Showing papers on "Histone binding published in 2012"

Identification and Characterization of Small Molecule Inhibitors of a Plant Homeodomain Finger

Abstract: A number of histone-binding domains are implicated in cancer through improper binding of chromatin. In a clinically reported case of acute myeloid leukemia (AML), a genetic fusion protein between nucleoporin 98 and the third plant homeodomain (PHD) finger of JARID1A drives an oncogenic transcriptional program that is dependent on histone binding by the PHD finger. By exploiting the requirement for chromatin binding in oncogenesis, therapeutics targeting histone readers may represent a new paradigm in drug development. In this study, we developed a novel small molecule screening strategy that utilizes HaloTag technology to identify several small molecules that disrupt binding of the JARID1A PHD finger to histone peptides. Small molecule inhibitors were validated biochemically through affinity pull downs, fluorescence polarization, and histone reader specificity studies. One compound was modified through medicinal chemistry to improve its potency while retaining histone reader selectivity. Molecular modelin...

Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy

Abstract: Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.

Two surfaces on the histone chaperone Rtt106 mediate histone binding, replication, and silencing

Abstract: The histone chaperone Rtt106 binds histone H3 acetylated at lysine 56 (H3K56ac) and facilitates nucleosome assembly during several molecular processes. Both the structural basis of this modification-specific recognition and how this recognition informs Rtt106 function are presently unclear. Guided by our crystal structure of Rtt106, we identified two regions on its double-pleckstrin homology domain architecture that mediated histone binding. When histone binding was compromised, Rtt106 localized properly to chromatin but failed to deliver H3K56ac, leading to replication and silencing defects. By mutating analogous regions in the structurally homologous chromatin-reorganizer Pob3, we revealed a conserved histone-binding function for a basic patch found on both proteins. In contrast, a loop connecting two β-strands was required for histone binding by Rtt106 but was dispensable for Pob3 function. Unlike Rtt106, Pob3 histone binding was modification-independent, implicating the loop of Rtt106 in H3K56ac-specific recognition in vivo. Our studies described the structural origins of Rtt106 function, identified a conserved histone-binding surface, and defined a critical role for Rtt106:H3K56ac-binding specificity in silencing and replication-coupled nucleosome turnover.

Novel Roles of Caenorhabditis elegans Heterochromatin Protein HP1 and Linker Histone in the Regulation of Innate Immune Gene Expression

Abstract: Linker histone (H1) and heterochromatin protein 1 (HP1) are essential components of heterochromatin which contribute to the transcriptional repression of genes. It has been shown that the methylation mark of vertebrate histone H1 is specifically recognized by the chromodomain of HP1. However, the exact biological role of linker histone binding to HP1 has not been determined. Here, we investigate the function of the Caenorhabditis elegans H1 variant HIS-24 and the HP1-like proteins HPL-1 and HPL-2 in the cooperative transcriptional regulation of immune-relevant genes. We provide the first evidence that HPL-1 interacts with HIS-24 monomethylated at lysine 14 (HIS-24K14me1) and associates in vivo with promoters of genes involved in antimicrobial response. We also report an increase in overall cellular levels and alterations in the distribution of HIS-24K14me1 after infection with pathogenic bacteria. HIS-24K14me1 localization changes from being mostly nuclear to both nuclear and cytoplasmic in the intestinal cells of infected animals. Our results highlight an antimicrobial role of HIS-24K14me1 and suggest a functional link between epigenetic regulation by an HP1/H1 complex and the innate immune system in C. elegans.

Multivalent recognition of histone tails by the PHD-fingers of CHD5

Abstract: The chromodomain, helicase, DNA-binding protein 5 (CHD5) is a chromatin remodeling enzyme which is implicated in tumor suppression. In this study, we demonstrate the ability of the CHD5 PHD fingers to specifically recognize the unmodified N-terminus of histone H3. We use two distinct modified peptide-library platforms (beads and glass slides) to determine the detailed histone binding preferences of PHD(1) and PHD(2) alone and the tandem PHD(1-2) construct. Both domains displayed similar binding preferences for histone H3, where modification (e.g., methylation, acetylation, and phosphorylation) at H3R2, H3K4, H3T3, H3T6, and H3S10 disrupts high-affinity binding, and the three most N-terminal amino acids (ART) are crucial for binding. The tandem CHD5-PHD(1-2) displayed similar preferences to those displayed by each PHD finger alone. Using NMR, surface plasmon resonance, and two novel biochemical assays, we demonstrate that CHD5-PHD(1-2) simultaneously engages two H3 N-termini and results in a 4-11-fold increase in affinity compared with either PHD finger alone. These studies provide biochemical evidence for the utility of tandem PHD fingers to recruit protein complexes at targeted genomic loci and provide the framework for understanding how multiple chromatin-binding modules function to interpret the combinatorial PTM capacity written in chromatin.

Crucial role of dynamic linker histone binding and divalent ions for DNA accessibility and gene regulation revealed by mesoscale modeling of oligonucleosomes.

Abstract: Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA–DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH–core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression.

The human histone chaperone sNASP interacts with linker and core histones through distinct mechanisms

Abstract: Somatic nuclear autoantigenic sperm protein (sNASP) is a human homolog of the N1/N2 family of histone chaperones. sNASP contains the domain structure characteristic of this family, which includes a large acidic patch flanked by several tetratricopeptide repeat (TPR) motifs. sNASP possesses a unique binding specificity in that it forms specific complexes with both histone H1 and histones H3/H4. Based on the binding affinities of sNASP variants to histones H1, H3.3, H4 and H3.3/H4 complexes, sNASP uses distinct structural domains to interact with linker and core histones. For example, one of the acidic patches of sNASP was essential for linker histone binding but not for core histone interactions. The fourth TPR of sNASP played a critical role in interactions with histone H3/H4 complexes, but did not influence histone H1 binding. Finally, analysis of cellular proteins demonstrated that sNASP existed in distinct complexes that contained either linker or core histones.

The Linker Histone Plays a Dual Role during Gametogenesis in Saccharomyces cerevisiae

Abstract: The differentiation of gametes involves dramatic changes to chromatin, affecting transcription, meiosis, and cell morphology. Sporulation in Saccharomyces cerevisiae shares many chromatin features with spermatogenesis, including a 10-fold compaction of the nucleus. To identify new proteins involved in spore nuclear organization, we purified chromatin from mature spores and discovered a significant enrichment of the linker histone (Hho1). The function of Hho1 has proven to be elusive during vegetative growth, but here we demonstrate its requirement for efficient sporulation and full compaction of the spore genome. Hho1 chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed increased genome-wide binding in mature spores and provides novel in vivo evidence of the linker histone binding to nucleosomal linker DNA. We also link Hho1 function to the transcription factor Ume6, the master repressor of early meiotic genes. Hho1 and Ume6 are depleted during meiosis, and analysis of published ChIP-chip data obtained during vegetative growth reveals a high binding correlation of both proteins at promoters of early meiotic genes. Moreover, Ume6 promotes binding of Hho1 to meiotic gene promoters. Thus, Hho1 may play a dual role during sporulation: Hho1 and Ume6 depletion facilitates the onset of meiosis via activation of Ume6-repressed early meiotic genes, whereas Hho1 enrichment in mature spores contributes to spore genome compaction.

Phosphorylation of the chromatin binding domain of KSHV LANA.

Abstract: The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1–329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3–21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function.

Direct interplay among histones, histone chaperones, and a chromatin boundary protein in the control of histone gene expression.

Abstract: In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.

Attenuation of the ubiquitin conjugate DNA damage signal by the proteasomal DUB POH1

Abstract: In mammalian cells, the response to double-strand DNA breaks (DSBs) is crucial to maintain cell viability and prevent oncogenic transformation. A complex mechanism has evolved to achieve a timely response to these lesions characterized by post-translational modification events. The modifications, including phosphorylation, ubiquitination and SUMOylation, contribute to the recruitment of factors, either through specific binding modules in DNA-damage response proteins, or by the unmasking of cryptic signals on chromatin to which the proteins bind. This mechanism amplifies the damage signal and offers an opportunity to the cell to regulate the strength, longevity and spread of the response. In a recent report we elucidated the means by which the mammalian proteasome plays a role in dampening DSB signaling and regulates the repair of DSBs.1 In considering the degree and complexity of Ub conjugation involved in the DSB response,2 we supposed that deubiqutinating ezymes (DUBs) not currently implicated would be likely to be important. To test this, we performed an siRNA screen to identify enzymes required to reduce Ub conjugates following release from exposure to hydroxyurea (HU), i.e., on recovery from S-phase DSBs. This identified POH1/PSMD14/rpn11, the obligate DUB of the 19S proteasome activating complex. The 19S activates the 20S core and is required to degrade Ub-modified proteins. In addition to an influence on global Ub conjugates, we found the enzymatic activity of POH1 was also required to restrict Ub accumulation at sites of DNA damage following HU and irradiation (IR). The Ub ligases RNF8 and RNF168 recruit to sites of DNA damage and are required for the subsequent accumulation of repair mediators 53BP1 and BRCA1 (in the BRCA1-A complex). 53BP1 binds to dimethylated histones but requires RNF8-mediated removal of competing histone binding proteins and RNF8-RNF168-mediated local generation of K63-linked poly-Ub to do so.3 The BRCA1-A complex contains the K63-Ub binding protein RAP80, which directs localization of the complex to sites of damage. This complex also contains K63-Ub-specific DUB, BRCC36, able to hydrolyse K63-chains and restrict the amount of 53BP1 at damaged chromatin.4 We found that while the ability of 53BP1 to form IR-induced foci (IRIF) was inhibited in cells with low expression of RNF8 or RNF168, they were permitted when POH1 was also depleted, and even 53BP1 expressed at very low levels could form IRIF when POH1 expression was reduced. Thus POH1 is a powerful antagonist to 53BP1, preventing 53BP1 from recruiting to DSB sites. The regulation was not at the level of protein expression of RNF8, RNF168 or 53BP1. Instead POH1 acted in both mechanisms associated with 53BP1 accumulation. It promoted the occupation of chromatin by JMJD2A, a protein that competes with 53BP1 for the dimethyl histone mark, and restricted the degree of K63-Ub at sites of damage. Intriguingly the 19S and BRCA1-A complexes have been likened due to the number of protein modules in common,6 and both POH1 and BRCC36 are Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) proteases with K63-Ub linkage specificity.5 Importantly co-depletion of POH1 and BRCC36 did not elicit 53BP1 foci larger than POH1 depletion alone, suggesting these DUBs act in the same mechanistic pathway to restrict 53BP1 assemblies. The influence of POH1 on DNA repair through non-homologous end joining correlated with its regulation of 53BP1 accumulation. Reduced DNA repair in RNF8-, RNF168- or 53BP1-depleted cells, in which no 53BP1 IRIF form, could be countered by co-depletion of POH1, which restored both repair and 53BP1 IRIF. Intriguingly, in cells depleted for POH1 and exhibiting enlarged 53BP1 foci end joining was reduced. This correlated with poor recruitment of the NHEJ factor Artemis. We speculate the block on DNA repair might be brought about by inappropriate proximity of 53BP1 to the DNA ends. These observations were all the more interesting when we examined the influence of POH1 depletion on BRCA1 and RAP80. We expected enlarged accumulations of these proteins, since the signal for their accumulation is also K63-Ub. However no increase was seen. The lack of BRCA1 spreading despite the increase in local K63-Ub, and, further, the inability to restore BRCA1 IRIF in RNF8-depleted cells by reduction of POH1, suggests that Ub-binding by RAP80 is insufficient for BRCA1 recruitment. Consistent with this conclusion RAP80 interaction with SUMO has recently been reported to be required for its recruitment.7 Thus, the cell is able to separate the regulation of the mediators, 53BP1 and BRCA1, despite a shared signal for their accumulation. K63-Ub binding proteins in the DSB response, such as 53BP1, inhibit DNA resection in homologous recombination (HR) repair. Thus we anticipated that HR might be defective in POH1-depleted cells in a manner dependent on 53BP1. However, although we demonstrated a requirement for POH1 in HR repair, this was not through, or not wholly through, 53BP1. Instead we found that overexpression of the small-nucleic acid-like protein, 19S component and BRCA2- co-factor, DSS1, could restore HR in cells with low POH1. DSS1 promotes BRCA2-medaited RAD51 loading,8 and we suggest that the 19S may have a role enriching DSS1 at sites of damage to improve HR-repair. Together with other recent reports of the proteasome at sites of DNA damage in mammalian cells (20S, 19S and other activators),9,10 these data elucidate a previously unappreciated role for the proteasome in DNA repair. Thus, to our appreciation of this complex as a protein macerator, we must now add the more subtle role of Ub-conjugate regulation. (Fig. 1) Figure 1. Model of POH1-mediated restriction of 53BP1 through K63- poly-Ub cleavage. RNF8/168 modify histones with K63-linked Ub. The combined activity of the removal of chromatin binding proteins (JMJD2A/B, not shown) and K63-poly-Ub generation ...

Validation of Histone-Binding Partners by Peptide Pull-Downs and Isothermal Titration Calorimetry

Abstract: In order to properly describe a chromatin-binding module and understand its biology, its binding interactions need to be specifically and explicitly defined. Tremendous gains in our understanding of the function, specificity, and concerted action of chromatin-binding complexes have been made through reductionist studies of chromatin-binding modules and posttranslationally modified histone peptides. Chromatin binding proteins often discriminate between histone posttranslational modifications and sequence contexts using subtle affinity differences that appear critical to their function. Biophysical measurements are best able to discern these minute binding energy distinctions and are increasingly important as the chromatin field endeavors to detail the unique molecular recognition of myriad chromatin states. We describe the theoretical basis and advantages of various biophysical measurements of binding affinity in the chromatin field, as well as proper experimental design and procedure for peptide pull-downs and isothermal titration calorimetry (ITC). Routine use of these techniques to characterize chromatin-binding proteins has the potential to profoundly advance our view of the molecular recognition of chromatin, allowing more quantitative comparisons across the chromatin field. Ultimately, precise determination of a binding affinity not only illuminates the biochemical and structural properties of an interface, but also informs investigation of function.

High-throughput strategy to identify inhibitors of histone-binding domains.

Abstract: Many epigenetic proteins recognize the posttranslational modification state of chromatin through their histone-binding domains and thereby recruit nuclear complexes to specific loci within the genome. A number of these domains have been implicated in cancer and other diseases through aberrant binding of chromatin; therefore, identifying small molecules that disrupt histone binding could be a powerful mechanism for disease therapy. We have developed a high-throughput assay for the detection of histone peptide–domain interactions utilizing AlphaScreen technology. Here, we describe how the assay can be first optimized and then performed for high-throughput screening of small molecule-binding inhibitors. We also describe strategies for biochemical validation of small molecules identified.

Novel functions of the MOZ double PHD finger domain

Abstract: Monocytic leukaemia zinc-finger protein (MOZ) is a histone acetyltransferase (HAT) implicated in haematopoiesis and acute myeloid leukaemia, as well as embryonic and postnatal development. MOZ contains multiple domains, including a MYST HAT domain and a double PHD finger domain (DPF) suggesting it interacts with histones. This work has established for the first time that the MOZ DPF exhibits dual functionality in establishing and sensing post-translational modifications (PTMs) of histones. Firstly, our data detected the direct interaction of MOZ with the N-terminal tails of histones H3 and H4 and shows that the MOZ DPF domain mediates such binding. Both PHD fingers are required and functionally cooperate to establish the DPF histone binding preference in terms of PTMs. We demonstrate that H3K4me3 prevents MOZ DPF association with H3, although H3K4me2 is tolerated. Similarly, H4Kac acts as a dominant exit signal that excludes MOZ from chromatin. This ability to sense H3K4 PTM status was confirmed in a collaborative effort establishing the crystal structure of MOZ DPF in complex with an unmodified H3 peptide. The H3 peptide adopted an α-helical conformation in the complex, which has not previously been observed. Secondly, we present novel data showing that the MOZ DPF domain exhibits a mild histone H3-specific acetyltransferase activity. This provides the first report of a possible enzymatic role in chromatin modification attributed to a PHD finger. Furthermore, the combined DPF and MYST domains were found to influence the reaction rate and substrate specificity of MOZ-induced histone acetylation. Our studies revealed that the MOZ DPF could associate with heterochromatic PTMs, namely H3K9me3. We report here that both the H3K9-specific methyltransferase SUV39H1 and heterochromatin protein 1 (HP1) form interactions with MOZ, implicating its function in both corepressor and coactivator complexes. Thus, our data suggest that like several other chromatin-associated proteins, MOZ is a multi-functional regulator of chromatin modification and gene expression.