Determination Of Primary Sequence Research Articles

U-CAN-seq: A Universal Competition Assay by Nanopore Sequencing.

RNA viruses quickly evolve subtle genotypic changes that can have major impacts on viral fitness and host range, with potential consequences for human health. It is therefore important to understand the evolutionary fitness of novel viral variants relative to well-studied genotypes of epidemic viruses. Competition assays are an effective and rigorous system with which to assess the relative fitness of viral genotypes. However, it is challenging to quickly and cheaply distinguish and quantify fitness differences between very similar viral genotypes. Here, we describe a protocol for using reverse transcription PCR in combination with commercial nanopore sequencing services to perform competition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay by Nanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used a well-studied N24A mutation in the chikungunya virus (CHIKV) nsp3 gene to confirm that we could detect a competitive disadvantage using U-CAN-seq. We also used this approach to show that mutations to the CHIKV 5' conserved sequence element that disrupt sequence but not structure did not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) did cause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent, primary sequence determinants in this loop play an important role in CHIKV biology. Our novel findings illustrate the utility of the U-CAN-seq competition assay.

Edman Degradation Reveals Unequivocal Analysis of the Disulfide Connectivity in Peptides and Proteins.

Disulfide bridges in peptides and proteins play an essential role in maintaining their conformation, structural integrity, and consequently function. Despite ongoing efforts, it is still not possible to detect disulfide bonds and the connectivity of multiply bridged peptides directly through a simple and sufficiently validated protein sequencing or peptide mapping method. Partial or complete reduction and chemical cysteine modification are required as initial steps, followed by the application of a proper detection method. Edman degradation (ED) has been used for primary sequence determination but is largely neglected since the establishment of mass spectrometry (MS)-based protein sequencing. Here, we evaluated and thoroughly characterized the phenyl thiohydantoin (PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl cysteine as bioanalytical standards for cysteine detection and quantification as well as for the elucidation of the disulfide connectivity in peptides by ED. Validation of the established derivatives was performed according to the guidelines of the International Committee of Harmonization on bioanalytical method validation, and their analytical properties were confirmed as reference standards. A series of model peptides was sequenced to test the usability of the PTH-Cys-derivatives as standards, whereas the native disulfide-bonded peptides CCAP-vil, μ-conotoxin KIIIA, and human insulin were used as case studies to determine their disulfide bond connectivity completely independent of MS analysis.

TGM2-mediated histone transglutamination is dictated by steric accessibility

Recent studies have identified serotonylation of glutamine-5 on histone H3 (H3Q5ser) as a novel posttranslational modification (PTM) associated with active transcription. While H3Q5ser is known to be installed by tissue transglutaminase 2 (TGM2), the substrate characteristics affecting deposition of the mark, at the level of both chromatin and individual nucleosomes, remain poorly understood. Here, we show that histone serotonylation is excluded from constitutive heterochromatic regions in mammalian cells. Biochemical studies reveal that the formation of higher-order chromatin structures associated with heterochromatin impose a steric barrier that is refractory to TGM2-mediated histone monoaminylation. A series of structure-activity relationship studies, including the use of DNA-barcoded nucleosome libraries, shows that steric hindrance also steers TGM2 activity at the nucleosome level, restricting monoaminylation to accessible sites within histone tails. Collectively, our data indicate that the activity of TGM2 on chromatin is dictated by substrate accessibility rather than by primary sequence determinants or by the existence of preexisting PTMs, as is the case for many other histone-modifying enzymes.

Unraveling the structure and function of CdcPDE: A novel phosphodiesterase from Crotalus durissus collilineatus snake venom

This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8–8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.

Evaluation of Sibling and Twin Fragment Ions Improves the Structural Characterization of Proteins by Top-Down MALDI In-Source Decay Mass Spectrometry.

Comprehensive determination of primary sequence and identification of post-translational modifications (PTMs) are key elements in protein structural analysis. Various mass spectrometry (MS) based fragmentation techniques are powerful approaches for mapping both the amino acid sequence and PTMs; one of these techniques is matrix-assisted laser desorption/ionization (MALDI), combined with in-source decay (ISD) fragmentation and Fourier-transform ion cyclotron resonance (FT-ICR) MS. MALDI-ISD MS protein analysis involves only minimal sample preparation and does not require spectral deconvolution. The resulting MALDI-ISD MS data is complementary to electrospray ionization-based MS/MS sequencing readouts, providing knowledge on the types of fragment ions is available. In this study, we evaluate the isotopic distributions of z′ ions in protein top-down MALDI-ISD FT-ICR mass spectra and show why these distributions can deviate from theoretical profiles as a result of co-occurring and isomeric z and y-NH3 ions. Two synthetic peptides, containing either normal or deuterated alanine residues, were used to confirm the presence and unravel the identity of isomeric z and y-NH3 fragment ions (“twins”). Furthermore, two reducing MALDI matrices, namely 1,5-diaminonaphthalene and N-phenyl-p-phenylenediamine were applied that yield ISD mass spectra with different fragment ion distributions. This study demonstrates that the relative abundance of isomeric z and y-NH3 ions requires consideration for accurate and confident assignments of z′ ions in MALDI-ISD FT-ICR mass spectra.

Myoglobin from common pheasant (Phasianus colchicus L.): Purification and primary structure characterization

Myoglobin (Mb) is a monomeric hemoprotein involved in dioxygen storage and transport in skeletal muscles and heart. Mb, containing the heme pigment, is present in large amount in meat, thus influencing meat color and consumer choice. Here, the primary structure of Mb isolated from muscle of Phasianus colchicus L. was determined by using a comparative peptide mapping approach based on MALDI-TOF mass spectrometry. This strategy allowed the determination of common pheasant Mb primary sequence, which resulted identical to the chicken Mb, as also confirmed by intact molecular mass determination by ESI/Q-TOF mass spectrometry. Indeed, the accurate molecular mass (17,290.50 Da) of common pheasant Mb was found to be in good agreement with that of chicken Mb (17,290.86 Da). Finally, the 3D model of common pheasant Mb was predicted by homology modeling. Overall, in this study we confirmed that chicken, turkey and common pheasant, belonging to the Galliformes order, share the same Mb sequence. Practical applications Considering the economic importance of game meat consumption, our result may be interesting for meat industry, providing useful information for the determination of species-specific differences in color and color stability compared to other poultry species.

Genetic variation and RNA structure regulate microRNA biogenesis

MiRNA biogenesis is highly regulated at the post-transcriptional level; however, the role of sequence and secondary RNA structure in this process has not been extensively studied. A single G to A substitution present in the terminal loop of pri-mir-30c-1 in breast and gastric cancer patients had been previously described to result in increased levels of mature miRNA. Here, we report that this genetic variant directly affects Drosha-mediated processing of pri-mir-30c-1 in vitro and in cultured cells. Structural analysis of this variant revealed an altered RNA structure that facilitates the interaction with SRSF3, an SR protein family member that promotes pri-miRNA processing. Our results are compatible with a model whereby a genetic variant in pri-mir-30c-1 leads to a secondary RNA structure rearrangement that facilitates binding of SRSF3 resulting in increased levels of miR-30c. These data highlight that primary sequence determinants and RNA structure are key regulators of miRNA biogenesis.

Prediction of Protein Lysine Acylation by Integrating Primary Sequence Information with Multiple Functional Features.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic methods have been widely used to identify lysine acylation proteins. However, these experimental approaches often fail to detect proteins that are in low abundance or absent in specific biological samples. To circumvent these problems, we developed a computational method to predict lysine acylation, including acetylation, malonylation, succinylation, and glutarylation. The prediction algorithm integrated flanking primary sequence determinants and evolutionary conservation of acylated lysine as well as multiple protein functional annotation features including gene ontology, conserved domains, and protein-protein interactions. The inclusion of functional annotation features increases predictive power oversimple sequence considerations for four of the acylation species evaluated. For example, the Matthews correlation coefficient (MCC) for the prediction of malonylation increased from 0.26 to 0.73. The performance of prediction was validated against an independent data set for malonylation. Likewise, when tested with independent data sets, the algorithm displayed improved sensitivity and specificity over existing methods. Experimental validation by Western blot experiments and LC-MS/MS detection further attested to the performance of prediction. We then applied our algorithm on to the mouse proteome and reported the global-scale prediction of lysine acetylation, malonylation, succinylation, and glutarylation, which should serve as a valuable resource for future functional studies.

Independent relationship between amyloid precursor protein (APP) dimerization and γ-secretase processivity.

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.

Stereochemical Determinants of C-terminal Specificity in PDZ Peptide-binding Domains: A NOVEL CONTRIBUTION OF THE CARBOXYLATE-BINDING LOOP

PDZ (PSD-95/Dlg/ZO-1) binding domains often serve as cellular traffic engineers, controlling the localization and activity of a wide variety of binding partners. As a result, they play important roles in both physiological and pathological processes. However, PDZ binding specificities overlap, allowing multiple PDZ proteins to mediate distinct effects on shared binding partners. For example, several PDZ domains bind the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), an epithelial ion channel mutated in CF. Among these binding partners, the CFTR-associated ligand (CAL) facilitates post-maturational degradation of the channel and is thus a potential therapeutic target. Using iterative optimization, we previously developed a selective CAL inhibitor peptide (iCAL36). Here, we investigate the stereochemical basis of iCAL36 specificity. The crystal structure of iCAL36 in complex with the CAL PDZ domain reveals stereochemical interactions distributed along the peptide-binding cleft, despite the apparent degeneracy of the CAL binding motif. A critical selectivity determinant that distinguishes CAL from other CFTR-binding PDZ domains is the accommodation of an isoleucine residue at the C-terminal position (P(0)), a characteristic shared with the Tax-interacting protein-1. Comparison of the structures of these two PDZ domains in complex with ligands containing P(0) Leu or Ile residues reveals two distinct modes of accommodation for β-branched C-terminal side chains. Access to each mode is controlled by distinct residues in the carboxylate-binding loop. These studies provide new insights into the primary sequence determinants of binding motifs, which in turn control the scope and evolution of PDZ interactomes.

HJURP Uses Distinct CENP-A Surfaces to Recognize and to Stabilize CENP-A/Histone H4 for Centromere Assembly

Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that whereas surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Furthermore, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.

The α-Helical Content of the Transmembrane Domain of the British Dementia Protein-2 (Bri2) Determines Its Processing by Signal Peptide Peptidase-like 2b (SPPL2b)

Regulated intramembrane proteolysis is a widely accepted concept describing the processing of various transmembrane proteins via ectodomain shedding followed by an intramembrane cleavage. The resulting cleavage products can be involved in reverse signaling. Presenilins, which constitute the active center of the γ-secretase complex, signal peptide peptidase (SPP), and its homologues, the SPP-like (SPPL) proteases are members of the family of intramembrane-cleaving aspartyl proteases of the GXGD-type. We recently demonstrated that Bri2 (itm2b) is a substrate for regulated intramembrane proteolysis by SPPL2a and SPPL2b. Intramembrane cleavage of Bri2 is triggered by an initial shedding event catalyzed by A Disintegrin and Metalloprotease 10 (ADAM10). Additionally primary sequence determinants within the intracellular domain, the transmembrane domain and the luminal juxtamembrane domain are required for efficient cleavage of Bri2 by SPPL2b. Using mutagenesis and circular dichroism spectroscopy we now demonstrate that a high α-helical content of the Bri2 transmembrane domain (TMD) reduces cleavage efficiency of Bri2 by SPPL2b, while the presence of a GXXXG dimerization motif influences the intramembrane cleavage only to a minor extent. Surprisingly, only one of the four conserved intramembrane glycine residues significantly affects the secondary structure of the Bri2 TMD and thereby its intramembrane cleavage. Other glycine residues do not influence the α-helical content of the transmembrane domain nor its intramembrane processing.

Modular Evolution and the Origins of Symmetry: Reconstruction of a Three-Fold Symmetric Globular Protein

The high frequency of internal structural symmetry in common protein folds is presumed to reflect their evolutionary origins from the repetition and fusion of ancient peptide modules, but little is known about the primary sequence and physical determinants of this process. Unexpectedly, a sequence and structural analysis of symmetric subdomain modules within an abundant and ancient globular fold, the β-trefoil, reveals that modular evolution is not simply a relic of the ancient past, but is an ongoing and recurring mechanism for regenerating symmetry, having occurred independently in numerous existing β-trefoil proteins. We performed a computational reconstruction of a β-trefoil subdomain module and repeated it to form a newly three-fold symmetric globular protein, ThreeFoil. In addition to its near perfect structural identity between symmetric modules, ThreeFoil is highly soluble, performs multivalent carbohydrate binding, and has remarkably high thermal stability. These findings have far-reaching implications for understanding the evolution and design of proteins via subdomain modules.

Book Review: Protein and Peptide Analysis by LC-MS—Experimental Strategies

Protein and Peptide Analysis by LC-MS—Experimental Strategies is a publication of the Royal Society of Chemistry Chromatography Monographs series (number 15). The book consists of 12 chapters authored by researchers at the coalface of LC-MS (liquid chromatography-mass spectrometry) applications directed towards investigations of peptides and proteins, their sequences, structures, biological interactions and functions. Chapter 1 (Lottspeich) sets the scene by introducing the concepts of top down and bottom up approaches with particular emphasis on quantitative analysis. The major isotopic labelling techniques are briefly compared and this serves as a platform for examples of experiments based on these techniques described in later chapters. Chapter 2 (Letzel) provides an excellent background to LC-MS for postgraduate students and researchers embarking on research that utilises this technique. The major types of ionisation methods are introduced and examples that highlight important considerations for designing the best analytical methods are provided. This is a particularly important chapter as it alerts those relatively new to LC-MS about the importance of the choice of ionisation mode, optimising experiments by careful choice of solvents, additives, spray conditions and instrumental parameters. Chapter 3 (Ohnuma and Fukamizo) provides a commentary on the utility of recombinant expression of proteins and peptides, and presents commonly used liquid chromatography resins and methods used in protein purification. A brief outline of the experimental procedure for purification of a protein (NtChiV) expressed in E. coli is given. While the procedure presented here does not provide a detailed commentary on the challenges that may be encountered, this section certainly provides a basis for relative newcomers to the methods to know what questions need to be asked and to understand what training may be required to achieve competency in the experimental protocols for protein expression and purification. The content of Chapters 4–12 reveals the scope of the application of LC-MS to protein/peptide research and goes well beyond applications for primary sequence determination. Liquid chromatography-mass spectrometry of intact proteins is described in Chapter 4 (Young and Garcia) and is a highlight of this book. The authors emphasise the importance of optimising experimental conditions for the particular protein under study and importantly have a good grasp of the advantages and limitations of the techniques as well as being able to present a big picture view of the research area. Chapters 5 (Cheison and Kulozik) and 6 (Hoos and Niessen) deal with different aspects of LC-MS analysis of peptides derived from proteolytic or chemical digestion of proteins. The focus of the former chapter is to describe an experimental set-up for the pH stat-controlled tryptic hydrolysis of proteins. In this way, a higher degree of reproducibility may be attained. The latter describes a general method for trypsin hydrolysis of Book Review

Primary sequence determination of a monoclonal antibody against α-synuclein using a novel mass spectrometry-based approach

A novel mass spectrometry-based workflow for de novo sequencing of a full-length monoclonal antibody (LB509) against α-synuclein is presented. This approach combines chemical modification of cysteine residues, multiple enzymatic digestion, stable isotope labeled amino acids in cell culture (SILAC), liquid chromatography coupled tandem mass spectrometry (LC/MS/MS) analysis including collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), and de novo sequencing software for data interpretation. The introduction of aminoethyl-8 (A-8) reagent which renders cysteine residues in the antibody to become substrates for trypsin digestion significantly increases the sequence coverage of the antibody, especially in the complementarity determining regions (CDRs). Incorporation of SILAC helps to distinguish minor sequence variations between original and deduced sequences. Methods in obtaining detailed sequence characterization are described, highlighting the advantage of using multiple fragmentation schemes, CID in ion trap and HCD in C-trap. Complete primary sequence for LB509 has been construed and the antibody generated exhibits specific binding to α-synuclein. The development and implementation of this technology enables an alternative approach for generating therapeutic candidate and reagent antibodies.

Mass Spectrometry and X-ray Diffraction Analysis of Two Crystal Types of Dioclea virgata Lectin: An Antinociceptive Protein Candidate to Structure/Function Analysis

The lectin from seeds of Dioclea virgata (DvirL) was purified in a single step affinity chromatography, sequenced by tandem mass spectrometry and submitted to crystallization and biological experiments. DvirL has a molecular mass of 25,412 ± 2 Da and the chains β and γ has 12,817 Da ± 2 and 12,612 Da ± 2, respectively. Primary sequence determination was assigned by tandem mass spectrometry and revealed a protein with 237 amino acids and 87% of identify with ConA. The protein crystals were obtained native and complexed with X-Man using vapor-diffusion method at a constant temperature of 293 K. A complete X-ray dataset was collected at 1.8 Å resolution. DvirL crystals were found to be orthorhombic, belonging to the space group I222, with a unit cell parameters a = 647.5 Å, b = 86.6 Å, c = 90.2 Å. Molecular replacement search found a solution with a correlation coefficient of 77.1% and an R(factor) of 44.6%. The present study also demonstrated that D. virgata lectin presents edematogenic and antinociceptive activities in rodents electing this protein as a candidate to structure/function analysis.

Resurrection of a clinical antibody: Template proteogenomic de novo proteomic sequencing and reverse engineering of an anti‐lymphotoxin‐α antibody

A mouse hybridoma antibody directed against a member of the tumour necrosis factor (TNF)-superfamily, lymphotoxin-alpha (LT-α), was isolated from stored mouse ascites and purified to homogeneity. After more than a decade of storage the genetic material was not available for cloning; however, biochemical assays with the ascites showed this antibody against LT-α (LT-3F12) to be a preclinical candidate for the treatment of several inflammatory pathologies. We have successfully rescued the LT-3F12 antibody by performing MS analysis, primary amino acid sequence determination by template proteogenomics, and synthesis of the corresponding recombinant DNA by reverse engineering. The resurrected antibody was expressed, purified and shown to demonstrate the desired specificity and binding properties in a panel of immuno-biochemical tests. The work described herein demonstrates the powerful combination of high-throughput informatic proteomic de novo sequencing with reverse engineering to reestablish monoclonal antibody-expressing cells from archived protein sample, exemplifying the development of novel therapeutics from cryptic protein sources.

Genetic Demonstration of a Redundant Role of Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Mitogen-Activated Protein Kinases in Promoting Fibroblast Proliferation

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway plays an important role in the proliferative response of mammalian cells to mitogens. However, the individual contribution of the isoforms ERK1 and ERK2 to cell proliferation control is unclear. The two ERK isoforms have similar biochemical properties and recognize the same primary sequence determinants on substrates. On the other hand, analysis of mice lacking individual ERK genes suggests that ERK1 and ERK2 may have evolved unique functions. In this study, we used a robust genetic approach to analyze the individual functions of ERK1 and ERK2 in cell proliferation using genetically matched primary embryonic fibroblasts. We show that individual loss of either ERK1 or ERK2 slows down the proliferation rate of fibroblasts to an extent reflecting the expression level of the kinase. Moreover, RNA interference-mediated silencing of ERK1 or ERK2 expression in cells genetically disrupted for the other isoform similarly reduces cell proliferation. We generated fibroblasts genetically deficient in both Erk1 and Erk2. Combined loss of ERK1 and ERK2 resulted in a complete arrest of cell proliferation associated with G(1) arrest and premature replicative senescence. Together, our findings provide compelling genetic evidence for a redundant role of ERK1 and ERK2 in promoting cell proliferation.

Direct detection of DNA methylation during single-molecule, real-time sequencing

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions.

Primary sequence and epigenetic determinants of in vivo occupancy of genomic DNA by GATA1

DNA sequence motifs and epigenetic modifications contribute to specific binding by a transcription factor, but the extent to which each feature determines occupancy in vivo is poorly understood. We addressed this question in erythroid cells by identifying DNA segments occupied by GATA1 and measuring the level of trimethylation of histone H3 lysine 27 (H3K27me3) and monomethylation of H3 lysine 4 (H3K4me1) along a 66 Mb region of mouse chromosome 7. While 91% of the GATA1-occupied segments contain the consensus binding-site motif WGATAR, only ∼0.7% of DNA segments with such a motif are occupied. Using a discriminative motif enumeration method, we identified additional motifs predictive of occupancy given the presence of WGATAR. The specific motif variant AGATAA and occurrence of multiple WGATAR motifs are both strong discriminators. Combining motifs to pair a WGATAR motif with a binding site motif for GATA1, EKLF or SP1 improves discriminative power. Epigenetic modifications are also strong determinants, with the factor-bound segments highly enriched for H3K4me1 and depleted of H3K27me3. Combining primary sequence and epigenetic determinants captures 52% of the GATA1-occupied DNA segments and substantially increases the specificity, to one out of seven segments with the required motif combination and epigenetic signals being bound.