Dorsal-Mediated Repression Requires the Formation of a Multiprotein Repression Complex at the Ventral Silencer

ABSTRACT Dorsal functions as both an activator and repressor of transcription to determine dorsoventral fate in the Drosophila melanogaster embryo. Repression by Dorsal requires the corepressor Groucho (Gro) and is mediated by silencers termed ventral repression regions (VRRs). A VRR in zerknüllt(zen) contains Dorsal binding sites as well as an essential element termed AT2. We have identified and purified an AT2 DNA binding activity in embryos and shown it to consist of cut(ct) and dead ringer (dri) gene products. Studies of loss-of-function mutations in ct anddri demonstrate that both genes are required for the activity of the AT2 site. Dorsal and Dri both bind Gro, acting cooperatively to recruit it to the DNA. Thus, ventral repression may require the formation of a multiprotein complex at the VRR. This complex includes Dorsal, Gro, and additional DNA binding proteins, which appear to convert Dorsal from an activator to a repressor by enabling it to recruit Gro to the template. By showing how binding site context can dramatically alter transcription factor function, these findings help clarify the mechanisms responsible for the regulatory specificity of transcription factors.

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

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab135 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.615 ◽

1988 ◽

Vol 8 (2) ◽

pp. 615-623 ◽

Cited By ~ 11

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.

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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

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.859-862.1990 ◽

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.

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

Development ◽

10.1242/dev.104.supplement.75 ◽

1988 ◽

Vol 104 (Supplement) ◽

pp. 75-83 ◽

Cited By ~ 2

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.

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Identification of Proteinase 3 (PR3) Gene Overexpresison and Protein Delocalization in Core Binding Factor Leukemias as a Mechanism Leading to Increased Chemosensitivity.

Blood ◽

10.1182/blood.v112.11.204.204 ◽

2008 ◽

Vol 112 (11) ◽

pp. 204-204

Author(s):

Daniela Cilloni ◽

Ilaria Defilippi ◽

Sonia Carturan ◽

Chiara Maffè ◽

Marisa Pautasso ◽

...

Keyword(s):

Dna Binding ◽

Cell Lines ◽

Mean Value ◽

Binding Activity ◽

Proteinase 3 ◽

Loss Of Function ◽

Core Binding Factor ◽

Secondary Aml ◽

Dna Binding Activity ◽

Binding Factor

Abstract Proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells. When abnormally expressed it confers factor-independent growth to hematopoietic cells. The aim of this study was to investigate the role of PR3 gene in leukemic haematopoiesis. We analyzed the expression levels of PR3 by RQ-PCR in 113 BM samples collected from AML patients at diagnosis. The FAB distribution was as follows: M0=5, M1=12, M2=38, M3=12, M4=37, M5=5, M6=4. 19 patients were characterized by t(8;21) and 16 by inv(16). PR3 expression level was also analyzed in 57 BM and 42 PB samples from 88 MDS patients (44 RA, 32 RAEB and 12 secondary-AML) and in 15 BM and 40 PB samples from healthy volunteers. PR3 protein was analyzed by western blot (WB) and its localization determined by immunofluorescence assay using specific antibodies. The transcription factor C/EBPα, which negatively regulates PR3 expression was studied in parallel at the RNA and protein level by RQ-PCR and WB. The DNA binding activity of C/EBPα was investigated by EMSA assay. Gain and loss of function experiments were performed by transfecting COS and 293T cell lines with a plasmid containing the full length PR3 sequence and HL60, Me-1, and Kasumi cell lines with specific shRNA. We found that PR3 is significantly overexpressed in AML samples. The median value of 2−Δ ΔCt is 740, (range 15-5043). Interestingly, patients affected by Core Binding Factor leukemias showed significantly higher PR3 values compared to patients with normal karyotypes (NK) (p<0,0002 for t(8;21), p<0,001 for inv16) and lower C/EPBα levels. EMSA assay demonstrated the absence of C/EBPα DNA binding activity in CBF AML cells but not in NK AML. In addition, PR3 overexpression was detected in 60% of RA patients (mean value: 10, range 3–268), and in all the cases of RAEB (mean value 201: range:128–803) and secondary AML (mean value 589, range 207–7131). WB demonstrated the correlation between the mRNA and protein amount. Interestingly, immunofluorescence demonstrated the de-localization of the protein within the nucleous in CBF AML but it is completely cytoplasmatic in leukemic cells with normal karyotype and in MDS. Transfection experiments with PR3 plasmid demonstrated that PR3 overexpression results into a significantly increased proliferation and reduced apoptosis. By contrast transfection with shRNA triggers apoptosis and cell growth inhibition. In addition, WB demonstrated that nuclear PR3 is able to cleavage the p65subunit of NF-kB into a p56 isoform which lacks any transcriptional activity as confirmed by EMSA. In conclusion, PR3 gene expression and protein are significantly increased in AML and MDS, particularly in CBF leukemias in which the protein is not only increased but also completely delocalized within the nucleous. PR3 overepression My be due to a significant downmodulation of C/EBPα. Ectopic expression of PR3 induces increased proliferation and apoptosis arrest. The abnormal nuclear localization of PR3 in CBF leukemias results into the loss of function of NF-kB thus representing one mechanism of chemo sensitivity in this group of patients.

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Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3633 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3633-3641 ◽

Cited By ~ 39

Author(s):

T Chakraborty ◽

T J Brennan ◽

L Li ◽

D Edmondson ◽

E N Olson

Keyword(s):

Dna Binding ◽

Gene Activation ◽

Binding Activity ◽

Specific Gene ◽

Gene Products ◽

Basic Domain ◽

High Affinity ◽

Dna Binding Activity

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

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

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.634 ◽

1996 ◽

Vol 16 (2) ◽

pp. 634-647 ◽

Cited By ~ 32

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8345-8351.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8345-8351 ◽

Cited By ~ 15

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.

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