UpSET recruits HDAC complexes and restricts chromatin accessibility and acetylation at promoter regions.

Developmental gene expression results from the orchestrated interplay between genetic and epigenetic mechanisms. Here, we describe upSET, a transcriptional regulator encoding a SET domain-containing protein recruited to active and inducible genes in Drosophila. However, unlike other Drosophila SET proteins associated with gene transcription, UpSET is part of an Rpd3/Sin3-containing complex that restricts chromatin accessibility and histone acetylation to promoter regions. In the absence of UpSET, active chromatin marks and chromatin accessibility increase and spread to genic and flanking regions due to destabilization of the histone deacetylase complex. Consistent with this, transcriptional noise increases, as manifest by activation of repetitive elements and off-target genes. Interestingly, upSET mutant flies are female sterile due to upregulation of key components of Notch signaling during oogenesis. Thus UpSET defines a class of metazoan transcriptional regulators required to fine tune transcription by preventing the spread of active chromatin.

Molecular Insights into Inhibition of the Methylated Histone-Plant Homeodomain Complexes by Calixarenes.

Plant homeodomain (PHD) finger-containing proteins are implicated in fundamental biological processes, including transcriptional activation and repression, DNA damage repair, cell differentiation, and survival. The PHD finger functions as an epigenetic reader that binds to posttranslationally modified or unmodified histone H3 tails, recruiting catalytic writers and erasers and other components of the epigenetic machinery to chromatin. Despite the critical role of the histone-PHD interaction in normal and pathological processes, selective inhibitors of this association have not been well developed. Here we demonstrate that macrocyclic calixarenes can disrupt binding of PHD fingers to methylated lysine 4 of histone H3 in vitro and in vivo. The inhibitory activity relies on differences in binding affinities of the PHD fingers for H3K4me and the methylation state of the histone ligand, whereas the composition of the aromatic H3K4me-binding site of the PHD fingers appears to have no effect. Our approach provides a novel tool for studying the biological roles of methyllysine readers in epigenetic signaling.

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.

CASK (LIN2) interacts with Cx43 in wounded skin and their coexpression affects cell migration.

Vertebrate gap junctions are composed of proteins from the connexin family. Co-immunoprecipitation, in vitro binding and far western experiments demonstrate that mammalian CASK (also known as LIN2) directly interacts with Cx43. Immunoprecipitation studies indicate that the CASK mainly interacts with the hypophosphorylated form of Cx43. Functional co-regulation of these proteins was found in MDCK cells migrating into a scratch wound, where expression of either protein individually inhibits migration but their coexpression abrogates this inhibitory effect. Immunofluorescence shows colocalization of Cx43 and CASK in mouse brain astrocytes and in response to wounding in human foreskin. During wounding, CASK is mobilized to the plasma membrane where it colocalizes with Cx43 and CADM1 1 hour after skin explant wounding. Together, these studies indicate that CASK interaction with Cx43 occurs relatively early in the connexin life cycle and imply a plasma membrane targeting role for the interaction that apparently affects cellular processes including cellular migration and wound healing.

Chicken alpha-globin switching depends on autonomous silencing of the embryonic pi globin gene by epigenetics mechanisms.

Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

A CTCF-dependent silencer located in the differentially methylated area may regulate expression of a housekeeping gene overlapping a tissue-specific gene domain.

The tissue-specific chicken alpha-globin gene domain represents one of the paradigms, in terms of its constitutively open chromatin conformation and the location of several regulatory elements within the neighboring housekeeping gene. Here, we show that an 0.2-kb DNA fragment located approximately 4 kb upstream to the chicken alpha-globin gene cluster contains a binding site for the multifunctional protein factor CTCF and possesses silencer activity which depends on CTCF binding, as demonstrated by site-directed mutagenesis of the CTCF recognition sequence. CTCF was found to be associated with this recognition site in erythroid cells but not in lymphoid cells where the site is methylated. A functional promoter directing the transcription of the apparently housekeeping ggPRX gene was found 120 bp from the CTCF-dependent silencer. The data are discussed in terms of the hypothesis that the CTCF-dependent silencer stabilizes the level of ggPRX gene transcription in erythroid cells where the promoter of this gene may be influenced by positive cis-regulatory signals activating alpha-globin gene transcription.

Sequential chromatin immunoprecipitation protocol: ChIP-reChIP.

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.

Epigenetic regulation of the human retinoblastoma tumor suppressor gene promoter by CTCF.

Epigenetic misregulation is a more common feature in human cancer than previously anticipated. In the present investigation, we identified CCCTC-binding factor (CTCF), the multivalent 11-zinc-finger nuclear factor, as a regulator that favors a particular local chromatin conformation of the human retinoblastoma gene promoter. We show that its binding contributes to Rb gene promoter epigenetic stability. Ablation of the CTCF binding site from the human Rb gene promoter induced a rapid epigenetic silencing of reporter gene expression in an integrated genome context. CTCF DNA binding is methylation sensitive, and the methylated Rb-CTCF site is recognized by the Kaiso methyl-CpG-binding protein. This is the first evidence suggesting that CTCF protects the Rb gene promoter, a classic CpG island, against DNA methylation, and when such control region is abnormally methylated Kaiso, and probably its associated repressor complex, induce epigenetic silencing of the promoter. Our results identify CTCF as a novel epigenetic regulator of the human retinoblastoma gene promoter.

Determination of Chromatin Accessibility in Drosophila MidgutEnterocytes by in situ 5mC Labeling.

Regulation of gene expression involves dynamic changes in chromatin organization, where in many cases open chromatin structure correlates with gene activation. Several methods enable monitoring changes in chromatin accessibility, including ATAC-seq, FAIRE-seq, MNase-seq and DNAse-seq methods, which involve Next-generation-sequencing (NGS). Focusing on the adult Drosophila differentiated gut enterocytes (ECs) we used a sequencing-free method that enables visualizing and semi-quantifying large-scale changes in chromatin structure using in vitro methylation assay with the bacterial CpG Methyltransferase, M. Sssl, that determine chromatin accessibility. In brief, as CpG methylation is minimal in differentiated somatic Drosophila cells, we used the bacterial M. SssI enzyme to methylate CpG dinucleotides in situ depending on their chromatin accessibility. The methylated dinucleotides are detected using 5mCytosine monoclonal antibody and nuclei are visualized microscopically. Thus, the 5mC method enables to monitor large-scale chromatin changes in heterogenic cellular tissue focusing on the cell type of interest and without the need for cell purification or NGS.

The transcription factor Hey and nuclear lamins specify and maintain cell identity.

The inability of differentiated cells to maintain their identity is a hallmark of age-related diseases. We found that the transcription factor Hey supervises the identity of differentiated enterocytes (ECs) in the adult Drosophila midgut. Lineage tracing established that Hey-deficient ECs are unable to maintain their unique nuclear organization and identity. To supervise cell identity, Hey determines the expression of nuclear lamins, switching from a stem-cell lamin configuration to a differentiated lamin configuration. Moreover, continued Hey expression is required to conserve large-scale nuclear organization. During aging, Hey levels decline, and EC identity and gut homeostasis are impaired, including pathological reprograming and compromised gut integrity. These phenotypes are highly similar to those observed upon acute targeting of Hey or perturbation of lamin expression in ECs in young adults. Indeed, aging phenotypes were suppressed by continued expression of Hey in ECs, suggesting that a Hey-lamin network safeguards nuclear organization and differentiated cell identity.

YY1 and GATA-1 interaction modulate the chicken 3'-side alpha-globin enhancer activity.

Studying the chicken alpha-globin domain as a model system of gene regulation, we have previously identified contiguous silencer-enhancer elements located on the 3'-side of the domain. To better characterize the enhancer we performed a systematic functional analysis to define its expression influence range and the ubiquitous and stage-specific transcriptional regulators interacting with this control element. In contrast to previous reports, we found that, in addition to a core element that includes three GATA-1 binding sites, the enhancer incorporates a 120 base-pair DNA fragment where EKLF, NF-E2 and a fourth GATA-1 factor could interact. Functional experiments demonstrate that the enhancer activity over the adult alpha(D) promoter is differentially regulated. We found that the transcriptional factor Ying Yang 1 (YY1) binds to the 120 base-pair DNA fragment and its effect over the enhancer activity is GATA-1-dependent. In addition, we characterize a novel physical interaction between GATA-1 and YY1 that influences the enhancer function. Experiments using a histone deacetylation inhibitor indicate that, in pre-erythroblasts, the enhancer down-regulation could be influenced by a closed chromatin conformation. Our observations show that the originally defined enhancer possesses a more complex composition than previously assumed. We propose that its activity is modulated through differential nuclear factor interactions and chromatin modifications at distinct erythroid stages.

Prelamin A processing, accumulation and distribution in normal cells and laminopathy disorders.

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.

Wash interacts with lamin and affects global nuclear organization.

The cytoplasmic functions of Wiskott-Aldrich syndrome family (WAS) proteins are well established and include roles in cytoskeleton reorganization and membrane-cytoskeletal interactions important for membrane/vesicle trafficking, morphogenesis, immune response, and signal transduction. Misregulation of these proteins is associated with immune deficiency and metastasis [1-4]. Cytoplasmic WAS proteins act as effectors of Rho family GTPases and polymerize branched actin through the Arp2/3 complex [1, 5]. Previously, we identified Drosophila washout (wash) as a new member of the WAS family with essential cytoplasmic roles in early development [6, 7]. Studies in mammalian cells and Dictyostelium suggest that WASH functions primarily in a multiprotein complex that regulates endosome shape and trafficking in an Arp2/3-dependent manner [8-11]. However, roles for classically cytoplasmic proteins in the nucleus are beginning to emerge, in particular, as participants in the regulation of gene expression [12, 13]. Here, we show that Drosophila Wash is present in the nucleus, where it plays a key role in global nuclear organization. wash mutant and knockdown nuclei disrupt subnuclear structures/organelles and exhibit the abnormal wrinkled morphology reminiscent of those observed in diverse laminopathies [14-16]. We find that nuclear Wash interacts with B-type Lamin (Lamin Dm0), and, like Lamin, Wash associates with constitutive heterochromatin. Wash knockdown increases chromatin accessibility of repressive compartments and results in a global redistribution of repressive histone modifications. Thus, our results reveal a novel role for Wash in modulating nucleus morphology and in the organization of both chromatin and non-chromatin nuclear sub-structures.

An acetyl-methyl switch drives a conformational change in p53.

Individual 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.

Sustained heterologous transgene expression in mammalian and avian cell lines.

Chromosomal position effects are the cause of variegation of transgene expression relating to the eukaryotic genome environment. These effects modify heterologous transgene expression, presumably due to dominant epigenetic marks that cause variegated or gradual silencing of transgene expression. As a solution, chromosomal insulators have arisen as a way to protect transgene expression against repressive marks (position effects), allowing homogeneous and consistent expression. Sustainable expression enables us to gain insight into in vivo protein function and protein stability, or to study position effects. Here, we show that the chicken cHS4 beta-globin insulator is able to protect transgene expression in cell lines, demonstrating its potential as a tool to improve the heterologous expression of any desired transgene. In addition, we show that green fluorescent protein (GFP) can be used as a reporter protein in the study of chromosomal position effects.

The histone-H3K4-specific demethylase KDM5B binds to its substrate and product through distinct PHD fingers.

The 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.

UpSET-ting the balance: modulating open chromatin features in metazoan genomes.

Appropriate gene expression relies on the sophisticated interplay between genetic and epigenetic factors. Histone acetylation and an open chromatin configuration are key features of transcribed regions and are mainly present around active promoters. Our recent identification of the SET-domain containing protein UpSET established a new functional link between the modulation of open chromatin features and active recruitment of well-known co-repressors in metazoans. Structurally, the SET domain of UpSET resembles H3K4 and H3K36 methyltransferases; however, it is does not confer histone methyltransferase activity. Rather than methylating histones to regulate gene expression like other SET domain-containing proteins, UpSET fine-tunes transcription by modulating the chromatin structure around active promoters resulting in suppression of expression of off-target genes or nearby repetitive elements. Chromatin modulation by UpSET occurs in part through its interaction with histone deacetylases. Here, we discuss the different scenarios in which UpSET could play key roles in modulating gene expression.

Kinase-independent feedback of the TAK1/TAB1 complex on BCL10 turnover and NF-κB activation.

Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.

Dido3 PHD modulates cell differentiation and division.

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.

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes.

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC.