scholarly journals Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

1993 ◽  
Vol 13 (4) ◽  
pp. 2354-2365 ◽  
Author(s):  
K M Catron ◽  
N Iler ◽  
C Abate
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Equilibrium Constants ◽  
Binding Specificity ◽  
Binding Activity ◽  
Selection Strategy ◽  
Homeodomain Proteins ◽  
Control Regions

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins

1993 ◽  
Vol 13 (4) ◽  
pp. 2354-2365
Author(s):  
K M Catron ◽  
N Iler ◽  
C Abate
Keyword(s):  
Dna Binding ◽  
Binding Sites ◽  
Target Genes ◽  
Equilibrium Constants ◽  
Binding Specificity ◽  
Binding Activity ◽  
Selection Strategy ◽  
Homeodomain Proteins ◽  
Control Regions

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.

scholarly journals In vitro DNA-binding profile of transcription factors: methods and new insights

2011 ◽  
Vol 210 (1) ◽  
pp. 15-27 ◽  
Author(s):  
Jinke Wang ◽  
Jie Lu ◽  
Guangming Gu ◽  
Yingxun Liu
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Target Genes ◽  
Binding Specificity ◽  
Cell Physiology ◽  
Binding Profile ◽  
Dna Binding Specificity

The DNA-binding specificity of transcription factors (TFs) has broad impacts on cell physiology, cell development and in evolution. However, the DNA-binding specificity of most known TFs still remains unknown. The specificity of a TF protein is determined by its relative affinity to all possible binding sites. In recent years, the development of several in vitro techniques permits high-throughput determination of relative binding affinity of a TF to all possible k bp-long DNA sequences, thus greatly promoting the characterization of DNA-binding specificity of many known TFs. All DNA sequences that can be bound by a TF with various binding affinities form their DNA-binding profile (DBP). The DBP is important to generate an accurate DNA-binding model, identify all DNA-binding sites and target genes of TFs in the whole genome, and build transcription regulatory network. This study reviewed these techniques, especially two master techniques: double-stranded DNA microarray and systematic evolution of ligands by exponential enrichment in combination with parallel DNA sequencing techniques (SELEX-seq).

scholarly journals Sequence-specific targeting of nuclear signal transduction pathways by homeodomain proteins.

1995 ◽  
Vol 15 (6) ◽  
pp. 3318-3326 ◽  
Author(s):  
D A Grueneberg ◽  
K J Simon ◽  
K Brennan ◽  
M Gilman
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Serum Response Factor ◽  
Binding Activity ◽  
Homeodomain Proteins ◽  
Extracellular Signals ◽  
Dna Binding Activity ◽  
Paired Class

Cells translate extracellular signals into specific programs of gene expression that reflect their developmental history or identity. We present evidence that one way this interpretation may be performed is by cooperative interactions between serum response factor (SRF) and certain homeodomain proteins. We show that human and Drosophila homeodomain proteins of the paired class have the ability to recruit SRF to DNA sequences not efficiently recognized by SRF on its own, thereby imparting to a linked reporter gene the potential to respond to polypeptide growth factors. This activity requires both the DNA-binding activity of the homeodomain and putative protein-protein contact residues on the exposed surfaces of homeodomain helices 1 and 2. The ability of the homeodomain to impart signal responsiveness is DNA sequence specific, and this specificity differs from the simple DNA-binding specificity of the homeodomain in vitro. The homeodomain imparts response to a spectrum of signals characteristic of the natural SRF-binding site in the c-fos gene. Response to some of these signals is dependent on the secondary recruitment of SRF-dependent ternary complex factors, and we show directly that a homeodomain can promote the recruitment of one such factor, Elk1. We infer that SRF and homeodomains interact cooperatively on DNA and that formation of SRF-homeodomain complexes permits the recruitment of signal-responsive SRF accessory proteins. The ability to route extracellular signals to specific target genes is a novel activity of the homeodomain, which may contribute to the identity function displayed by many homeodomain genes.

scholarly journals The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

2018 ◽  
Vol 39 (3) ◽  
Author(s):  
Kyle T. Helzer ◽  
Mary Szatkowski Ozers ◽  
Mark B. Meyer ◽  
Nancy A. Benkusky ◽  
Natalia Solodin ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Interactions ◽  
Posttranslational Modifications ◽  
Binding Sites ◽  
Protein Function ◽  
Estrogen Receptor Α ◽  
Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

scholarly journals Adenovirus E1B 55-Kilodalton Oncoprotein Inhibits p53 Acetylation by PCAF

2000 ◽  
Vol 20 (15) ◽  
pp. 5540-5553 ◽  
Author(s):  
Yue Liu ◽  
April L. Colosimo ◽  
Xiang-Jiao Yang ◽  
Daiqing Liao
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Binding Activity ◽  
Physical Interaction ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Adenovirus E1b

ABSTRACT The adenovirus E1B 55-kDa protein binds to cellular tumor suppressor p53 and inactivates its transcriptional transactivation function. p53 transactivation activity is dependent upon its ability to bind to specific DNA sequences near the promoters of its target genes. It was shown recently that p53 is acetylated by transcriptional coactivators p300, CREB bidning protein (CBP), and PCAF and that acetylation of p53 by these proteins enhances p53 sequence-specific DNA binding. Here we show that the E1B 55-kDa protein specifically inhibits p53 acetylation by PCAF in vivo and in vitro, while acetylation of histones and PCAF autoacetylation is not affected. Furthermore, the DNA-binding activity of p53 is diminished in cells expressing the E1B 55-kDa protein. PCAF binds to the E1B 55-kDa protein and to a region near the C terminus of p53 encompassing Lys-320, the specific PCAF acetylation site. We further show that the E1B 55-kDa protein interferes with the physical interaction between PCAF and p53, suggesting that the E1B 55-kDa protein inhibits PCAF acetylase function on p53 by preventing enzyme-substrate interaction. These results underscore the importance of p53 acetylation for its function and suggest that inhibition of p53 acetylation by viral oncoproteins prevent its activation, thereby contributing to viral transformation.

scholarly journals BCL11A promotes myeloid leukemogenesis by repressing PU.1 target genes

2021 ◽  
Author(s):  
Yoshitaka Sunami ◽  
Takashi Yokoyama ◽  
Seiko Yoshino ◽  
Tomoko Takahara ◽  
Yukari Yamazaki ◽  
...  
Keyword(s):  
Dna Binding ◽  
Myeloid Leukemia ◽  
Target Genes ◽  
Cell Interaction ◽  
Binding Activity ◽  
Hemoglobin Switching ◽  
Dna Binding Activity ◽  
Adult Hemoglobin

The transcriptional repressor, BCL11A, is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase, Trib1, in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells both in vitro and in vivo. ChIP-seq analysis showed that upon DNA-binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by both sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components, HDAC and LSD1, reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor, pracinostat, and LSD1 inhibitor, GSK2879552, resulted in growth inhibition both in vitro and in vivo. High BCL11A expression is associated with worse prognosis in human AML patients. Blocking of BCL11A expression upregulates the expression of PU.1 target genes, and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

scholarly journals Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

1996 ◽  
Vol 16 (4) ◽  
pp. 1479-1489 ◽  
Author(s):  
M Ubeda ◽  
X Z Wang ◽  
H Zinszner ◽  
I Wu ◽  
J F Habener ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Binding Activity ◽  
Control Element ◽  
Core Element ◽  
Transcriptional Activation Domain ◽  
Dna Binding Activity ◽  
Basic Region

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

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.

Sign in / Sign up

Export Citation Format

Share Document