scholarly journals Functional Characterization of a Testis-Specific DNA Binding Activity at the H19/Igf2 Imprinting Control Region

2003 ◽  
Vol 23 (22) ◽  
pp. 8345-8351 ◽  
Author(s):  
Aaron B. Bowman ◽  
John M. Levorse ◽  
Robert S. Ingram ◽  
Shirley M. Tilghman
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Control Region ◽  
Binding Sites ◽  
Germ Line ◽  
Binding Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Epigenetic State ◽  
Imprinting Control Region

ABSTRACT The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.

Androgen-dependent and DNA binding-independent association of androgen receptor with chromatic regions coding androgen-induced non-coding RNAs

2021 ◽  
Author(s):  
Takahiro Sawada ◽  
Koichi Nishimura ◽  
Jinichi Mori ◽  
Yoshiaki Kanemoto ◽  
Alexander Kouzmenko ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Specific Antigen ◽  
Binding Activity ◽  
Lncap Cells ◽  
Coding Regions ◽  
Dna Binding Activity ◽  
Genome Wide ◽  
Non Coding Rnas ◽  
Independent Association

Abstract Androgen induces the binding of its receptor (AR) to androgen-responsive elements (AREs), while genome-wide studies showed that most androgen-induced AR binding sites on chromatin were unrelated to AREs. Enhancer RNAs (eRNAs), a class of non-coding RNAs(ncRNAs), are transcribed from super-enhancers (SEs), and trigger the formation of large ribonucleoprotein (RNP) condensates of transcription factors. By in silico search, an SE is found to be located on the locus of KLK3 that encodes prostate specific antigen (PSA). On the KLK3 SE, androgen-induced expression of ncRNAs was detected and designated as KLK3eRNAs in LNCaP cells, and androgen-induced association of AR and FOXA1 on the KLK3eRNA coding regions was detected. Such androgen-induced association of an AR mutant lacking DNA binding activity on the KLK3eRNA coding regions was undetectable on an exogenous ARE. Thus, the present findings suggest a molecular basis of androgen-induced association of AR with chromatin on ARE-unrelated sequences.

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.

Analysis Of E2A Point Mutations In B Cell Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4850-4850
Author(s):  
Eroica Soans ◽  
John K Choi
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Binding Activity ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Cell Leukemia ◽  
Wild Type ◽  
Dna Binding Activity ◽  
E Box ◽  
B Cell Leukemia

Abstract Introduction TCF3 encodes for E2A protein, which belongs to the helix loop helix transcription factor family. E2A activates transcription of downstream genes by binding to E-box motifs as a homo or hetero dimer. E2A plays an important role in B lymphocyte development. Therefore deletion or mutations in TCF3 or even lowered activity of E2A are causes of B cell leukemia and lymphomas. Recently, three mutations V557E, D561E and N551K in E2A were isolated in Burkitt’s lymphoma (Schmitz, Young et al. 2012). The first two mutations are present in the homo dimerization region of E2A while N551K is present in the DNA binding region. Though the paper enumerated role of TCF3 in Burkitt’s lymphoma but the significance of these TCF3 mutations or mechanism needed further characterization. We hypothesized that these TCF3 mutations have an alternate mechanism as compared to wild type TCF3 and therefore may affect B cell development. Methods We characterized three TCF3 mutants by cloning them into in MIGR1 backbone using TOPO cloning. E2A activity was measured using an E2A-specific luciferase reporter assay in 293T cells. DNA binding activity was measured using a DNA protein binding colorimetric assay. Results V557E and D561E mutants have lower activity as compared to wild type E2A as studied using E2A-specific luciferase reporter assay; while N551K showed no activity in the same assay as compared to wild type E2A activity. Similarly V557E and D561 form weaker bonds with the E box motifs while N551K showed no DNA binding activity as studied using colorimetric DNA-protein binding assay. The plasmid expressions were verified using western blot analysis. Conclusion Our findings suggest mutations V557E and D561E may follow a similar pathway as wild-type E2A but have lower activity. The N551K mutation has an alternate pathway to wild type TCF3 that may impact B cell proliferation, survival and development. Disclosures: No relevant conflicts of interest to declare.

scholarly journals DNA-binding properties of the Drosophila melanogaster zeste gene product.

1988 ◽  
Vol 8 (2) ◽  
pp. 615-623 ◽  
Author(s):  
A Mansukhani ◽  
A Crickmore ◽  
P W Sherwood ◽  
M L Goldberg
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Fusion Proteins ◽  
Genetic Interaction ◽  
Binding Activity ◽  
Transcriptional Unit ◽  
Dna Binding Activity ◽  
Zinc Finger Motifs ◽  
White Locus

The ability of the zeste moiety of beta-galactosidase-zeste fusion proteins synthesized in Escherichia coli to bind specific DNA sequences was examined. Such fusion proteins recognize a region of the white locus upstream of the start of transcription; this region has previously been shown to be required for genetic interaction between the zeste and white loci. Another strong binding site was localized to a region between 50 and 205 nucleotides before the start of the Ubx transcriptional unit; expression of the bithorax complex is also known to be influenced by the zeste locus. Weaker binding sites were also seen in the vicinity of the bxd and Sgs-4 genes, but it is currently unclear whether these binding sites play a role in transvection effects. The DNA-binding activity of the zeste protein is restricted to a domain of approximately 90 amino acids near the N terminus. This domain does not appear to contain homeobox or zinc finger motifs found in other DNA-binding proteins. The DNA-binding domain is not disrupted by any currently characterized zeste mutations.

scholarly journals Cu,Zn-superoxide dismutase deficiency in mice leads to organ-specific increase in oxidatively damaged DNA and NF-κB1 protein activity.

2010 ◽  
Vol 57 (4) ◽  
Author(s):  
Agnieszka Siomek ◽  
Kamil Brzoska ◽  
Barbara Sochanowicz ◽  
Daniel Gackowski ◽  
Rafal Rozalski ◽  
...  
Keyword(s):  
Dna Damage ◽  
Superoxide Dismutase ◽  
Dna Binding ◽  
Urinary Excretion ◽  
Oxidative Dna Damage ◽  
Binding Activity ◽  
Wild Type ◽  
Dna Binding Activity ◽  
Organ Specific ◽  
Damaged Dna

Earlier experimental studies have demonstrated that: i) Cu,Zn-superoxide dismutase deficiency leads to oxidative stress and carcinogenesis; ii) dysregulation of NF-κB pathway can mediate a wide variety of diseases, including cancer. Therefore, we decided, for the first time, to examine the level of oxidative DNA damage and the DNA binding activity of NF-κB proteins in SOD1 knockout, heterozygous and wild-type mice. Two kinds of biomarkers of oxidatively damaged DNA: urinary excretion of 8-oxodG and 8-oxoGua, and the level of oxidatively damaged DNA were analysed using HPLC-GC-MS and HPLC-EC. The DNA binding activity of p50 and p65 proteins in a nuclear extracts was assessed using NF-κB p50/p65 EZ-TFA transcription factor assay. These parameters were determined in the brain, liver, kidney and urine of SOD1 knockout, heterozygous and wild-type mice. The level of 8-oxodG in DNA was higher in the liver and kidney of knockout mice than in wild type. No differences were found in urinary excretion of 8-oxoGua and 8-oxodG between wild type and the SOD1-deficient animals. The activity of the p50 protein was higher in the kidneys, but surprisingly not in the livers of SOD1-deficient mice, whereas p65 activity did not show any variability. Our results indicate that in Cu,Zn-SOD-deficient animals the level of oxidative DNA damage and NF-κB1 activity are elevated in certain organs only, which may provide some explanation for organ-specific ROS-induced carcinogenesis.

Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites

1990 ◽  
Vol 10 (2) ◽  
pp. 859-862
Author(s):  
G M Santangelo ◽  
J Tornow
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Heterologous Gene ◽  
Dna Binding Activity ◽  
Glycolytic Gene

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences

Development ◽  
1988 ◽  
Vol 104 (Supplement) ◽  
pp. 75-83 ◽  
Author(s):  
Allen Laughon ◽  
William Howell ◽  
Matthew P. Scott
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Binding Activity ◽  
Regulatory Function ◽  
Temperature Sensitive ◽  
Structural Domain ◽  
Dna Binding Activity ◽  
Dna Restriction Fragments

The ANT-C gene cluster is part of a network of genes that govern pattern formation in the development of Drosophila. The ANT-C genes encode proteins that contain a conserved 60 amino acid sequence, the homeodomain. Here we show that the homeodomains encoded by two of the ANT-C loci confer sequencespecific DNA-binding activity. The DNA sequence specificities of the Dfd and ftz homeodomains appear to overlap completely in vitro, indicating that differences in regulatory specificity among ANT-C and BX-C proteins (assuming that differences exist) must be a consequence of the nonconserved protein sequences found outside of the homeodomains. Deletions that remove sequences from either end of the ftz homeodomain abolish DNA-binding activity, consistent with the commonly held assumption that the homeodomain is a structural domain. The relevance of in vitro DNA-binding experiments to the regulatory function of ftz is supported by our finding that a temperature-sensitive ftz mutation that causes a pairwise fusion of embryonic segments also reduces the affinity of the ftz homeodomain for DNA. Restriction fragments containing ftz homeodomain binding sites were identified within a 90 kb stretch of DNA extending the Antp P1 and P2 promoters. Binding sites appear to be clustered near the P1 promoter but also occur near P2 and in the region between the two. The task remains of determining which of these sequences mediate regulation of Antp by ftz or by other genes that encode closely related homeodomains.

scholarly journals Protein-DNA interactions at the major and minor promoters of the divergently transcribed dhfr and rep3 genes during the Chinese hamster ovary cell cycle.

1996 ◽  
Vol 16 (2) ◽  
pp. 634-647 ◽  
Author(s):  
J Wells ◽  
P Held ◽  
S Illenye ◽  
N H Heintz
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Dna Binding ◽  
Binding Sites ◽  
Binding Activity ◽  
Dnase I ◽  
S Phase ◽  
Ovary Cell ◽  
Dna Binding Activity ◽  
Genomic Footprinting

In mammals, two TATA-less bidirectional promoters regulate expression of the divergently transcribed dihydrofolate reductase (dhfr) and rep3 genes. In CHOC 400 cells, dhfr mRNA levels increase about fourfold during the G1-to-S phase transition of the cell cycle, whereas the levels of rep3 transcripts vary less than twofold during this time. To assess the role of DNA-binding proteins in transcriptional regulation of the dhfr and rep3 genes, the major and minor dhfr-rep3 promoter regions were analyzed by high-resolution genomic footprinting during the cell cycle. At the major dhfr promoter, prominent DNase I footprints over four upstream Sp1 binding sites did not vary throughout G1 and entry into the S phase. Genomic footprinting revealed that a protein is constitutively bound to the overlapping E2F sites throughout the G1-to-S phase transition, an interaction that is most evident on the transcribed template strand. On the nontranscribed strand, multiple changes in the DNase I cleavage pattern are observed during transit through G1 and entry into the S phase. By using gel mobility shift assays and a series of sequence-specific probes, two different species of E2F were shown to interact with the dhfr promoter during the cell cycle. The DNA binding activity of one E2F species, which preferentially recognizes the sequence TTTGGCGC, did not vary significantly during the cell cycle. The DNA binding activity of the second E2F species, which preferentially recognizes the sequence TTTCGCGC, increased during the G1-to-S phase transition. Together, these results indicate that Sp1 and the species of E2F that binds TTTGGCGC participate in the formation of a basal transcription complex, while the species of E2F that binds TTTCGCGC regulates dhfr gene expression during the G1-to-S phase transition. At the minor promoter, DNase I footprints at a consensus c-Myc binding site and three Sp1 binding sites showed little variation during the G1-to-S phase transition. In addition to protein binding at sequences known to be involved in the regulation of transcription, genomic footprinting of the entire promoter region also showed that a protein factor is constitutively bound to the first intron of the rep3 gene.

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