scholarly journals The Vitamin B 12 -Dependent Photoreceptor AerR Relieves Photosystem Gene Repression by Extending the Interaction of CrtJ with Photosystem Promoters

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Mingxu Fang ◽  
Carl E. Bauer
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Light Absorption ◽  
Growth Conditions ◽  
Binding Activity ◽  
Regulatory Function ◽  
Vitamin B ◽  
Dna Binding Activity

ABSTRACT Purple nonsulfur bacteria adapt their physiology to a wide variety of environmental conditions often through the control of transcription. One of the main transcription factors involved in controlling expression of the Rhodobacter capsulatus photosystem is CrtJ, which functions as an aerobic repressor of photosystem genes. Recently, we reported that a vitamin B 12 binding antirepressor of CrtJ called AerR is required for anaerobic expression of the photosystem. However, the mechanism whereby AerR regulates CrtJ activity is unclear. In this study, we used a combination of next-generation sequencing and biochemical methods to globally identify genes under control of CrtJ and the role of AerR in controlling this regulation. Our results indicate that CrtJ has a much larger regulon than previously known, with a surprising regulatory function under both aerobic and anaerobic photosynthetic growth conditions. A combination of in vivo chromatin immunoprecipitation-DNA sequencing (ChIP-seq) and ChIP-seq and exonuclease digestion (ChIP-exo) studies and in vitro biochemical studies demonstrate that AerR forms a 1:2 complex with CrtJ (AerR-CrtJ 2 ) and that this complex binds to many promoters under photosynthetic conditions. The results of in vitro and in vivo DNA binding studies indicate that AerR-CrtJ 2 anaerobically forms an extended interaction with the bacteriochlorophyll bchC promoter to relieve repression by CrtJ. This is contrasted by aerobic growth conditions where CrtJ alone functions as an aerobic repressor of bchC expression. These results indicate that the DNA binding activity of CrtJ is modified by interacting with AerR in a redox-regulated manner and that this interaction alters CrtJ’s function. IMPORTANCE Photoreceptors control a wide range of physiology often by regulating downstream gene expression in response to light absorption via a bound chromophore. Different photoreceptors are known to utilize a number of different compounds for light absorption, including the use of such compounds as flavins, linearized tetrapyrroles (bilins), and carotenoids. Recently, a novel class of photoreceptors that use vitamin B 12 (cobalamin) as a blue-light-absorbing chromophore have been described. In this study, we analyzed the mechanism by which the vitamin B 12 binding photoreceptor AerR controls the DNA binding activity of the photosystem regulator CrtJ. This study shows that a direct interaction between the vitamin B 12 binding photoreceptor AerR with CrtJ modulates CrtJ binding to DNA and importantly, the regulatory outcome of gene expression, as shown here with photosystem promoters.

The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽  
1991 ◽  
Vol 113 (1) ◽  
pp. 245-255 ◽  
Author(s):  
M. Van Doren ◽  
H.M. Ellis ◽  
J.W. Posakony
Keyword(s):  
Dna Binding ◽  
Cell Fate ◽  
Protein Complexes ◽  
Competitive Inhibitor ◽  
Binding Activity ◽  
Regulatory Function ◽  
Biochemical Basis ◽  
Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

scholarly journals NAD+ Modulates p53 DNA Binding Specificity and Function

2004 ◽  
Vol 24 (22) ◽  
pp. 9958-9967 ◽  
Author(s):  
Kevin G. McLure ◽  
Masatoshi Takagi ◽  
Michael B. Kastan
Keyword(s):  
Dna Damage ◽  
Dna Binding ◽  
Binding Specificity ◽  
Binding Activity ◽  
Vitamin B ◽  
Cancer Therapies ◽  
Dna Binding Activity ◽  
Dna Binding Specificity

ABSTRACT DNA damage induces p53 DNA binding activity, which affects tumorigenesis, tumor responses to therapies, and the toxicities of cancer therapies (B. Vogelstein, D. Lane, and A. J. Levine, Nature 408:307-310, 2000; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Both transcriptional and transcription-independent activities of p53 contribute to DNA damage-induced cell cycle arrest, apoptosis, and aneuploidy prevention (M. B. Kastan et al., Cell 71:587-597, 1992; K. H. Vousden and X. Lu, Nat. Rev. Cancer 2:594-604, 2002). Small-molecule manipulation of p53 DNA binding activity has been an elusive goal, but here we show that NAD+ binds to p53 tetramers, induces a conformational change, and modulates p53 DNA binding specificity in vitro. Niacinamide (vitamin B3) increases the rate of intracellular NAD+ synthesis, alters radiation-induced p53 DNA binding specificity, and modulates activation of a subset of p53 transcriptional targets. These effects are likely due to a direct effect of NAD+ on p53, as a molecule structurally related to part of NAD+, TDP, also inhibits p53 DNA binding, and the TDP precursor, thiamine (vitamin B1), inhibits intracellular p53 activity. Niacinamide and thiamine affect two p53-regulated cellular responses to ionizing radiation: rereplication and apoptosis. Thus, niacinamide and thiamine form a novel basis for the development of small molecules that affect p53 function in vivo, and these results suggest that changes in cellular energy metabolism may regulate p53.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

Extinction of insulin gene expression in hybrids between beta cells and fibroblasts is accompanied by loss of the putative beta-cell-specific transcription factor IEF1

1991 ◽  
Vol 11 (3) ◽  
pp. 1547-1552
Author(s):  
D Leshkowitz ◽  
M D Walker
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Hybrid Cells ◽  
Dna Binding Activity ◽  
Insulin Producing Cells ◽  
Insulin Gene Expression ◽  
Insulinoma Cells

Insulin-producing cells and fibroblasts were fused to produce hybrid lines. In hybrids derived from both hamster and rat insulinoma cells, no insulin mRNA could be detected in any of seven lines examined by Northern (RNA) analysis despite the presence in each line of the insulin genes of both parental cells. Hybrid cells were transfected with recombinant chloramphenicol acetyltransferase plasmids containing defined segments of the rat insulin I gene 5' flank. We observed no transcriptional activity of the intact insulin enhancer or of IEB2, a critical cis-acting element of the insulin enhancer. IEB2 has previously been shown to interact in vitro with IEF1, a DNA-binding activity observed selectively in insulin-producing cells. Hybrid cells showed no detectable IEF1 activity. Furthermore, the insulin enhancer was unable to reduce transcription directed by the Moloney sarcoma virus enhancer in a double-enhancer construct. Thus, extinction of insulin gene expression in the hybrids apparently does not operate through a direct action of repressors on the insulin enhancer; rather, extinction is accompanied by, and may be caused by, reduced DNA-binding activity of the putative transcriptional activator IEF1.

Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

1989 ◽  
Vol 9 (6) ◽  
pp. 2464-2476
Author(s):  
M Cockell ◽  
B J Stevenson ◽  
M Strubin ◽  
O Hagenbüchle ◽  
P K Wellauer
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Binding Activity ◽  
Alpha Amylase ◽  
Dna Binding Activity ◽  
A Cell ◽  
Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

scholarly journals The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication

2004 ◽  
Vol 85 (7) ◽  
pp. 2001-2013 ◽  
Author(s):  
Koen W. R. van Cleef ◽  
Wendy M. A. Scaf ◽  
Karen Maes ◽  
Suzanne J. F. Kaptein ◽  
Erik Beuken ◽  
...  
Keyword(s):  
Dna Binding ◽  
Virus Replication ◽  
Deletion Mutant ◽  
Nuclear Protein ◽  
Human Herpesvirus ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Double Stranded Dna

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas.

1989 ◽  
Vol 9 (6) ◽  
pp. 2464-2476 ◽  
Author(s):  
M Cockell ◽  
B J Stevenson ◽  
M Strubin ◽  
O Hagenbüchle ◽  
P K Wellauer
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Binding Activity ◽  
Alpha Amylase ◽  
Dna Binding Activity ◽  
A Cell ◽  
Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

scholarly journals Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

1995 ◽  
Vol 15 (10) ◽  
pp. 5552-5562 ◽  
Author(s):  
E Roulet ◽  
M T Armentero ◽  
G Krey ◽  
B Corthésy ◽  
C Dreyer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
New Members ◽  
Binding Domains ◽  
Dna Binding Activity

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

scholarly journals Reduced sulphydryl groups are required for DNA binding of Ku protein

1993 ◽  
Vol 293 (3) ◽  
pp. 769-774 ◽  
Author(s):  
W W Zhang ◽  
M Yaneva
Keyword(s):  
Dna Binding ◽  
Protein A ◽  
Binding Activity ◽  
Heat Inactivation ◽  
Cysteine Residues ◽  
Dna Binding Activity ◽  
Sulphydryl Groups ◽  
Ku Protein

The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.

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