here, we identify the PHD6 finger of MLL4 (MLL4-PHD6) as a selective reader of the epigenetic modification H4K16ac. The solution NMR structure of MLL4-PHD6 in complex with a H4K16ac peptide along with binding and mutational analyses reveal unique mechanistic features underlying recognition of H4K16ac. Genomic studies show that one third of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions in vivo and that MLL4 occupancy in a set of genomic targets depends on the acetyltransferase activity of MOF, a H4K16ac-specific acetyltransferase. The recognition of H4K16ac is conserved in the PHD7 finger of paralogous MLL3. Together, our findings reveal a previously uncharacterized acetyllysine reader and suggest that selective targeting of H4K16ac by MLL4 provides a direct functional link between MLL4, MOF and H4K16 acetylation., Histone methyltransferase MLL4 is a transcriptional regulator. Here the authors identify the PHD6 finger of MLL4 as a selective reader of the epigenetic modification H4K16ac and show that a subset of MLL4 chromatin binding sites overlap with H4K16ac-enriched regions, which depends on MOF activity.

Summary UHRF1 facilitates the establishment and maintenance of DNA methylation patterns in mammalian cells. The establishment domains are defined, including E3 ligase function, but the maintenance domains are poorly characterized. Here, we demonstrate that UHRF1 histone- and hemimethylated DNA binding functions, but not E3 ligase activity, maintain cancer-specific DNA methylation in human colorectal cancer (CRC) cells. Disrupting either chromatin reader activity reverses DNA hypermethylation, reactivates epigenetically silenced tumor suppressor genes (TSGs), and reduces CRC oncogenic properties. Moreover, an inverse correlation between high UHRF1 and low TSG expression tracks with CRC progression and reduced patient survival. Defining critical UHRF1 domain functions and its relationship with CRC prognosis suggests directions for, and value of, targeting this protein to develop therapeutic DNA demethylating agents.

MORC3 is linked to inflammatory myopathies and cancer, however the precise role of MORC3 in normal cell physiology and disease remains poorly understood. Here, we present detailed genetic, biochemical, and structural analyses of MORC3. We demonstrate that MORC3 is significantly upregulated in Down syndrome, and that genetic abnormalities in MORC3 are associated with cancer. The CW domain of MORC3 binds to the methylated histone H3K4 tail, and this interaction is essential for the recruitment of MORC3 to chromatin and accumulation in nuclear bodies. We show that MORC3 possesses intrinsic ATPase activity that requires DNA however is negatively regulated by CW, which interacts with the ATPase domain. Natively linked CW impedes binding of the ATPase domain to DNA, resulting in a decrease in the DNA-stimulated enzymatic activity. Collectively, our studies provide a molecular framework detailing newly identified functions of MORC3 and suggest that its modulation may contribute to human disease.

Histone acetylation levels are regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) that antagonistically control the overall balance of this post-translational modification. HDAC inhibitors (HDACi) are potent agents that disrupt this balance and are used clinically to treat diseases including cancer. Despite their use, little is known about their effects on chromatin regulators, particularly those that signal through lysine acetylation. We apply quantitative genomic and proteomic approaches to demonstrate that HDACi robustly increases a low-abundance histone 4 polyacetylation state, which serves as a preferred binding substrate for several bromodomain-containing proteins, including BRD4. Increased H4 polyacetylation occurs in transcribed genes and correlates with the targeting of BRD4. Collectively, these results suggest that HDAC inhibition functions, at least in part, through expansion of a rare histone acetylation state, which then retargets lysine-acetyl readers associated with changes in gene expression, partially mimicking the effect of bromodomain inhibition.

Mapping lysine methyltransferase substrate selectivity reveals gaps in the proteome-wide curation of lysine methylomes., Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.

DNA methylation by de novo DNA methyltransferases 3A (DNMT3A) and 3B (DNMT3B) at cytosines is essential for genome regulation and development. Dysregulation of this process is implicated in various diseases, notably cancer. However, the mechanisms underlying DNMT3 substrate recognition and enzymatic specificity remain elusive. Here we report a 2.65-angstrom crystal structure of the DNMT3A-DNMT3L-DNA complex in which two DNMT3A monomers simultaneously attack two cytosine-phosphate-guanine (CpG) dinucleotides, with the target sites separated by 14 base pairs within the same DNA duplex. The DNMT3A-DNA interaction involves a target recognition domain, a catalytic loop, and DNMT3A homodimeric interface. Arg836 of the target recognition domain makes crucial contacts with CpG, ensuring DNMT3A enzymatic preference towards CpG sites in cells. Haematological cancer-associated somatic mutations of the substrate-binding residues decrease DNMT3A activity, induce CpG hypomethylation, and promote transformation of haematopoietic cells. Together, our study reveals the mechanistic basis for DNMT3A-mediated DNA methylation and establishes its aetiological link to human disease.

The key sensor of energy status in mammalian cells, AMP-activated protein kinase (AMPK), can also be activated by the AMP analog aminoimidazolecarboxamide nucleoside monophosphate (ZMP) generated directly from aminoimidazolecarboxamide ribonucleoside (AICAR) or from inhibition of purine synthesis by the antifolate pemetrexed (PTX), a drug used extensively in the treatment of lung cancers. Despite this common mechanism, signaling downstream of AMPK activated by PTX or AICAR differed. AICAR-activated AMPK inhibited mTORC1 both directly by phosphorylation of the mTORC1 subunit Raptor and indirectly by phosphorylation of the regulator TSC2. In contrast, PTX-activated AMPK inhibited mTORC1 solely through Raptor phosphorylation. This dichotomy was due to p53 function. Transcription of p53 target genes, including TSC2, was activated by AICAR but not by PTX. Although both PTX and AICAR stabilized p53, only AICAR activated Chk2 phosphorylation, stimulating p53-dependent transcription. However, Raptor phosphorylation by AMPK was independent of p53 and was sufficient, after PTX treatment, to inhibit mTORC1. We concluded that PTX effects on mTORC1 were independent of TSC2 and p53 and that the activation of a p53 transcriptional response by AICAR was due to an activation of Chk2 that was not elicited by PTX.

SummaryThe protein stability and chromatin functions of UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) are regulated in a cell-cycle-dependent manner. We report a structural characterization of the complex between UHRF1 and the deubiquitinase USP7. The first two UBL domains of USP7 bind to the polybasic region (PBR) of UHRF1, and this interaction is required for the USP7-mediated deubiquitination of UHRF1. Importantly, we find that the USP7-binding site of the UHRF1 PBR overlaps with the region engaging in an intramolecular interaction with the N-terminal tandem Tudor domain (TTD). We show that the USP7-UHRF1 interaction perturbs the TTD-PBR interaction of UHRF1, thereby shifting the conformation of UHRF1 from a TTD-“occluded” state to a state open for multivalent histone binding. Consistently, introduction of a USP7-interaction-defective mutation to UHRF1 significantly reduces its chromatin association. Together, these results link USP7 interaction to the dynamic deubiquitination and chromatin association of UHRF1.

Histone post-translational modifications (PTMs) are important genomic regulators often studied by chromatin immunoprecipitation (ChIP), whereby their locations and relative abundance are inferred by antibody capture of nucleosomes and associated DNA. However, the specificity of antibodies within these experiments have not been systematically studied. Here, we use histone peptide arrays and internally calibrated ChIP (ICeChIP) to characterize 52 commercial antibodies purported to distinguish the H3K4 methylforms (me1, me2, and me3, with each ascribed distinct biological functions). We find that many widely-used antibodies poorly distinguish the methylforms and that high- and low-specificity reagents can yield dramatically different biological interpretations, resulting in substantial divergence from the literature for numerous H3K4 methylform paradigms. Using ICeChIP, we also discern quantitative relationships between enhancer H3K4 methylation and promoter transcriptional output and can measure global PTM abundance changes. Our results illustrate how poor antibody specificity contributes to the “reproducibility crisis,” demonstrating the need for rigorous, platform-appropriate validation.

Mitosis encompasses key molecular changes including chromatin condensation, nuclear envelope breakdown, and reduced transcription levels. Immediately after mitosis, the interphase chromatin structure is reestablished and transcription resumes. The reestablishment of the interphase chromatin is probably achieved by ‘bookmarking’,i.e., the retention of at least partial information during mitosis. Yet, while recent studies demonstrate that chromatin accessibility is generally preserved during mitosis and is only locally modulated, the exact details of the bookmarking process and its components are still unclear. To gain a deeper understanding of the mitotic bookmarking process, we merged proteomics, immunofluorescence, and ChIP-seq approaches to study the mitotic and interphase genomic organization of human cells. We focused on key histone modifications and employed HeLa-S3 cells as a model system. Generally, we observed a global concordance between the genomic organization of histone modifications in interphase and mitosis, yet the abundance of the two types of modifications we investigated was different. Whereas histone methylation patterns remain highly similar, histone acetylation patterns show a general reduction while maintaining their genomic organization. These results demonstrate that the epigenomic landscape can serve as a major component of the mitotic bookmarking process. Next, to further investigate mitosis-associated chromatin changes, we followed up on previous studies that showed that nucleosome depleted regions (NDRs) become occupied by a nucleosome during mitosis. Surprisingly, we observed that the nucleosome introduced into the NDR during mitosis encompasses a distinctive set of histone modifications, differentiating it from the surrounding nucleosomes. We show that the nucleosomes near the NDR appear to both shift into the NDR during mitosis and adopt a unique modification pattern. HDAC inhibition by the small molecule TSA reverts this pattern. These results provide evidence for a mitotic deposition and change in the modifications of the nucleosomes surrounding the NDR. Altogether, by merging multiple approaches, our study provides evidence to support a model where mitotic bookmarking is achieved by histone modifications and uncovers new insights into the deposition of nucleosomes during mitosis.

Rice D53 repression motif links two sites of TPL corepressors to mediate TPL oligomerization and nucleosome association., TOPLESS are tetrameric plant corepressors of the conserved Tup1/Groucho/TLE (transducin-like enhancer of split) family. We show that they interact through their TOPLESS domains (TPDs) with two functionally important ethylene response factor–associated amphiphilic repression (EAR) motifs of the rice strigolactone signaling repressor D53: the universally conserved EAR-3 and the monocot-specific EAR-2. We present the crystal structure of the monocot-specific EAR-2 peptide in complex with the TOPLESS-related protein 2 (TPR2) TPD, in which the EAR-2 motif binds the same TPD groove as jasmonate and auxin signaling repressors but makes additional contacts with a second TPD site to mediate TPD tetramer-tetramer interaction. We validated the functional relevance of the two TPD binding sites in reporter gene assays and in transgenic rice and demonstrate that EAR-2 binding induces TPD oligomerization. Moreover, we demonstrate that the TPD directly binds nucleosomes and the tails of histones H3 and H4. Higher-order assembly of TPD complexes induced by EAR-2 binding markedly stabilizes the nucleosome-TPD interaction. These results establish a new TPD-repressor binding mode that promotes TPD oligomerization and TPD-nucleosome interaction, thus illustrating the initial assembly of a repressor-corepressor-nucleosome complex.

SUMMARY A role for cancer cell epithelial-to-mesenchymal transition (EMT) in cancer is well established. Here, we show that, in addition to cancer cell EMT, ovarian cancer cell metastasis relies on an epigenomic mesenchymal-to-epithelial transition (MET) in host mesenchymal stem cells (MSCs). These reprogrammed MSCs, termed carcinoma-associated MSCs (CA-MSCs), acquire pro-tumorigenic functions and directly bind cancer cells to serve as a metastatic driver/chaperone. Cancer cells induce this epigenomic MET characterized by enhancer-enriched DNA hypermethylation, altered chromatin accessibility, and differential histone modifications. This phenomenon appears clinically relevant, as CA-MSC MET is highly correlated with patient survival. Mechanistically, mirroring MET observed in development, MET in CA-MSCs is mediated by WT1 and EZH2. Importantly, EZH2 inhibitors, which are clinically available, significantly inhibited CA-MSC-mediated metastasis in mouse models of ovarian cancer., Graphical Abstract, In Brief Fan et al. demonstrate that ovarian cancer reprograms the epigenome of stromal cells, inducing a mesenchymal-to-epithelial transition (MET) to form carcinoma-associated mesenchymal stem cells (CA-MSCs). This MET, mediated by WT1 and EZH2, enables CA-MSC:cancer cell co-metastasis. EZH2 inhibition decreases CA-MSC-mediated metastasis, presenting a potential therapeutic opportunity in ovarian cancer.

A major mechanism regulating the accessibility and function of eukaryotic genomes are the covalent modifications to DNA and histone proteins that dependably package our genetic information inside the nucleus of every cell. Formally postulated over a decade ago, it is becoming increasingly clear that post-translational modifications (PTMs) on histones act singly and in combination to form a language or 'code' that is read by specialized proteins to facilitate downstream functions in chromatin. Underappreciated at the time was the level of complexity harbored both within histone PTMs and their combinations, as well as within the proteins that read and interpret the language. In addition to histone PTMs, newly-identified DNA modifications that can recruit specific effector proteins have raised further awareness that histone PTMs operate within a broader language of epigenetic modifications to orchestrate the dynamic functions associated with chromatin. Here, we highlight key recent advances in our understanding of the epigenetic language encompassing histone and DNA modifications and foreshadow challenges that lie ahead as we continue our quest to decipher the fundamental mechanisms of chromatin regulation. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

Chromatin-modifying enzymes are predominantly nuclear; however, these factors are also localized to the cytoplasm, and very little is known about their role in this compartment. In this report, we reveal a non-chromatin-linked role for the lysine-specific demethylase KDM4A. We demonstrate that KDM4A interacts with the translation initiation complex and affects the distribution of translation initiation factors within polysome fractions. Furthermore, KDM4A depletion reduced protein synthesis and enhanced the protein synthesis suppression observed with mTOR inhibitors, which paralleled an increased sensitivity to these drugs. Finally, we demonstrate that JIB-04, a JmjC demethylase inhibitor, suppresses translation initiation and enhances mTOR inhibitor sensitivity. These data highlight an unexpected cytoplasmic role for KDM4A in regulating protein synthesis and suggest novel potential therapeutic applications for this class of enzyme.This report documents an unexpected cytoplasmic role for the lysine demethylase KDM4A. We demonstrate that KDM4A interacts with the translation initiation machinery, regulates protein synthesis and, upon coinhibition with mTOR inhibitors, enhances the translation suppression and cell sensitivity to these therapeutics.

The human mixed-lineage leukemia 5 (MLL5) protein mediates hematopoietic cell homeostasis, cell cycle, and survival; however, the molecular basis underlying MLL5 activities remains unknown. Here, we show that MLL5 is recruited to gene-rich euchromatic regions via the interaction of its plant homeodomain finger with the histone mark H3K4me3. The 1.48-Å resolution crystal structure of MLL5 plant homeodomain in complex with the H3K4me3 peptide reveals a noncanonical binding mechanism, whereby K4me3 is recognized through a single aromatic residue and an aspartate. The binding induces a unique His–Asp swapping rearrangement mediated by a C-terminal α-helix. Phosphorylation of H3T3 and H3T6 abrogates the association with H3K4me3 in vitro and in vivo, releasing MLL5 from chromatin in mitosis. This regulatory switch is conserved in the Drosophila ortholog of MLL5, UpSET, and suggests the developmental control for targeting of H3K4me3. Together, our findings provide first insights into the molecular basis for the recruitment, exclusion, and regulation of MLL5 at chromatin.

Histone post-translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains. While significant progress has been made characterizing individual effector domains, the role of paired domains and how they function in a combinatorial fashion within chromatin are poorly defined. Here we show that the linked tandem Tudor and plant homeodomain (PHD) of UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1) operates as a functional unit in cells, providing a defined combinatorial readout of a heterochromatin signature within a single histone H3 tail that is essential for UHRF1-directed epigenetic inheritance of DNA methylation. These findings provide critical support for the “histone code” hypothesis, demonstrating that multivalent histone engagement plays a key role in driving a fundamental downstream biological event in chromatin.

Polycomb repressive complex 2 (PRC2) regulates pluripotency, differentiation and tumorigenesis through catalysis of histone H3 lysine 27 trimethylation (H3K27me3) on chromatin. However, the mechanisms that underlie PRC2 recruitment and spreading on chromatin remain unclear. Here we report that histone H3 lysine 36 trimethylation (H3K36me3)-binding activity is harbored in the Tudor motifs of PRC2-associated polycomblike (PCL) proteins PHF1/PCL1 and PHF19/PCL3. Ectopically expressed PHF1 induced Tudor-dependent stabilization of PRC2 complexes on bulk chromatin and mediated spreading of PRC2 and H3K27me3 into H3K36me3-containing chromatin regions. In murine pluripotent stem cells, we identified coexistence of H3K36me3, H3K27me3, and PHF19/PCL3 at a subset of ‘poised’ developmental genes, and demonstrated that PHF19/PCL3 Tudor function is required for optimal H3K27me3 and repression of these loci. Collectively, our data suggest that PCL recognition of H3K36me3 promotes intrusion of PRC2 complexes into active chromatin regions to promote gene silencing and modulate the chromatin landscape during development.

BackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: they are non-renewable, vary in performance between lots, and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform as well as polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

The epigenetic inheritance of DNA methylation requires UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that recruits DNMT1 to sites of newly replicated DNA through ubiquitylation of histone H3. UHRF1 binds DNA with selectivity towards hemi-methylated CpGs (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. HeDNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies are the first demonstrations of a DNA-protein interaction and an epigenetic modification directly regulating E3 ubiquitin ligase activity. They also define an orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance. DOI: http://dx.doi.org/10.7554/eLife.17101.001, eLife digest Cells are able to regulate the activity of their genes in response to different cues. Genetic information is encoded in DNA and one way to regulate gene activity is to modify the DNA by attaching chemical “epigenetic” markers to it. When a cell divides, these epigenetic markers can be inherited by the daughter cells so that they share the same patterns of gene activity as the parent cell. When the DNA of the parent cell is copied prior to cell division, the epigenetic markers are also copied onto the new DNA. Mistakes in this process are linked to a wide range of diseases in humans, such as cancer and neurological disorders. One type of epigenetic marker is known as a methyl tag and it is added to DNA by certain enzymes in a process called DNA methylation. A protein called UHRF1 is required for human cells to inherit patterns of DNA methylation through cell division. This protein binds to newly copied DNA that lacks some methyl tags as well as to another protein associated with DNA called histone H3. UHRF1 modifies histone H3 by attaching a small protein molecule called ubiquitin to it. This helps to recruit a DNA methylation enzyme to place methyl tags on the newly copied DNA. However, it was not clear how the various properties of UHRF1 allow it to control how DNA methylation is inherited. Harrison et al. addressed this question by studying purified proteins and DNA fragments outside of living cells. The results show that UHRF1 binding to DNA and histone H3 work together to bring UHRF1 to the sites on DNA that require methylation. Further experiments revealed that the methylation pattern on newly copied DNA is able to activate the ability of UHRF1 to place ubiquitin on histone H3. The findings of Harrison et al. reveal a new mechanism by which dividing cells control how DNA methylation is inherited by their daughter cells. A future challenge will be to find out how attaching ubiquitin to histone H3 activates DNA methylation. DOI: http://dx.doi.org/10.7554/eLife.17101.002

G9a and GLP lysine methyltransferases form a heterodimeric complex that is responsible for the majority of histone H3 lysine 9 mono- and di-methylation (H3K9me1/me2). Widely interspaced zinc finger (WIZ) associates with the G9a-GLP protein complex, but its role in mediating lysine methylation is poorly defined. Here, we show that WIZ regulates global H3K9me2 levels by facilitating the interaction of G9a with chromatin. Disrupting the association of G9a-GLP with chromatin by depleting WIZ resulted in altered gene expression and protein-protein interactions that were distinguishable from that of small molecule-based inhibition of G9a/GLP, supporting discrete functions of the G9a-GLP-WIZ chromatin complex in addition to H3K9me2 methylation.

Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.

Access to high-quality antibodies is a necessity for the study of histones and their posttranslational modifications (PTMs). Here we debut the Histone Antibody Specificity Database (http://www.histoneantibodies.com), an online and expanding resource cataloging the behavior of widely used, commercially available histone antibodies by peptide microarray. This interactive web portal provides a critical resource to the biological research community that routinely uses these antibodies as detection reagents for a wide range of applications.

SummaryIndividual posttranslational modifications (PTMs) of p53 mediate diverse p53-dependent responses; however, much less is known about the combinatorial action of adjacent modifications. Here, we describe crosstalk between the early DNA damage response mark p53K382me2 and the surrounding PTMs that modulate binding of p53 cofactors, including 53BP1 and p300. The 1.8 Å resolution crystal structure of the tandem Tudor domain (TTD) of 53BP1 in complex with p53 peptide acetylated at K381 and dimethylated at K382 (p53K381acK382me2) reveals that the dual PTM induces a conformational change in p53. The α-helical fold of p53K381acK382me2 positions the side chains of R379, K381ac, and K382me2 to interact with TTD concurrently, reinforcing a modular design of double PTM mimetics. Biochemical and nuclear magnetic resonance analyses show that other surrounding PTMs, including phosphorylation of serine/threonine residues of p53, affect association with TTD. Our findings suggest a novel PTM-driven conformation switch-like mechanism that may regulate p53 interactions with binding partners.

SummaryThe histone lysine demethylase KDM5B regulates gene transcription and cell differentiation and is implicated in carcinogenesis. It contains multiple conserved chromatin-associated domains, including three PHD fingers of unknown function. Here, we show that the first and third, but not the second, PHD fingers of KDM5B possess histone binding activities. The PHD1 finger is highly specific for unmodified histone H3 (H3K4me0), whereas the PHD3 finger shows preference for the trimethylated histone mark H3K4me3. RNA-seq analysis indicates that KDM5B functions as a transcriptional repressor for genes involved in inflammatory responses, cell proliferation, adhesion, and migration. Biochemical analysis reveals that KDM5B associates with components of the nucleosome remodeling and deacetylase (NuRD) complex and may cooperate with the histone deacetylase 1 (HDAC1) in gene repression. KDM5B is downregulated in triple-negative breast cancer relative to estrogen-receptor-positive breast cancer. Overexpression of KDM5B in the MDA-MB 231 breast cancer cells suppresses cell migration and invasion, and the PHD1-H3K4me0 interaction is essential for inhibiting migration. These findings highlight tumor-suppressive functions of KDM5B in triple-negative breast cancer cells and suggest a multivalent mechanism for KDM5B-mediated transcriptional regulation.

In this communication we introduce an efficient implementation of adaptive biasing that greatly improves the speed of free energy computation in molecular dynamics simulations. We investigated the use of accelerated simulations to inform on compound design using a recently reported and clinically relevant inhibitor of the chromatin regulator BRD4 (bromodomain-containing protein 4). Benchmarking on our local compute cluster, our implementation achieves up to 2.5 times more force calls per day than plumed2. Results of five 1 μs-long simulations are presented, which reveal a conformational switch in the BRD4 inhibitor between a binding competent and incompetent state. Stabilization of the switch led to a −3 kcal/mol improvement of absolute binding free energy. These studies suggest an unexplored ligand design principle and offer new actionable hypotheses for medicinal chemistry efforts against this druggable epigenetic target class.

Histone posttranslational modifications (PTMs) play a pivotal role in regulating the dynamics and function of chromatin. Supported by an increasing body of literature, histone PTMs such as methylation and acetylation function together in the context of a “histone code,” which is read, or interpreted, by effector proteins that then drive a functional output in chromatin (e.g., gene transcription). A growing number of domains that interact with histones and/or their PTMs have been identified. While significant advances have been made in our understanding of how these domains interact with histones, a wide number of putative histone-binding motifs have yet to be characterized, and undoubtedly, novel domains will continue to be discovered. In this chapter, we provide a detailed method for the construction of combinatorially modified histone peptides, microarray fabrication using these peptides, and methods to characterize the interaction of effector proteins, antibodies, and the substrate specificity of histone-modifying enzymes. We discuss these methods in the context of other available technologies and provide a user-friendly approach to enable the exploration of histone–protein–enzyme interactions and function.

A fundamental challenge in mammalian biology has been the elucidation of mechanisms linking DNA methylation and histone post-translational modifications. Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has multiple domains that bind chromatin, and it is implicated genetically in the maintenance of DNA methylation. However, molecular mechanisms underlying DNA methylation regulation by UHRF1 are poorly defined. Here we show that UHRF1 association with methylated histone H3 Lys9 (H3K9) is required for DNA methylation maintenance. We further show that UHRF1 association with H3K9 methylation is insensitive to adjacent H3 S10 phosphorylation--a known mitotic 'phospho-methyl switch'. Notably, we demonstrate that UHRF1 mitotic chromatin association is necessary for DNA methylation maintenance through regulation of the stability of DNA methyltransferase-1. Collectively, our results define a previously unknown link between H3K9 methylation and the faithful epigenetic inheritance of DNA methylation, establishing a notable mitotic role for UHRF1 in this process.

Antibodies specific for histone post-translational modifications (PTMs) have been central to our understanding of chromatin biology. Here, we describe an unexpected and novel property of histone H4 site-specific acetyl antibodies in that they prefer poly-acetylated histone substrates. By all current criteria, these antibodies have passed specificity standards. However, we find these site-specific histone antibodies preferentially recognize chromatin signatures containing two or more adjacent acetylated lysines. Significantly, we find that the poly-acetylated epitopes these antibodies prefer are evolutionarily conserved and are present at levels that compete for these antibodies over the intended individual acetylation sites. This alarming property of acetyl-specific antibodies has far-reaching implications for data interpretation and may present a challenge for the future study of acetylated histone and non-histone proteins.

MORF [MOZ (monocytic leukemia zinc-finger protein)-related factor] and MOZ are catalytic subunits of histone acetyltransferase (HAT) complexes essential in hematopoiesis, neurogenesis, skeletogenesis and other developmental programs and implicated in human leukemias. The canonical HAT domain of MORF/MOZ is preceded by a tandem of plant homeodomain (PHD) fingers whose biological roles and requirements for MORF/MOZ activity are unknown. Here, we demonstrate that the tandem PHD1/2 fingers of MORF recognize the N-terminal tail of histone H3. Acetylation of Lys9 (H3K9ac) or Lys14 (H3K14ac) enhances binding of MORF PHD1/2 to unmodified H3 peptides twofold to threefold. The selectivity for acetylated H3 tail is conserved in the double PHD1/2 fingers of MOZ. This interaction requires the intact N-terminus of histone H3 and is inhibited by trimethylation of Lys4. Biochemical analysis using NMR, fluorescence spectroscopy and mutagenesis identified key amino acids of MORF PHD1/2 necessary for the interaction with histones. Fluorescence microscopy and immunoprecipitation experiments reveal that both PHD fingers are required for binding to H3K14ac in vivo and localization to chromatin. The HAT assays indicate that the interaction with H3K14ac may promote enzymatic activity in trans. Together, our data suggest that the PHD1/2 fingers play a role in MOZ/MORF HATs association with the chromatic regions enriched in acetylated marks.

The chemotherapeutic drug pemetrexed, an inhibitor of thymidylate synthase, has an important secondary target in human leukemic cells, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of purine biosynthesis. The purine intermediate aminoimidazolecarboxamide ribonucleotide (ZMP), which accumulates behind this block, transmits an inhibitory signal to the mTORC1 complex via activation of the cellular energy sensor AMP-activated kinase (AMPK). Given that the PI3K-AKT-mTOR pathway is frequently deregulated during carcinogenesis, we asked whether the indirect activation of AMPK by pemetrexed offers an effective therapeutic strategy for carcinomas with defects in this pathway. Activation of AMPK by ZMP in pemetrexed-treated colon and lung carcinoma cells and the downstream consequences of this activation were strikingly more robust than previously seen in leukemic cells. Genetic experiments demonstrated the intermediacy of AICART inhibition and the centrality of AMPK activation in these effects. Whereas AMPK activation resulted in marked inhibition of mTORC1, other targets of AMPK were phosphorylated that were not mTORC1-dependent. Whereas AMPK activation is thought to require AMPKα T172 phosphorylation, pemetrexed also activated AMPK in carcinoma cells null for LKB1, the predominant AMPKα T172 kinase whose deficiency is common in lung adenocarcinomas. Like rapamycin analogs, pemetrexed relieved feedback suppression of PI3K and AKT, but the prolonged accumulation of unphosphorylated 4E-BP1, a tight-binding inhibitor of cap-dependent translation, was seen following AMPK activation. Our findings indicate that AMPK activation by pemetrexed inhibits mTORC1-dependent and -independent processes that control translation and lipid metabolism, identifying pemetrexed as a targeted therapeutic agent for this pathway that differs significantly from rapamycin analogs. Cancer Res; 70(24); 10299–309. ©2010 AACR.

Summaryp53 is dynamically regulated through various posttranslational modifications (PTMs), which differentially modulate its function and stability. The dimethylated marks p53K370me2 and p53K382me2 are associated with p53 activation or stabilization and both are recognized by the tandem Tudor domain (TTD) of 53BP1, a p53 cofactor. Here we detail the molecular mechanisms for the recognition of p53K370me2 and p53K382me2 by 53BP1. The solution structures of TTD in complex with the p53K370me2 and p53K382me2 peptides show a remarkable plasticity of 53BP1 in accommodating these diverse dimethyllysine-containing sequences. We demonstrate that dimeric TTDs are capable of interacting with the two PTMs on a single p53K370me2K382me2 peptide, greatly strengthening the 53BP1-p53 interaction. Analysis of binding affinities of TTD toward methylated p53 and histones reveals strong preference of 53BP1 for p53K382me2, H4K20me2, and H3K36me2 and suggests a possible role of multivalent contacts of 53BP1 in p53 targeting to and accumulation at the sites of DNA damage.

Death Inducer Obliterator 3 (Dido3) is implicated in the maintenance of stem cell genomic stability and tumorigenesis. Here, we show that Dido3 regulates the expression of stemness genes in embryonic stem cells through its plant homeodomain (PHD) finger. Binding of Dido3 PHD to histone H3K4me3 is disrupted by threonine phosphorylation that triggers Dido3 translocation from chromatin to the mitotic spindle. The crystal structure of Dido3 PHD in complex with H3K4me3 reveals an atypical aromatic-cage-like binding site that contains a histidine residue. Biochemical, structural, and mutational analyses of the binding mechanism identified the determinants of specificity and affinity and explained the inability of homologous PHF3 to bind H3K4me3. Together, our findings reveal a link between the transcriptional control in embryonic development and regulation of cell division., This research is supported by NIH grants GM096863 and GM101664 (to T.G.K.), GM068088 (to B.D.S.), and HL65440 (to M.G.) and Spanish government grants PS09/00572 (to K.H.M.v.W) and SAF2010-21295, PIB2010BZ-00564, and MITIC (S2010/BMD-2502) (to C.M.A.). K.H.M.v.W. is supported by a JAE-doc fellowship from the Spanish National Research Council (CSIC). S.B.R. is supported by the UNC Lineberger Comprehensive Cancer Center Basic Sciences Training Program (T32CA09156) and a Postdoctoral Fellowship from the American Cancer Society (PF-13-085-01-DMC). Financial support for NSLS comes principally from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy and from the National Center for Research Resources of the National Institutes of Health (NIH).

Background Histone posttranslational modifications (PTMs) function to regulate chromatin structure and function in part through the recruitment of effector proteins that harbor specialized “reader” domains. Despite efforts to elucidate reader domain–PTM interactions, the influence of neighboring PTMs and the target specificity of many reader domains is still unclear. The aim of this study was to use a high-throughput histone peptide microarray platform to interrogate 83 known and putative histone reader domains from the chromo and Tudor domain families to identify their interactions and characterize the influence of neighboring PTMs on these interactions. Results Nearly a quarter of the chromo and Tudor domains screened showed interactions with histone PTMs by peptide microarray, revealing known and several novel methyllysine interactions. Specifically, we found that the CBX/HP1 chromodomains that recognize H3K9me also recognize H3K23me2/3—a poorly understood histone PTM. We also observed that, in addition to their interaction with H3K4me3, Tudor domains of the Spindlin family also recognized H4K20me3—a previously uncharacterized interaction. Several Tudor domains also showed novel interactions with H3K4me as well. Conclusions These results provide an important resource for the epigenetics and chromatin community on the interactions of many human chromo and Tudor domains. They also provide the basis for additional studies into the functional significance of the novel interactions that were discovered. Electronic supplementary material The online version of this article (doi:10.1186/s13072-017-0117-5) contains supplementary material, which is available to authorized users.