Site-specific DNA-binding Activity Research Articles

Epigenetic Dynamics in the Function of T-Lineage Regulatory Factor Bcl11b.

The transcription factor Bcl11b is critically required to support the development of diverse cell types, including T lymphocytes, type 2 innate lymphoid cells, neurons, craniofacial mesenchyme and keratinocytes. Although in T cell development its onset of expression is tightly linked to T-lymphoid lineage commitment, the Bcl11b protein in fact regulates substantially different sets of genes in different lymphocyte populations, playing strongly context-dependent roles. Somewhat unusually for lineage-defining transcription factors with site-specific DNA binding activity, much of the reported chromatin binding of Bcl11b appears to be indirect, or guided in large part by interactions with other transcription factors. We describe evidence suggesting that a further way in which Bcl11b exerts such distinct stage-dependent functions is by nucleating changes in regional suites of epigenetic modifications through recruitment of multiple families of chromatin-modifying enzyme complexes. Herein we explore what is - and what remains to be - understood of the roles of Bcl11b, its cofactors, and how it modifies the epigenetic state of the cell to enforce its diverse set of context-specific transcriptional and developmental programs.

Nonspecific DNA binding by P1 ParA determines the distribution of plasmid partition and repressor activities

The faithful segregation, or "partition," of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a "super-repressor." In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.

Impact of cadmium, cobalt and nickel on sequence-specific DNA binding of p63 and p73 in vitro and in cells

Site-specific DNA recognition and binding activity belong to common attributes of all three members of tumor suppressor p53 family proteins: p53, p63 and p73. It was previously shown that heavy metals can affect p53 conformation, sequence-specific binding and suppress p53 response to DNA damage. Here we report for the first time that cadmium, nickel and cobalt, which have already been shown to disturb various DNA repair mechanisms, can also influence p63 and p73 sequence-specific DNA binding activity and transactivation of p53 family target genes. Based on results of electrophoretic mobility shift assay and luciferase reporter assay, we conclude that cadmium inhibits sequence-specific binding of all three core domains to p53 consensus sequences and abolishes transactivation of several promoters (e.g. BAX and MDM2) by 50μM concentrations. In the presence of specific DNA, all p53 family core domains were partially protected against loss of DNA binding activity due to cadmium treatment. Effective cadmium concentration to abolish DNA–protein interactions was about two times higher for p63 and p73 proteins than for p53. Furthermore, we detected partial reversibility of cadmium inhibition for all p53 family members by EDTA. DTT was able to reverse cadmium inhibition only for p53 and p73. Nickel and cobalt abolished DNA–p53 interaction at sub-millimolar concentrations while inhibition of p63 and p73 DNA binding was observed at millimolar concentrations. In summary, cadmium strongly inhibits p53, p63 and p73 DNA binding in vitro and in cells in comparison to nickel and cobalt. The role of cadmium inhibition of p53 tumor suppressor family in carcinogenesis is discussed.

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.

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.

Repression of the miR-17-92 cluster by p53 has an important function in hypoxia-induced apoptosis

We here report that miR-17-92 cluster is a novel target for p53-mediated transcriptional repression under hypoxia. We found the expression levels of miR-17-92 cluster were reduced in hypoxia-treated cells containing wild-type p53, but were unchanged in hypoxia-treated p53-deficient cells. The repression of miR-17-92 cluster under hypoxia is independent of c-Myc. Luciferase reporter assays mapped the region responding to p53-mediated repression to a p53-binding site in the proximal region of the miR-17-92 promoter. Chromatin immunoprecipitation (ChIP), Re-ChIP and gel retardation assays revealed that the binding sites for p53- and the TATA-binding protein (TBP) overlap within the miR-17-92 promoter; these proteins were found to compete for binding. Finally, we show that pri-miR-17-92 expression correlated well with p53 status in colorectal carcinomas. Over-express miR-17-92 cluster markedly inhibits hypoxia-induced apoptosis, whereas blocked miR-17-5p and miR-20a sensitize the cells to hypoxia-induced apoptosis. These data indicated that p53-mediated repression of miR-17-92 expression likely has an important function in hypoxia-induced apoptosis, and thus further our understanding of the tumour suppressive function of p53.

Spectrometric Studies on Interaction Between Metal Ions and DNA-Binding Domain of Tumor Suppressor Protein p53

Abstract Site-specific recognition and DNA-binding activity of tumor suppressor protein p53 are crucial for its tumor suppressor function. Previous reports have shown that metal ions could affect specific recognition and DNA-binding activity of p53 DNA-binding domain (p53DBD). In this study, we investigated the binding reaction between p53DBD and nine kinds of metal ions by fluorescence titration method, the binding affinity of metal ions to p53DBD is Fe 3+ > Zn 2+ > Cu 2+ > Ca 2+ > Mg 2+ > Ba 2+ > Mn 2+ > Ni 2+ > Co 2+ . Analysis of the far-UV CD data suggested that the binding of Ba 2+ , Ca 2+ , Co 2+ , Mn 2+ and Ni 2+ did not induce changes in protein secondary structure. The binding of Zn 2+ , Mg 2+ and Fe 3+ induced a subtle conformational change, whereas the binding of Cu 2+ resulted in a lot of loss in its helical content. Analysis of 8-anilino-1-naphthalenesulfonic acid binding data showed that the binding of Mg 2+ enhanced hydrophobic exposure on protein surface such as Zn 2+ , whereas Fe 3+ decreased hydrophobic exposure. Therefore, Mg 2+ and Fe 3+ may be one of potential factors to affect or regulate the transactivation of p53.

Influence of Magnesium Ion on the Binding of p53 DNA-Binding Domain to DNA-Response Elements

Site-specific recognition and DNA-binding activity of p53 are crucial for its tumour suppressor function. Previous reports have shown that metal ions can affect the specific recognition and DNA-binding activity of p53DBD. Here we firstly report that magnesium ion can bind to the protein and influence its DNA-binding activity. To elucidate the nature and the effect of metal ions in the reaction chemistry, we utilized endogenous tryptophan fluorescence to quantitate the interaction between p53DBD and metal ions. The K(a) value for the binding of Mg(2+) to the protein is 1.88 x 10(3) M(-1). Analysis of the CD data clearly suggested that the binding of magnesium ion induced a subtle conformational change rather than a radical modification of the overall protein architecture. Based on the results of electrophoretic mobility shift assays and fluorescence experiments, we concluded that the binding of Mg(2+) significantly stimulated the binding of the protein to DNA in a sequence-independent manner, which differed from that of zinc ions in a sequence-specific manner. Based on these results and the fact that Mg(2+) exists at relatively high concentration in the cell, we propose that Mg(2+) is one of potential factors to affect or regulate the transactivation of p53.

EBV Is Necessary for Proliferation of Dually Infected Primary Effusion Lymphoma Cells

Epstein Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are found together in approximately 80% of primary effusion lymphomas (PEL), but their contribution to these cancers is unclear. We found that dominant-negative derivatives of EBNA1 inhibited EBV-positive PEL cells from forming colonies. Those rare PEL cells that proliferated after expression of the dominant-negative derivatives usually expressed these derivatives at low or undetectable levels and continued to maintain their EBV genomes. Those proliferating cells expressing higher levels of the derivatives expressed mutant derivatives that could not bind DNA. These findings indicate that EBV is required to sustain proliferation, as measured by colony formation of dually infected PEL cells. The dominant-negative derivatives of EBNA1 had no effect on the colony-forming ability of five EBV-negative, KSHV-negative hematopoietic cell lines. Surprisingly, they did inhibit the colony-forming ability of EBV-negative, KSHV-positive PEL cells. The small fraction of cells that continued to proliferate expressed only mutants of the EBNA1 derivatives that could no longer bind DNA. These findings indicate that the site-specific DNA-binding activity of EBNA1 or its derivatives when expressed efficiently in EBV-negative, KSHV-positive PEL cells inhibits their colony formation possibly through their binding to the KSHV genome.

RNA-mediated response to heat shock in mammalian cells

The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and a previously unknown non-coding RNA that we term HSR1 (heat shock RNA-1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation.

Regulation of Iron Transport inStreptococcus pneumoniaeby RitR, an Orphan Response Regulator

RitR (formerly RR489) is an orphan two-component signal transduction response regulator in Streptococcus pneumoniae that has been shown to be required for lung pathogenicity. In the present study, by using the rough strain R800, inactivation of the orphan response regulator gene ritR by allele replacement reduced pathogenicity in a cyclophosphamide-treated mouse lung model but not in a thigh model, suggesting a role for RitR in regulation of tissue-specific virulence factors. Analysis of changes in genome-wide transcript mRNA levels associated with the inactivation of ritR compared to wild-type cells was performed by the use of high-density DNA microarrays. Genes with a change in transcript abundance associated with inactivation of ritR included piuB, encoding an Fe permease subunit, and piuA, encoding an Fe carrier-binding protein. In addition, a dpr ortholog, encoding an H(2)O(2) resistance protein that has been shown to reduce synthesis of reactive oxygen intermediates, was activated in the wild-type (ritR(+)) strain. Microarray experiments suggested that RitR represses Fe uptake in vitro by negatively regulating the Piu hemin-iron transport system. Footprinting experiments confirmed site-specific DNA-binding activity for RitR and identified three binding sites that partly overlap the +1 site for transcription initiation upstream of piuB. Transcripts belonging to other gene categories found to be differentially expressed in our array studies include those associated with (i) H(2)O(2) resistance, (ii) repair of DNA damage, (iii) sugar transport and capsule biosynthesis, and (iv) two-component signal transduction elements. These observations suggest that RitR is an important response regulator whose primary role is to maintain iron homeostasis in S. pneumoniae. The name ritR (repressor of iron transport) for the orphan response regulator gene, rr489, is proposed.

Polyomavirus hr-t mutant-specific induction of a G2/M cell-cycle arrest that is not overcome by the expression of middle T and/or small T

The polyomavirus hr-t class of mutants has served as a major prototype to study the function of middle T + small T in the virus lytic cycle (T.L. Benjamin, 1982, Biochem. Biophys. Acta 695 (2), 69–95). The properties of these middle T + small T defective mutants were defined by comparisons with “wild-type” strains reconstructed by marker rescue. Similar comparisons in the A2 genetic background have revealed a number of differences (L. Chen and M. Fluck, 2001b, J. Virol. 75, 8380–8389). Here we describe a major divergence in their effects on cell-cycle progression of both permissive mouse NIH3T3 cells and semipermissive Fischer rat FR3T3 cells. Infection of NIH3T3 or FR3T3 cells in serum-rich medium with wild-type A2 (WTA2) or WTA2-derived middle T + small T-defective mutants did not perturb cell cycling, tested up to entry into the third cycle. In contrast, infection with four hr-t mutants analyzed, examined in detail with mutant B2, resulted in an accumulation of cells in G2/M in a dose-dependent and serum-independent manner. The arrest began in the first cell cycle. At multiplicities of infection above 10 PFU/cell, 50–80% of the cell population became arrested by the end of the second cycle. FR3T3 arrested cells detached from the monolayer with a rounded up morphology. Three other hr-t mutants investigated were also found to arrest cells in G2/M. Expression of middle T and/or small T either in trans or in cis did not abrogate this cell-cycle arrest, as demonstrated in the latter case with the middle T + small T expressing strain “wtB2” obtained by repair of the B2 deletion. In FR3T3 cells, the induction of a cell-cycle arrest by wtB2 was accompanied by a severe delay and reduction in neoplastic transformation relative to WTA2 used at equal dose. Mutation(s) in the C-terminal domain of large T antigen, upstream of the site-specific DNA binding activity, is necessary for the cell-cycle block. The possible causes for the cell-cycle block are discussed.

Structure, Function, and Aggregation of the Zinc-Free Form of the p53 DNA Binding Domain

The p53 DNA binding domain (DBD) contains a single bound zinc ion that is essential for activity. Zinc remains bound to wild-type DBD at temperatures below 30 degrees C; however, it rapidly dissociates at physiological temperature. The resulting zinc-free protein (apoDBD) is folded and stable. NMR spectra reveal that the DNA binding surface is altered in the absence of Zn(2+). Fluorescence anisotropy studies show that Zn(2+) removal abolishes site-specific DNA binding activity, although full nonspecific DNA binding affinity is retained. Surprisingly, the majority of tumorigenic mutations that destabilize DBD do not appreciably destabilize apoDBD. The R175H mutation instead substantially accelerates the rate of Zn(2+) loss. A considerable fraction of cellular p53 may therefore exist in the folded zinc-free form, especially when tumorigenic mutations are present. ApoDBD appears to promote aggregation of zinc-bound DBD via a nucleation-growth process. These data provide an explanation for the dominant negative phenotype exhibited by many mutations. Through a combination of induced p53 aggregation and diminished site-specific DNA binding activity, Zn(2+) loss may represent a significant inactivation pathway for p53 in the cell.

Identification of Active Site Residues of the Adeno-associated Virus Type 2 Rep Endonuclease

Adeno-associated virus type 2 Rep endonuclease activity is necessary for both viral DNA replication and site-specific integration of the viral genome into human chromosome 19. The biochemical activities required for site-specific endonuclease activity (namely specific DNA binding and transesterification activity) have been mapped to the amino-terminal domain of the AAV2 Rep protein. The amino-terminal 208 amino acids are alone sufficient for site-specific endonuclease activity, and nicking by this domain is metal-dependent. To identify this metal-binding site, we have employed a cysteine mutagenesis approach that targets conserved acidic amino acids. By using this technique, we provide functional biochemical data supporting a role for glutamate 83 in the coordination of metal ions in the context of Rep endonuclease activity. In addition, our biochemical data suggest that glutamate 164, although not involved in the coordination of metal ions, is closely associated with the active site. Thus, in lieu of a crystal structure for the AAV type 2 amino-terminal domain, our data corroborate the recently published structural studies of the AAV type 5 endonuclease and suggest that although the two enzymes are not highly conserved with respect to the AAV family, their active sites are highly conserved.

P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities.

The partition system of P1 plasmids is composed of two proteins, ParA and ParB, and a cis-acting site parS. parS is wrapped around ParB and Escherichia coli IHF protein in a higher order nucleoprotein complex called the partition complex. ParA is an ATPase that autoregulates the expression of the par operon and has an essential but unknown function in the partition process. In this study we demonstrate a direct interaction between ParA and the P1 partition complex. The interaction was strictly dependent on ParB and ATP. The consequence of this interaction depended on the ParB concentration. At high ParB levels, ParA was recruited to the partition complex via a ParA-ParB interaction, but at low ParB levels, ParA removed or disassembled ParB from the partition complex. ADP could not support these interactions, but could promote the site-specific DNA binding activity of ParA to parOP, the operator of the par operon. Conversely, ATP could not support a stable interaction of ParA with parOP in this assay. Our data suggest that ParA-ADP is the repressor of the par operon, and ParA-ATP, by interacting with the partition complex, plays a direct role in partition. Therefore, one role of adenine nucleotide binding and hydrolysis by ParA is that of a molecular switch controlling entry into two separate pathways in which ParA plays different roles.

DNA damage activates p53 through a phosphorylation-acetylation cascade.

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

The papillomavirus E1 protein forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities.

The E1 protein from bovine papillomavirus has site-specific DNA binding activity, DNA helicase activity, and DNA-dependent ATPase activity consistent with the properties of an initiator protein. Here we have identified and characterized a novel oligomeric form of E1 that is associated with the ATPase and DNA helicase activities and whose formation is strongly stimulated by single-stranded DNA. This oligomeric form corresponds to a hexamer of E1.

DNA binding by the KP repressor protein inhibits P-element transposase activity in vitro.

P elements are a family of mobile DNA elements found in Drosophila. P-element transposition is tightly regulated, and P-element-encoded repressor proteins are responsible for inhibiting transposition in vivo. To investigate the molecular mechanisms by which one of these repressors, the KP protein, inhibits transposition, a variety of mutant KP proteins were prepared and tested for their biochemical activities. The repressor activities of the wild-type and mutant KP proteins were tested in vitro using several different assays for P-element transposase activity. These studies indicate that the site-specific DNA-binding activity of the KP protein is essential for repressing transposase activity. The DNA-binding domain of the KP repressor protein is also shared with the transposase protein and resides in the N-terminal 88 amino acids. Within this region, there is a C2HC putative metal-binding motif that is required for site-specific DNA binding. In vitro the KP protein inhibits transposition by competing with the transposase enzyme for DNA-binding sites near the P-element termini.

A transcriptional partner for MAD proteins in TGF-beta signalling.

The transforming-growth-factor-beta (TGF-beta) superfamily is critical for establishing mesoderm during early embryogenesis in Xenopus. The transcriptional activation of Mix.2, an immediate-early response gene specific to activin-like members of the TGF-beta superfamily, is associated with the rapid appearance of a site-specific DNA-binding activity that recognizes a fifty-base-pair regulatory element known as ARE in the Mix.2 promoter. Cloning of the site-specific DNA-binding component of this activity revealed it to be a new winged-helix transcription factor and a direct target for signalling by the TGF-beta superfamily. XMAD2, a recently identified TGF-beta signal transducer, forms a complex with the transcription factor in an activin-dependent fashion to generate an activated ARE-binding complex. A model is proposed to explain how TGF-beta superfamily signals might regulate the expression of specific genes in the early embryo.

The p53-mediated G1 checkpoint is retained in tumorigenic rat embryo fibroblast clones transformed by the human papillomavirus type 16 E7 gene and EJ-ras.

Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.