Tcf1 and Lef1 transcription factors establish CD8(+) T cell identity through intrinsic HDAC activity.

The CD4(+) and CD8(+) T cell dichotomy is essential for effective cellular immunity. How individual T cell identity is established remains poorly understood. Here we show that the high-mobility group (HMG) transcription factors Tcf1 and Lef1 are essential for repressing CD4(+) lineage-associated genes including Cd4, Foxp3 and Rorc in CD8(+) T cells. Tcf1- and Lef1-deficient CD8(+) T cells exhibit histone hyperacetylation, which can be ascribed to intrinsic histone deacetylase (HDAC) activity in Tcf1 and Lef1. Mutation of five conserved amino acids in the Tcf1 HDAC domain diminishes HDAC activity and the ability to suppress CD4(+) lineage genes in CD8(+) T cells. These findings reveal that sequence-specific transcription factors can utilize intrinsic HDAC activity to guard cell identity by repressing lineage-inappropriate genes.

Histone Tail Conformations: A Fuzzy Affair with DNA.

The core histone tails are critical in chromatin structure and signaling. Studies over the past several decades have provided a wealth of information on the histone tails and their interaction with chromatin factors. However, the conformation of the histone tails in a chromatin relevant context has remained elusive. Only recently has enough evidence emerged to start to build a structural model of the tails in the context of nucleosomes and nucleosome arrays. Here, we review these studies and propose that the histone tails adopt a high-affinity fuzzy complex with DNA, characterized by robust but dynamic association. Furthermore, we discuss how these DNA-bound conformational ensembles promote distinct chromatin structure and signaling, and that their fuzzy nature is important in transitioning between functional states.

PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here, we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

Single-Molecule Characterization of Cy3.5 -Cy5.5 Dye Pair for FRET Studies of Nucleic Acids and Nucleosomes.

Single molecule FRET (Forster resonance energy transfer) is very powerful method for studying biomolecular binding dynamics and conformational transitions. Only a few donor - acceptor dye pairs have been characterized for use in single-molecule FRET (smFRET) studies. Hence, introducing and characterizing additional FRET dye pairs is important in order to widen the scope of applications of single-molecule FRET in biomolecular studies. Here we characterize the properties of the Cy3.5 and Cy5.5 dye pair under FRET at the single-molecule level using naked double-stranded DNA (dsDNA) and the nucleosome. We show that this pair of dyes is photostable for ~ 5 min under continuous illumination. We also report Cy3.5-Cy5.5 FRET proximity dependence and stability in the presence of several biochemical buffers and photoprotective reagents in the context of double-stranded DNA. Finally, we demonstrate compatibility of the Cy3.5-Cy5.5 pair for smFRET in vitro studies of nucleosomes.

Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility.

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.

Engagement of DNA and H3K27me3 by the CBX8 chromodomain drives chromatin association.

Polycomb repressive complex 1 (PRC1) is critical for mediating gene repression during development and adult stem cell maintenance. Five CBX proteins, CBX2,4,6,7,8, form mutually exclusive PRC1 complexes and are thought to play a role in the association of PRC1 with chromatin. Specifically, the N-terminal chromodomain (CD) in the CBX proteins is thought to mediate specific targeting to methylated histones. For CBX8, however, the chromodomain has demonstrated weak affinity and specificity for methylated histones in vitro, leaving doubt as to its role in CBX8 chromatin association. Here, we investigate the function of the CBX8 CD in vitro and in vivo. We find that the CD is in fact a major driver of CBX8 chromatin association and determine that this is driven by both histone and previously unrecognized DNA binding activity. We characterize the structural basis of histone and DNA binding and determine how they integrate on multiple levels. Notably, we find that the chromatin environment is critical in determining the ultimate function of the CD in CBX8 association.

The PHD finger of Spp1 mediates histone modification cross-talk.

Binding of the Spp1 PHD finger to histone H3K4me3 is sensitive to adjacent post-translational modifications in the histone tail. This commentary discusses the findings of He and colleagues [Biochem. J.476, 1957-1973] which show that the PHD finger binds to H3K4me3 in a selective manner which is conserved in the Saccharomyces pombe and mammalian orthologues of Spp1.

A histone reader becomes the readout.

Chromatin signaling events are critical for the dynamic regulation of the genome. Although much has been learned about histone modification in these events, the modification of nonhistone chromatin regulators and cross-talk between these pathways is less well understood. Here, Appikonda et al. demonstrate that the transcription co-factor and oncoprotein TRIM24 (tripartite motif-containing protein 24) is SUMOylated upon association with a specific histone modification signature, which regulates the transcription of genes involved in cell adhesion. These data extend our understanding of signaling cascades in the nucleus and offer new insights for cancer drug development.

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity.

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not been developed. Here, we identify endonuclease I as a robust biomarker for E. coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E. coli clones revealed that endonuclease I is the predominant DNase in E. coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E. coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3 hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E. coli.

Reading More than Histones: The Prevalence of Nucleic Acid Binding among Reader Domains.

The eukaryotic genome is packaged into the cell nucleus in the form of chromatin, a complex of genomic DNA and histone proteins. Chromatin structure regulation is critical for all DNA templated processes and involves, among many things, extensive post-translational modification of the histone proteins. These modifications can be "read out" by histone binding subdomains known as histone reader domains. A large number of reader domains have been identified and found to selectively recognize an array of histone post-translational modifications in order to target, retain, or regulate chromatin-modifying and remodeling complexes at their substrates. Interestingly, an increasing number of these histone reader domains are being identified as also harboring nucleic acid binding activity. In this review, we present a summary of the histone reader domains currently known to bind nucleic acids, with a focus on the molecular mechanisms of binding and the interplay between DNA and histone recognition. Additionally, we highlight the functional implications of nucleic acid binding in chromatin association and regulation. We propose that nucleic acid binding is as functionally important as histone binding, and that a significant portion of the as yet untested reader domains will emerge to have nucleic acid binding capabilities.

Towards understanding methyllysine readout.

BACKGROUND: Lysine methylation is the most versatile covalent posttranslational modification (PTM) found in histones and non-histone proteins. Over the past decade a number of methyllysine-specific readers have been discovered and their interactions with histone tails have been structurally and biochemically characterized. More recently innovative experimental approaches have emerged that allow for studying reader interactions in the context of the full nucleosome and nucleosomal arrays. SCOPE OF REVIEW: In this review we give a brief overview of the known mechanisms of histone lysine methylation readout, summarize progress recently made in exploring interactions with methylated nucleosomes, and discuss the latest advances in the development of small molecule inhibitors of the methyllysine-specific readers. MAJOR CONCLUSIONS: New studies reveal various reader-nucleosome contacts outside the methylated histone tail, thus offering a better model for association of histone readers to chromatin and broadening our understanding of the functional implications of these interactions. In addition, some progress has been made in the design of antagonists of these interactions. GENERAL SIGNIFICANCE: Specific lysine methylation patterns are commonly associated with certain chromatin states and genomic elements, and are linked to distinct biological outcomes such as transcription activation or repression. Disruption of patterns of histone modifications is associated with a number of diseases, and there is tremendous therapeutic potential in targeting histone modification pathways. Thus, investigating binding of readers of these modifications is not only important for elucidating fundamental mechanisms of chromatin regulation, but also necessary for the design of targeted therapeutics. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

Inhibition of histone binding by supramolecular hosts.

The tandem PHD (plant homeodomain) fingers of the CHD4 (chromodomain helicase DNA-binding protein 4) ATPase are epigenetic readers that bind either unmodified histone H3 tails or H3K9me3 (histone H3 trimethylated at Lys9). This dual function is necessary for the transcriptional and chromatin remodelling activities of the NuRD (nucleosome remodelling and deacetylase) complex. In the present paper, we show that calixarene-based supramolecular hosts disrupt binding of the CHD4 PHD2 finger to H3K9me3, but do not affect the interaction of this protein with the H3K9me0 (unmodified histone H3) tail. A similar inhibitory effect, observed for the association of chromodomain of HP1γ (heterochromatin protein 1γ) with H3K9me3, points to a general mechanism of methyl-lysine caging by calixarenes and suggests a high potential for these compounds in biochemical applications. Immunofluorescence analysis reveals that the supramolecular agents induce changes in chromatin organization that are consistent with their binding to and disruption of H3K9me3 sites in living cells. The results of the present study suggest that the aromatic macrocyclic hosts can be used as a powerful new tool for characterizing methylation-driven epigenetic mechanisms.

Bivalent recognition of nucleosomes by the tandem PHD fingers of the CHD4 ATPase is required for CHD4-mediated repression.

CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis for their association with chromatin. We found that PHD1/2 targets nucleosomes in a multivalent manner, concomitantly engaging two histone H3 tails. This robust synergistic interaction displaces HP1γ from pericentric sites, inducing changes in chromatin structure and leading to the dispersion of the heterochromatic mark H3K9me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.

The structural basis of urea-induced protein unfolding in ß-catenin.

Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of ß-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic interactions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation.

Photoactive Spatial Proximity Probes for Binding Pairs with Epigenetic Marks.

A new strategy for encoding polypeptide libraries with photolabile tags is developed. The photoassisted assay, based on conditional release of encoding tags only from bound pairs, can differentiate between peptides which have minor differences in a form of post-translational modifications with epigenetic marks. The encoding strategy is fully compatible with automated peptide synthesis. The encoding pendants are compact and do not perturb potential binding interactions.

Polyamines regulate cell fate by altering the activity of histone-modifying enzymes.

Polyamines are polycationic alkyl-amines abundant in proliferating stem and cancer cells. How these metabolites influence numerous cellular functions remains unclear. Here we show that polyamine levels decrease during differentiation and that inhibiting polyamine synthesis leads to a differentiated-like cell state. Polyamines concentrate in the nucleus and are further enriched in the nucleoli of cells in culture and in vivo . Loss of polyamines drives changes in chromatin accessibility that correlate with altered histone post-translational modifications. Polyamines interact electrostatically with DNA on the nucleosome core, stabilizing histone tails in conformations accessible to modifying enzymes. These data reveal a mechanism by which an abundant metabolite influences chromatin structure and function in a non-sequence specific manner, facilitating chromatin remodeling during reprogramming and limiting it during fate commitment.

Nucleosome conformation dictates the histone code.

Histone post-translational modifications (PTMs) play a critical role in chromatin regulation. It has been proposed that these PTMs form localized 'codes' that are read by specialized regions (reader domains) in chromatin-associated proteins (CAPs) to regulate downstream function. Substantial effort has been made to define [CAP: histone PTM] specificities, and thus decipher the histone code and guide epigenetic therapies. However, this has largely been done using the reductive approach of isolated reader domains and histone peptides, which cannot account for any higher-order factors. Here, we show that the [BPTF PHD finger and bromodomain: histone PTM] interaction is dependent on nucleosome context. The tandem reader selectively associates with nucleosomal H3K4me3 and H3K14ac or H3K18ac, a combinatorial engagement that despite being in cis is not predicted by peptides. This in vitro specificity of the BPTF tandem reader for PTM-defined nucleosomes is recapitulated in a cellular context. We propose that regulatable histone tail accessibility and its impact on the binding potential of reader domains necessitates we refine the 'histone code' concept and interrogate it at the nucleosome level.

Handpicking epigenetic marks with PHD fingers.

Plant homeodomain (PHD) fingers have emerged as one of the largest families of epigenetic effectors capable of recognizing or 'reading' post-translational histone modifications and unmodified histone tails. These interactions are highly specific and can be modulated by the neighboring epigenetic marks and adjacent effectors. A few PHD fingers have recently been found to also associate with non-histone proteins. In this review, we detail the molecular mechanisms and biological outcomes of the histone and non-histone targeting by PHD fingers. We discuss the significance of crosstalk between the histone modifications and consequences of combinatorial readout for selective recruitment of the PHD finger-containing components of chromatin remodeling and transcriptional complexes.

Protocol to prepare doubly labeled fluorescent nucleosomes for single-molecule fluorescence microscopy.

Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the basic building unit of chromatin. Here, we present a protocol for preparing doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E. coli and purification of recombinant human core histones. We then detail fluorescent labeling of histones and nucleosomal double-stranded DNA followed by octamer refolding and nucleosome reconstitution.

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

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.