Scott B. Rothbart

An H3K36 Methylation-Engaging Tudor Motif of Polycomb-like Proteins Mediates PRC2 Complex Targeting

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 polycomb-like (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.

Therapeutics by Cytotoxic Metabolite Accumulation: Pemetrexed Causes ZMP Accumulation, AMPK Activation, and Mammalian Target of Rapamycin Inhibition

Pemetrexed represents the first antifolate cancer drug to be approved by the Food and Drug Administration in 20 years; it is currently in widespread use for first line therapy of mesothelioma and non-small cell lung cancer. Pemetrexed has more than one site of action; the primary site is thymidylate synthase. We now report that the secondary target is the downstream folate-dependent enzyme in de novo purine synthesis, aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART). The substrate of the AICART reaction, ZMP, accumulated in intact pemetrexed-inhibited tumor cells, identifying AICART as the step in purine synthesis that becomes rate-limiting after drug treatment. The accumulating ZMP causes an activation of AMP-activated protein kinase with subsequent inhibition of the mammalian target of rapamycin (mTOR) and hypophosphorylation of the downstream targets of mTOR that control initiation of protein synthesis and cell growth. We suggest that the activity of pemetrexed against human cancers is a reflection of its direct inhibition of folate-dependent target proteins combined with prolonged inhibition of the mTOR pathway secondary to accumulation of ZMP.

A Histone Methylation Network Regulates Transgenerational Epigenetic Memory in C. elegans

How epigenetic information is transmitted from generation to generation remains largely unknown. Deletion of the C. elegans histone H3 lysine 4 dimethyl (H3K4me2) demethylase spr-5 leads to inherited accumulation of the euchromatic H3K4me2 mark and progressive decline in fertility. Here, we identified multiple chromatin-modifying factors, including H3K4me1/me2 and H3K9me3 methyltransferases, an H3K9me3 demethylase, and an H3K9me reader, which either suppress or accelerate the progressive transgenerational phenotypes of spr-5 mutant worms. Our findings uncover a network of chromatin regulators that control the transgenerational flow of epigenetic information and suggest that the balance between euchromatic H3K4 and heterochromatic H3K9 methylation regulates transgenerational effects on fertility.

An Interactive Database for the Assessment of Histone Antibody Specificity

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.

Tandem PHD Fingers of MORF/MOZ Acetyltransferases Display Selectivity for Acetylated Histone H3 and Are Required for the Association with Chromatin

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.

Molecular basis for chromatin binding and regulation of MLL5

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.

Neuronal Stress Pathway Mediating a Histone Methyl/Phospho Switch Is Required for Herpes Simplex Virus Reactivation

Herpes simplex virus (HSV) reactivation from latent neuronal infection requires stimulation of lytic gene expression from promoters associated with repressive heterochromatin. Various neuronal stresses trigger reactivation, but how these stimuli activate silenced promoters remains unknown. We show that a neuronal pathway involving activation of c-Jun N-terminal kinase (JNK), common to many stress responses, is essential for initial HSV gene expression during reactivation. This JNK activation in neurons is mediated by dual leucine zipper kinase (DLK) and JNK-interacting protein 3 (JIP3), which direct JNK toward stress responses instead of other cellular functions. Surprisingly, JNK-mediated viral gene induction occurs independently of histone demethylases that remove repressive lysine modifications. Rather, JNK signaling results in a histone methyl/phospho switch on HSV lytic promoters, a mechanism permitting gene expression in the presence of repressive lysine methylation. JNK is present on viral promoters during reactivation, thereby linking a neuronal-specific stress pathway and HSV reactivation from latency.

Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1

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.

Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies

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.

Hemi-methylated DNA regulates DNA methylation inheritance through allosteric activation of H3 ubiquitylation by UHRF1.

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