Direct protein-DNA Interactions Research Articles

The AP2 transcription factors TOE1/TOE2 convey Arabidopsis age information to ethylene signaling in plant de novo root regeneration.

We reveal that transcription factors TOE1 and TOE2 directly inhibit the transcription of EIN3. Ethylene triggers leaf abscission and senescence during plant aging. Previous studies have shown that the transcription of ETHYLENE INSENSITIVE 3 (EIN3), which encodes a key transcription factor in ethylene signaling, is gradually upregulated during plant aging. However, it is still unknown how plants transmit their age information to achieve transcriptional control of EIN3. Here, we report that the EAR-like motif-containing transcription factors TARGET OF EAT 1 (TOE1) and its homolog TOE2 directly associated with the EIN3 promoter. The transcription of EIN3 is further enhanced in mutants of toe1 toe2 during plant aging. TOE1/TOE2 are tightly controlled by canonical microRNA 172 (miR172)-mediated plant aging signaling, which result in a decline in TOE1/TOE2 expression during aging. These results illustrate that during plant aging, the reduced expressions of TOE1/TOE2 trigger an upregulation of EIN3. Next, we took advantage of EIN3-regulated de novo root regeneration (DNRR) as an age-controlled phenotype to dissect the biological function of this regulatory circuit. The DNRR rates in toe1 toe2 are more severely decreased with plant aging; however, the simultaneous loss of ein3 and eil1 (toe1 toe2 ein3 eil1 quadruple mutants) almost completely rescued the DNRR defects. Taken together, our findings show that the plant age-regulated TOE transcription factors precisely integrate plant age information and developmental programs through direct protein-DNA interactions.

A functional screen identifies transcriptional networks that regulate HIV-1 and HIV-2

The molecular networks involved in the regulation of HIV replication, transcription, and latency remain incompletely defined. To expand our understanding of these networks, we performed an unbiased high-throughput yeast one-hybrid screen, which identified 42 human transcription factors and 85 total protein-DNA interactions with HIV-1 and HIV-2 long terminal repeats. We investigated a subset of these transcription factors for transcriptional activity in cell-based models of infection. KLF2 and KLF3 repressed HIV-1 and HIV-2 transcription in CD4+ T cells, whereas PLAGL1 activated transcription of HIV-2 through direct protein-DNA interactions. Using computational modeling with interacting proteins, we leveraged the results from our screen to identify putative pathways that define intrinsic transcriptional networks. Overall, we used a high-throughput functional screen, computational modeling, and biochemical assays to identify and confirm several candidate transcription factors and biochemical processes that influence HIV-1 and HIV-2 transcription and latency.

High-intensity UV laser ChIP-seq for the study of protein-DNA interactions in living cells

Genome-wide mapping of transcription factor binding is generally performed by chemical protein–DNA crosslinking, followed by chromatin immunoprecipitation and deep sequencing (ChIP-seq). Here we present the ChIP-seq technique based on photochemical crosslinking of protein–DNA interactions by high-intensity ultraviolet (UV) laser irradiation in living mammalian cells (UV-ChIP-seq). UV laser irradiation induces an efficient and instant formation of covalent “zero-length” crosslinks exclusively between nucleic acids and proteins that are in immediate contact, thus resulting in a “snapshot” of direct protein–DNA interactions in their natural environment. Here we show that UV-ChIP-seq, applied for genome-wide profiling of the sequence-specific transcriptional repressor B-cell lymphoma 6 (BCL6) in human diffuse large B-cell lymphoma (DLBCL) cells, produces sensitive and precise protein–DNA binding profiles, highly enriched with canonical BCL6 DNA sequence motifs. Using this technique, we also found numerous previously undetectable direct BCL6 binding sites, particularly in condensed, inaccessible areas of chromatin.

Barley plants over-expressing the NAC transcription factor gene HvNAC005 show stunting and delay in development combined with early senescence.

The plant-specific NAC transcription factors have attracted particular attention because of their involvement in stress responses, senescence, and nutrient remobilization. The HvNAC005 gene of barley encodes a protein belonging to subgroup NAC-a6 of the NAC family. This study shows that HvNAC005 is associated with developmental senescence. It was significantly up-regulated following ABA treatment, supported by ABA-responsive elements in its promoter, but it was not up-regulated during dark-induced senescence. The C-termini of proteins closely related to HvNAC005 showed overall high divergence but also contained conserved short motifs. A serine- and leucine-containing central motif was essential for transcriptional activity of the HvNAC005 C-terminus in yeast. Over-expression of HvNAC005 in barley resulted in a strong phenotype with delayed development combined with precocious senescence. The over-expressing plants showed up-regulation of genes involved with secondary metabolism, hormone metabolism, stress, signalling, development, and transport. Up-regulation of senescence markers and hormone metabolism and signalling genes supports a role of HvNAC005 in the cross field of different hormone and signalling pathways. Binding of HvNAC005 to promoter sequences of putative target genes containing the T[G/A]CGT core motif was shown by direct protein-DNA interactions of HvNAC005 with promoters for two of the up-regulated genes. In conclusion, HvNAC005 was shown to be a strong positive regulator of senescence and so is an obvious target for the fine-tuning of gene expression in future attempts to improve nutrient remobilization related to the senescence process in barley.

Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein � DNA Interactions in Magnaporthe oryzae

Chromatin Immunoprecipitation (ChIP) Assay for Detecting Direct and Indirect Protein � DNA Interactions in Magnaporthe oryzae

Gli regulates MUC5AC transcription in human gastrointestinal cells.

MUC5AC is a well-known gastric differentiation marker, which has been frequently used for the classification of stomach cancer. Immunohistochemistry revealed that expression of MUC5AC decreases accompanied with increased malignant property of gastric mucosa, which further suggests the importance of MUC5AC gene regulation. Alignment of the 5′-flanking regions of MUC5AC gene of 13 mammal species denoted high homology within 200 bp upstream of the coding region. Luciferase activities of the deletion constructs containing upstream 451 bp or shorter fragments demonstrated that 15 bp region between −111 and −125 bp plays a critical role on MUC5AC promoter activity in gastrointestinal cells. We found a putative Gli-binding site in this 15 bp sequence, and named this region a highly conserved region containing a Gli-binding site (HCR-Gli). Overexpression of Gli homologs (Gli1, Gli2, and Gli3) clearly enhanced MUC5AC promoter activity. Exogenous modulation of Gli1 and Gli2 also affected the endogenous MUC5AC gene expression in gastrointestinal cells. Chromatin immunoprecipitation assays demonstrated that Gli1 directly binds to HCR-Gli: Gli regulates MUC5AC transcription via direct protein-DNA interaction. Conversely, in the 30 human cancer cell lines and various normal tissues, expression patterns of MUC5AC and Gli did not coincide wholly: MUC5AC showed cell line-specific or tissue-specific expression whereas Gli mostly revealed ubiquitous expression. Luciferase promoter assays suggested that the far distal MUC5AC promoter region containing upstream 4010 bp seems to have several enhancer elements for gene transcription. In addition, treatments with DNA demethylation reagent and/or histone deacetylase inhibitor induced MUC5AC expression in several cell lines that were deficient in MUC5AC expression. These results indicated that Gli is necessary but not sufficient for MUC5AC expression: namely, the multiple regulatory mechanisms should work in the distal promoter region of MUC5AC gene.

High-resolution mapping of transcription factor binding sites on native chromatin

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

High-resolution mapping of transcription factor binding sites on native chromatin

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.

Histone exchange and histone modifications during transcription and aging

The organization of the eukaryotic genome into chromatin enables DNA to fit inside the nucleus while also regulating the access of proteins to the DNA to facilitate genomic functions such as transcription, replication and repair. The basic repeating unit of chromatin is the nucleosome, which includes 147bp of DNA wrapped 1.65 times around an octamer of core histone proteins comprising two molecules each of H2A, H2B, H3 and H4 [1]. Each nucleosome is a highly stable unit, being maintained by over 120 direct protein–DNA interactions and several hundred water mediated ones [1]. Accordingly, there is considerable interest in understanding how processive enzymes such as RNA polymerases manage to pass along the coding regions of our genes that are tightly packaged into arrays of nucleosomes. Here we present the current mechanistic understanding of this process and the evidence for profound changes in chromatin dynamics during aging. This article is part of a Special Issue entitled: Histone chaperones and Chromatin assembly.

Coupling Sequence-specific Recognition to DNA Modification

Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is approximately 28 A away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.

Detailed mechanism for transposition by TnpA transposase involves DNA shape rather than direct protein-DNA recognition to generate an active nucleoprotein complex

A series of single-crystal structures determined by Barabas and colleagues provides a detailed mechanism for how the TnpA transposase from Helicobacter pylori recognizes, cleaves, and integrates the IS200/IS605 class of transposable elements. An interesting aspect of the mechanism is that the transposase recognizes the transposon through the unique fold-back structure adopted by the sequences of the DNA components, rather than through direct protein-DNA interactions. This is an example of indirect readout that is reminiscent of how four-stranded junctions are recognized by recombination proteins, but is also analogous to ribonucleoproteins, in that the DNA facilitates formation of an active nucleic acid-protein complex.

Predicting transcription factor specificity with all-atom models

The binding of a transcription factor (TF) to a DNA operator site can initiate or repress the expression of a gene. Computational prediction of sites recognized by a TF has traditionally relied upon knowledge of several cognate sites, rather than an ab initio approach. Here, we examine the possibility of using structure-based energy calculations that require no knowledge of bound sites but rather start with the structure of a protein–DNA complex. We study the PurR Escherichia coli TF, and explore to which extent atomistic models of protein–DNA complexes can be used to distinguish between cognate and noncognate DNA sites. Particular emphasis is placed on systematic evaluation of this approach by comparing its performance with bioinformatic methods, by testing it against random decoys and sites of homologous TFs. We also examine a set of experimental mutations in both DNA and the protein. Using our explicit estimates of energy, we show that the specificity for PurR is dominated by direct protein–DNA interactions, and weakly influenced by bending of DNA.

Ang II‐Inducible PDGF‐D Transcription Requires Ser/Thr Residues In The Second Zinc Finger Region of Sp1: Novel Phosphospecific Antibodies Demonstrate that Sp1 is Phosphorylated by SMC Injury

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein‐DNA interactions, protein‐protein interactions, chromatin remodelling and maintenance of methylation‐free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation and phosphorylation by kinases such as the atypical protein kinase C‐zeta. Although Sp1 controls the basal and inducible regulation of many genes, the post‐translational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670 and Thr681) in the zinc finger (ZNF) domain of Sp1 that are modified by PKC‐zeta, and have generated novel anti‐peptide antibodies recognising the PKC‐zeta‐phosphorylated form of Sp1. Angiotensin II (Ang II), which activates PKC‐zeta phosphorylation (at Thr410) via the Ang II Type I receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the PDGF‐D promoter. All 3 residues in Sp1 (Thr668, Ser670 and Thr681) are required for Sp1‐dependent PDGF‐D activation in response to Ang II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in SMCs of human atherosclerotic plaques, and is dynamically expressed together with PDGF‐D in SMCs of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC‐zeta‐phospho‐Sp1 axis and Ang II‐inducible gene expression.

Angiotensin II–Inducible Platelet-Derived Growth Factor-D Transcription Requires Specific Ser/Thr Residues in the Second Zinc Finger Region of Sp1

Sp1, the first identified and cloned transcription factor, regulates gene expression via multiple mechanisms including direct protein-DNA interactions, protein-protein interactions, chromatin remodeling, and maintenance of methylation-free CpG islands. Sp1 is itself regulated at different levels, for example, by glycosylation, acetylation, and phosphorylation by kinases such as the atypical protein kinase C-zeta. Although Sp1 controls the basal and inducible regulation of many genes, the posttranslational processes regulating its function and their relevance to pathology are not well understood. Here we have used a variety of approaches to identify 3 amino acids (Thr668, Ser670, and Thr681) in the zinc finger domain of Sp1 that are modified by PKC-zeta and have generated novel anti-peptide antibodies recognizing the PKC-zeta-phosphorylated form of Sp1. Angiotensin II, which activates PKC-zeta phosphorylation (at Thr410) via the angiotensin II type 1 receptor, stimulates Sp1 phosphorylation and increases Sp1 binding to the platelet-derived growth factor-D promoter. All 3 residues in Sp1 (Thr668, Ser670, and Thr681) are required for Sp1-dependent platelet-derived growth factor-D activation in response to angiotensin II. Immunohistochemical analysis revealed that phosphorylated Sp1 is expressed in smooth muscle cells of human atherosclerotic plaques and is dynamically expressed together with platelet-derived growth factor-D in smooth muscle cells of the injured rat carotid artery wall. This study provides new insights into the regulatory mechanisms controlling the PKC-zeta-phospho-Sp1 axis and angiotensin II-inducible gene expression.

Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis

Expression of the gene cluster icaADBC is necessary for biofilm production in Staphylococcus epidermidis. The ica operon is negatively controlled by the repressor IcaR. Here, the crystal structure of IcaR was determined and the refined structure revealed a homodimer comprising entirely α-helices, typical of the tetracycline repressor protein family for gene regulations. The N-terminal domain contains a conserved helix-turn-helix DNA-binding motif with some conformational variations, indicating flexibility in this region. The C-terminal domain shows a complementary surface charge distribution about the dyad axis, ideal for efficient and specific dimer formation. The results of the electrophoretic mobility shift assay and isothermal titration calorimetry suggested that a 28 bp core segment of the ica operator is implicated in the cooperative binding of two IcaR dimers on opposite sides of the duplex DNA. Computer modeling based on the known DNA-complex structure of QacR and site-specific mutagenesis experiments showed that direct protein–DNA interactions are mostly conserved, but with slight variations for recognizing the different sequences. By interfering with the binding of IcaR to DNA, aminoglycoside gentamicin and other antibiotics may activate the icaADBC genes and elicit biofilm production in S. epidermidis, and likely S. aureus, as a defense mechanism.

Regulation of Human MC2-R Gene Expression by CREB, CREM, and ICER in the Adrenocortical Cell Line Y1

The MC2-Receptor (melanocortin 2 receptor, MC2-R) is a Gs-protein coupled receptor that is upregulated by its own ligand ACTH and by forskolin. The mechanisms regulating MC2-R expression are still unclear. We therefore investigated the role of the stimulatory transcription factors CREB and CREM and the inhibitory factor ICER for regulation of human MC2-R expression. We cotransfected mouse adrenocortical Y1 cells with luciferase reporter gene vectors containing full length and deleted human MC2-R promoter constructs with expression plasmids for CREB, CREBS133A, CREMtau, CREMtauS117A, or ICER. Direct protein-DNA interaction was investigated by EMSA. Wild type CREB did not significantly affect promoter activity due to high endogenous CREB activity. However, CREBS133A decreased forskolin stimulated MC2-R promoter activity by 48+/-5% (mean+/-SEM) while unstimulated values remained unchanged. CREMtau moderately increased basal and forskolin stimulated luciferase activity in a dose-dependent manner (maximum effect 252+/-24% and 186+/-13% VS. control vector, respectively). While this effect required the full length promoter, cAMP stimulation was retained in shorter constructs. ICER reduced basal luciferase activity in Y1 cells by 17+/-28%, but completely abolished forskolin stimulation. Although 5'-deletion constructs mapped the minimum promoter region required for ICER effect to the shortest -64/+40 construct, direct protein DNA interaction in this promoter region could not be identified by EMSA. Moreover, mutation of the SF-1 binding sites, which retained ICER dependent inhibition, excluded SF-1 to be required for this effect. We conclude from these data that transcription factors of the CREB/CREM/ATF family have a moderate effect on human MC2-R promoter activity, but seem to play a minor role in transmitting stimulation of the cAMP pathway to increased MC2-R expression.

Interfacial Water as a “Hydration Fingerprint” in the Noncognate Complex of BamHI

The molecular code of specific DNA recognition by proteins as a paradigm in molecular biology remains an unsolved puzzle primarily because of the subtle interplay between direct protein-DNA interaction and the indirect contribution from water and ions. Transformation of the nonspecific, low affinity complex to a specific, high affinity complex is accompanied by the release of interfacial water molecules. To provide insight into the conversion from the loose to the tight form, we characterized the structure and energetics of water at the protein-DNA interface of the BamHI complex with a noncognate sequence and in the specific complex. The fully hydrated models were produced with Grand Canonical Monte Carlo simulations. Proximity analysis shows that water distributions exhibit sequence dependent variations in both complexes and, in particular, in the noncognate complex they discriminate between the correct and the star site. Variations in water distributions control the number of water molecules released from a given sequence upon transformation from the loose to the tight complex as well as the local entropy contribution to the binding free energy. We propose that interfacial waters can serve as a “hydration fingerprint” of a given DNA sequence.

ChIPOTle: a user-friendly tool for the analysis of ChIP-chip data

ChIPOTle is a new software tool designed specifically for the analysis of ChIP-chip data.

The Campylobacter jejuni dccRS two‐component system is required for optimal in vivo colonization but is dispensable for in vitro growth

A Campylobacter jejuni two-component signal transduction system (TCSTS), designated dccR-dccS (diminished capacity to colonize; Cj1223c-Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A DeltadccR response regulator mutant generated using the virulent strain 81-176 background exhibited significantly reduced colonization of immunocompetent limited flora (I-LF) mice, severe combined immunodeficient limited flora (SCID-LF) mice, and 1-day-old chicks. A DeltadccS sensor kinase mutant was likewise defective for colonization in the I-LF mouse model. DeltadccR-infected SCID-LF mice also exhibited dramatically reduced inflammation relative to wild type-infected SCID-LF mice. Despite this diminished colonization capacity, the DeltadccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two-component system; binding of purified His-tagged DccR to the promoter region of two of these genes supports a direct protein-DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two-component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.

Structural basis of transcription activation: the CAP-alpha CTD-DNA complex.

The Escherichia coli catabolite activator protein (CAP) activates transcription at P(lac), P(gal), and other promoters through interactions with the RNA polymerase alpha subunit carboxyl-terminal domain (alphaCTD). We determined the crystal structure of the CAP-alphaCTD-DNA complex at a resolution of 3.1 angstroms. CAP makes direct protein-protein interactions with alphaCTD, and alphaCTD makes direct protein-DNA interactions with the DNA segment adjacent to the DNA site for CAP. There are no large-scale conformational changes in CAP and alphaCTD, and the interface between CAP and alphaCTD is small. These findings are consistent with the proposal that activation involves a simple "recruitment" mechanism.