Site-specific DNA Binding Research Articles

Wanted: A New Model for Glucocorticoid Receptor Transactivation and Transrepression

Wanted: A New Model for Glucocorticoid Receptor Transactivation and Transrepression

Aptamer-guided gene targeting in yeast and human cells

Gene targeting is a genetic technique to modify an endogenous DNA sequence in its genomic location via homologous recombination (HR) and is useful both for functional analysis and gene therapy applications. HR is inefficient in most organisms and cell types, including mammalian cells, often limiting the effectiveness of gene targeting. Therefore, increasing HR efficiency remains a major challenge to DNA editing. Here, we present a new concept for gene correction based on the development of DNA aptamers capable of binding to a site-specific DNA binding protein to facilitate the exchange of homologous genetic information between a donor molecule and the desired target locus (aptamer-guided gene targeting). We selected DNA aptamers to the I-SceI endonuclease. Bifunctional oligonucleotides containing an I-SceI aptamer sequence were designed as part of a longer single-stranded DNA molecule that contained a region with homology to repair an I-SceI generated double-strand break and correct a disrupted gene. The I-SceI aptamer-containing oligonucleotides stimulated gene targeting up to 32-fold in yeast Saccharomyces cerevisiae and up to 16-fold in human cells. This work provides a novel concept and research direction to increase gene targeting efficiency and lays the groundwork for future studies using aptamers for gene targeting.

Mechanosensing of DNA bending in a single specific protein-DNA complex

Many crucial biological processes are regulated by mechanical stimuli. Here, we report new findings that pico-Newton forces can drastically affect the stability of the site-specific DNA binding of a single transcription factor, the E. coli integration host factor (IHF), by stretching a short ~150 nm DNA containing a single IHF binding site. Dynamic binding and unbinding of single IHF were recorded and analyzed for the force-dependent stability of the IHF-DNA complex. Our results demonstrate that the IHF-DNA interaction is fine tuned by force in different salt concentration and temperature over physiological ranges, indicating that, besides other physiological factors, force may play equally important role in transcription regulation. These findings have broad implications with regard to general mechanosensitivity of site-specific DNA bending proteins.

Cytosolic HMGB1 expression in human renal clear cell cancer indicates higher pathological T classifications and tumor grades.

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 functions as a proinflammatory cytokine. Here, we studied expressions of HMGB1 and HMGB2 in human renal cancer. Immunohistological expressions of HMGB1 and HMGB2 were assessed in 39 surgically resected human renal cancer specimens. HMGB1 was expressed in the nucleus in 37 out of 39 (94.9%) renal clear cell cancers,while its expression in the cytosol was noted in 19 cases (48.7%). Cytosolic HMGB1 is expressed more frequently in cancers beyond the pT1b classification than in those at the pT1a classification. Higher tumor grades (≥ G2) were also significantly linked with the cytosolic expression of HMGB1. HMGB2 was expressed in the nucleus in 35 of 39 (89.7%) renal clear cell cancers, while its expression in the cytosol was observed in only 7 case (17.9%). Linkage between cytosolic expression of HMGB2 and T classifications was weakly observed, while that between nuclear expression and T classifications was not. HMGB1 expressed in the cytoplasm may be an effective marker indicating higher T classifications and tumor grades.

Genome Engineering with TAL-Effector Nucleases and Alternative Modular Nuclease Technologies

Over three years following the discovery of the TAL code, artificial TAL effector DNA binding domains have emerged as the premier platform for building site-specific DNA binding polypeptides for use in biological research. Here, we provide an overview of TAL effector and alternative modular DNA binding domain (mDBD) technologies, focusing on their use in established and emerging architectures for building site-specific endonucleases for genome engineering applications. We also discuss considerations for choosing TAL effector/mDBD or alternative nuclease technologies for genome engineering projects ranging from basic laboratory gene editing of cultured cell lines to therapeutics. Finally, we highlight how the rapid pace of development of mDBD-based, such as monomeric TALENs (I-TevI-TAL), and more recently RNA-guided nucleases (CRISPR-Cas9) has led to a transition in the field of genome engineering towards development of the next generation of technologies aimed at controlling events that occur after targeted DNA breaks are made.

The Influence of Spatial Variation in Chromatin Density Determined by X-Ray Tomograms on the Time to Find DNA Binding Sites

In this work, we examine how volume exclusion caused by regions of high chromatin density might influence the time required for proteins to find specific DNA binding sites. The spatial variation of chromatin density within mouse olfactory sensory neurons is determined from soft X-ray tomography reconstructions of five nuclei. We show that there is a division of the nuclear space into regions of low-density euchromatin and high-density heterochromatin. Volume exclusion experienced by a diffusing protein caused by this varying density of chromatin is modeled by a repulsive potential. The value of the potential at a given point in space is chosen to be proportional to the density of chromatin at that location. The constant of proportionality, called the volume exclusivity, provides a model parameter that determines the strength of volume exclusion. Numerical simulations demonstrate that the mean time for a protein to locate a binding site localized in euchromatin is minimized for a finite, nonzero volume exclusivity. For binding sites in heterochromatin, the mean time is minimized when the volume exclusivity is zero (the protein experiences no volume exclusion). An analytical theory is developed to explain these results. The theory suggests that for binding sites in euchromatin there is an optimal level of volume exclusivity that balances a reduction in the volume searched in finding the binding site, with the height of effective potential barriers the protein must cross during the search process.

Dissection of the ATPase Active Site of P1 ParA Reveals Multiple Active Forms Essential for Plasmid Partition

The segregation, or partition, of bacterial plasmids is driven by the action of plasmid-encoded partition ATPases, which work to position plasmids inside the cell. The most common type of partition ATPase, generally called ParA, is represented by the P1 plasmid ParA protein. ParA interacts with P1 ParB (the site-specific DNA binding protein that recognizes the parS partition site), and interacts with the bacterial chromosome via an ATP-dependent nonspecific DNA binding activity. ParA also regulates expression of the par genes by acting as a transcriptional repressor. ParA requires ATP for multiple steps and in different ways during the partition process. Here, we analyze the properties of mutations in P1 ParA that are altered in a key lysine in the Walker A motif of the ATP binding site. Four different residues at this position (Lys, Glu, Gln, Arg) result in four different phenotypes in vivo. We focus particularly on the arginine substitution (K122R) because it results in a worse-than-null and dominant-negative phenotype called ParPD. We show that ParAK122R binds and hydrolyzes ATP, although the latter activity is reduced compared with wild-type. ParAK122R interacts with ParB, but the consequences of the interaction are damaged. The ability of ParB to stimulate the ATPase activity of ParA in vitro and its repressor activity in vivo is defective. The K122R mutation specifically damages the disassembly of ParA-ParB-DNA partition complexes, which we believe explains the ParPD phenotype in vivo.

CK2 Phosphorylation Inactivates DNA Binding by the Papillomavirus E1 and E2 Proteins

Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1.

ZASC1 knockout mice exhibit an early bone marrow-specific defect in murine leukemia virus replication

BackgroundZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010).MethodsTo determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies.ResultsMice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection.ConclusionsThese studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.

Bayesian hierarchical model of protein-binding microarrayk-mer data reduces noise and identifies transcription factor subclasses and preferredk-mers

Sequence-specific transcription factors (TFs) regulate the expression of their target genes through interactions with specific DNA-binding sites in the genome. Data on TF-DNA binding specificities are essential for understanding how regulatory specificity is achieved. Numerous studies have used universal protein-binding microarray (PBM) technology to determine the in vitro binding specificities of hundreds of TFs for all possible 8 bp sequences (8mers). We have developed a Bayesian analysis of variance (ANOVA) model that decomposes these 8mer data into background noise, TF familywise effects and effects due to the particular TF. Adjusting for background noise improves PBM data quality and concordance with in vivo TF binding data. Moreover, our model provides simultaneous identification of TF subclasses and their shared sequence preferences, and also of 8mers bound preferentially by individual members of TF subclasses. Such results may aid in deciphering cis-regulatory codes and determinants of protein-DNA binding specificity. Source code, compiled code and R and Python scripts are available from http://thebrain.bwh.harvard.edu/hierarchicalANOVA. Supplementary data are available at Bioinformatics online.

Probing the N-terminus of ParB using cysteine-scanning mutagenesis and thiol modification

Plasmid partition systems require site-specific DNA binding proteins to recognize the plasmid partition site, or centromere. When bound to the centromere, these proteins, typically called ParB, interact with the ParA ATPases, which in turn promote the proper positioning of plasmids prior to cell division. P1 ParB is a typical member of a major class of ParB-like proteins that are dimeric helix-turn-helix DNA binding proteins. The N-terminus of ParB contains the region that interacts with ParA and with itself, but it has been difficult to study because this region of the protein is flexible in solution. Here we describe the use of cysteine-scanning mutagenesis and thiol modification of the N-terminus of ParB to create tools to probe the interactions of ParB with itself, with ParA and with DNA. We introduce twelve single-cysteine substitutions across the N-terminus of ParB and show that most do not compromise the function of ParB and that none completely inactivate the protein in vivo. We test three of these ParB variants in vitro and show that they do not alter ParB function, measured by its ability to stimulate ParA ATPase activity and its site-specific DNA binding activity. We discuss that this approach will be generally applicable to the ParB-like proteins in this class of partition systems because of their natural low content of cysteines, and because our evidence suggests that many residues in the N-terminus are amenable to substitution by cysteine.

How DNA-Binding Proteins find their Target Sites in Human Cells

Precise and timely expression of specific genes is fundamental for the viability of any organism. Specific DNA-binding proteins (transcription factors, TFs) transiently associate to promoter sequences on the genome to activate or repress transcription of specific genes. The way TFs explore the nuclear space, find, and bind to their target sequences is the key to decipher transcriptional control but is still subject of debate. We exploit a live-cell model system to identify what factors primarily orchestrate TFs target search. Our assay is based on human cells containing, at a single locus, specific DNA binding sites (‘target') for an exogenous DNA-binding protein (TetR, ‘searcher'). First, single-particle-tracking experiments revealed that individual TetR proteins move in a very composite way. They transiently interact with nonspecific DNA sequences, via their DNA-binding-domain, with residence times ranging from hundreds of millisecond up to few seconds. Furthermore, a subset of proteins shows confinement within micron-sized regions even when DNA-binding is impaired, which suggests that the organization of mammalian nuclei plays a role in search dynamics. Next, we directly quantified the association rate constant of proteins to their targets in living cells. Based on our binding assay, the measured rate constant resulted much slower than expected and local decondensation of chromatin, at the target site locus, did not affect the association rate. In other words, the effective size of a DNA binding site is much smaller than the size of the molecular interaction. The picture emerging from our experiments shows that, in human cells, specific DNA-binding proteins move in a complex environment where they may remain locally confined. They interact transiently also with nonspecific DNA sequences and their target search mechanism is consistent with a non-compact exploration of the nuclear space with very inefficient binding.

Contributions of the Histone Arginine Methyltransferase PRMT6 to the Epigenetic Function of RUNX1

Hematopoietic differentiation is directed by transcription factors such as RUNX1. RUNX1 binds to specific DNA binding sites in regulatory elements of genes and recruits epigenetic cofactors to target loci. In this way histone modification patterns and the chromatin environment are altered, which results in adjusted gene expression. The process of transcription factor binding and cofactor recruitment is dynamic and strongly influenced by specific posttranslational modifications, which are triggered by signaling. In this way cellular signaling is integrated at the epigenetic level by transcription factors. The identification of epigenetic cofactors and the study of their epigenetic influence on transcription is crucial for the understanding of transcription factor function in differentiation and disease. In this article, the recent observation that RUNX1 is associated with the protein arginine methyltransferase 6 will be reviewed. PRMT6 triggers H3R2me2a at RUNX1 target genes; this histone modification negatively influences the positive H3K4me3 mark and this way acts repressive. The RUNX1/PRMT6 association has an impact on bivalent histone marks. Upon differentiation, a RUNX1 corepressor complex with PRMT6 is exchanged with a RUNX1 coactivator complex. Furthermore, the potential cross talk of transcription factors and epigenetic cofactors with histone marks will be discussed.

Identification of functional cis-regulatory elements by sequential enrichment from a randomized synthetic DNA library

BackgroundThe identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified.ResultsHere we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli.ConclusionsSuccessful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II.

E. Coli RNA Polymerase Searches for Promoters through 3D Diffusion

Gene expression, DNA replication, and genome maintenance all start with site-specific DNA binding proteins, which must recognize specific targets from among a vast excess of nonspecific DNA. For example, to initiate transcription, E. coli RNA polymerase (RNAP) must locate promoter sequences, which comprise <2% of the bacterial genome. This promoter search problem remains one of the least understood aspects of gene expression, largely due to the transient nature of intermediates involved in the search process. Here we use single-molecule microscopy to visualize RNAP in real time as it searches for promoters, and we develop a theoretical framework that allows us to analyze target searches at the submicroscopic scale based on single-molecule promoter association rates. Contrary to long-held assumptions, we demonstrate that the promoter search by E. coli RNAP is dominated entirely by 3D diffusion, which has direct implications for understanding how E. coli RNAP and other proteins locate their targets within physiological settings.

HMGB1 and HMGB2 Expression Patterns in Human Papillary Renal Cancers

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 has a dual function, a cytokine in addition to a nuclear protein. The increased expression of HMGB1 has been reported for several different tumors. Here, we assessed HMGB1 and HMGB2 expressions in two cases of papillary renal cell carcinoma. One case with pT1a, Grade 2 showed HMGB1 expression in the nucleus and cytosol and HMGB2 expression in the nucleus, but not in the cytosol. In the other case, there were three renal tumors, one of which was clear cell renal cell carcinoma with pT1a, Grade 3 and two were papillary renal cell carcinomas, Grade 2 (5 mmand2 mmin the diameter). Both HMGB1 and HMGB2 were expressed in the nucleus and cytosol of papillary carcinoma. In the clear cell carcinoma of this case, HMGB1 expression was stained both in the nucleus and cytosol, while HMGB2 was observed in the nucleus, but not in the cytosol. More samples need to be further investigated in order to draw conclusions concerning HMGB expressions in papillary renal cell carcinomas.

The Sequence and Function Relationship in Ligand-Induced Folding

The Escherichia coli biotin repressor, BirA, the central protein of the E. coli Biotin Regulatory System serves as a metabolic enzyme and the transcription repressor of the biotin biosynthetic operon. For both functions, the active BirA species is bound to bio-5'-AMP, which it synthesizes from substrates biotin and ATP. As an enzyme, BirA catalyzes post-translational biotin linkage to a specific lysine residue of the biotin carboxyl carrier protein (BCCP), a subunit of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid biosynthesis. Transcription repression requires self-association of the adenylate-bound BirA followed by site-specific DNA binding to the biotin operator of the biotin biosynthetic operon. Both functions require folding of two flexible surface loops including the biotin-binding loop (BBL), comprised of residues 116-124, that folds over the biotin moiety and the adenylate-binding loop (ABL), comprised of residues 212-234, that folds over the adenine moiety of the adenylate. In the BirA: adenylate structure side chains of residues F124 and P126 of the BBL and M211 and V218 of the ABL bridge the two loops. Combined kinetic and equilibrium measurements of the consequences of single alanine substitutions of these bridging residues indicate heterogeneity in the effects on the multiple BirA functions. Isothermal titration calorimetry and stopped-flow fluorescence measurements reveal that the substitutions significantly compromise both bio-5'-AMP binding and synthesis. By contrast, these same substitutions have modest effects on homodimerization and biotin transfer to BCCP. These results in combination with previous studies of amino acid replacements in three ABL residues that form a hydrophobic cluster concomitant with adenylate binding indicate a ligand-linked folding process that is characterized by a complex relationship between sequence and function.

HMGB1 and HMGB2 Expression Patterns in Human Papillary Renal Cancers

High mobility group box (HMGB) proteins are nuclear nonhistone chromosomal proteins that bend DNA, bind preferentially to distorted DNA structures, and promote the assembly of site-specific DNA binding proteins. Recent reports indicate that HMGB1 has a dual function, a cytokine in addition to a nuclear protein. The increased expression of HMGB1 has been reported for several different tumors. Here, we assessed HMGB1 and HMGB2 expressions in two cases of papillary renal cell carcinoma. One case with pT1a, Grade 2 showed HMGB1 expression in the nucleus and cytosol and HMGB2 expression in the nucleus, but not in the cytosol. In the other case, there were three renal tumors, one of which was clear cell renal cell carcinoma with pT1a, Grade 3 and two were papillary renal cell carcinomas, Grade 2 (5 mmand2 mmin the diameter). Both HMGB1 and HMGB2 were expressed in the nucleus and cytosol of papillary carcinoma. In the clear cell carcinoma of this case, HMGB1 expression was stained both in the nucleus and cytosol, while HMGB2 was observed in the nucleus, but not in the cytosol. More samples need to be further investigated in order to draw conclusions concerning HMGB expressions in papillary renal cell carcinomas.

The DNA binding site specificity and antiproliferative property of ternary Pt(ii) and Zn(ii) complexes of phenanthroline and N,N′-ethylenediaminediacetic acid

The binding site specificity of the ternary complexes, [M(II)(phen)(edda)] (M(II) = Pt(2+) and Zn(2+); phen = 1,10-phenanthroline; edda = N,N'-ethylenediaminediacetic acid), for the self-complementary oligonucleotides (ODNs), ds(C(1)G(2)C(3)G(4)A(5)A(6)T(7)T(8)C(9)G(10)C(11)G(12))(2) (ODN1) and ds(C(1)G(2)C(3)G(4)T(5)A(6)T(7)A(8)C(9)G(10)C(11)G(12))(2) (ODN2), was studied by NMR measurements. The results indicated that [Pt(ii)(phen)(edda)] was partially intercalated between C(3)/G(10) and G(4)/C(9) base pairs of ODN1 and ODN2 in the major grooves, whereas [Zn(II)(phen)(edda)] was bound specifically to the TATA region of ODN2 in the minor groove and to the terminal G(2)/C(11) base pair of ODN1 in the major groove. The preference for the TATA sequence over the AATT sequence in the binding of [Zn(phen)(edda)] was attributed to the wider minor groove width of the TATA sequence. The bindings of the complexes to ct-DNA were also studied by UV, CD, and fluorescence spectroscopy. Additionally, the antiproliferative property of [Pt(II)(phen)(edda)] towards MCF7 breast cancer cells and normal MCF10-A cells was compared with that of [Zn(II)(phen)(edda)].

Reversible cycling between cysteine persulfide-ligated [2Fe-2S] and cysteine-ligated [4Fe-4S] clusters in the FNR regulatory protein

Fumarate and nitrate reduction (FNR) regulatory proteins are O(2)-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions [4Fe-4S](2+)-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S](2+) cluster undergoes a rapid conversion to a [2Fe-2S](2+) cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between [4Fe-4S](2+) and [2Fe-2S](2+) clusters in Escherichia coli FNR. Selective (34)S labeling of the bridging sulfides in the [4Fe-4S](2+) cluster-bound form of FNR facilitated identification of resonantly enhanced Cys(32)S-(34)S stretching modes in the resonance Raman spectrum of the O(2)-exposed [2Fe-2S](2+) cluster-bound form of FNR. This result indicates O(2)-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S](2+) cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O(2) exposure of [4Fe-4S](2+)-FNR. The [4Fe-4S](2+) cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S](2+) cluster by anaerobic incubation with DTT and Fe(2+) ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O(2)-exposed forms of O(2)-sensitive [4Fe-4S] clusters in radical S-adenosylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O(2) sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters.