Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence

Letovsky, John; Dynan, William S.

doi:10.1093/nar/17.7.2639

Cited by 228 publications

(155 citation statements)

References 23 publications

Supporting

Mentioning

139

Contrasting

Unclassified

Order By: Relevance

“…4A). The choices for nucleotide substitutions were based on previous studies in which either factor binding or functional activity of the particular binding site was destroyed (3,23,24,38). These mutant enhancer fragments were subcloned into the luciferase expression vector with the minimal CD4 promoter and transiently transfected into D10 T cells, and the enhancer activity was then compared with activities unmutated enhancer constructs (Fig.…”

Section: Resultsmentioning

confidence: 99%

Elf-1 binds to a critical element in a second CD4 enhancer.

Wurster¹,

Siu²,

Leiden³

et al. 1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

The coordinated expression of CD4 and CD8 during T-cell development is tightly coupled with the maturation state of the T cell. Additionally, the mutually exclusive expression of these receptors in mature T cells is representative of the functional T-cell subclasses (CD4+ helper T cells versus CD8+ cytotoxic T cells). We have studied the regulation CD4 gene transcription during T-cell development in an attempt to gain an understanding of the molecular mechanisms involved in T-cell development and differentiation. Here we present the identification of a second transcriptional enhancer in the murine CD4 locus 24 kb upstream of the CD4 promoter. This enhancer is active in mature T cells and is especially active in CD4+ helper T cells. A number of nuclear proteins bind to elements in the minimal CD4 enhancer that includes consensus sites for AP-1, Spl, Gata, and Ets transcription factor families. We find that the Ets consensus site is crucial for enhancer activity and that the recently identified Ets factor, Elf-i, which is expressed at high levels in T cells and involved in the regulation of several other T-cell-specific genes, is a dominant protein in T-cell nuclear extracts that binds to this site.T-cell development involves the differentiation of functionally immature pre-T cells into mature, antigen-reactive effector cells (reviewed in reference 61). This process includes selection events in which the maturing T-cell population is depleted of self-reactive clones and enriched for those that can recognize foreign peptides bound by self-major histocompatibility complex (MHC). The development and selection of T-cell precursors occur in the thymus and are characterized by a coordinated expression of tissue-specific genes. Some of these genes encode cell surface molecules that directly promote T-cell differentiation and selection events. Understanding the molecular mechanisms responsible for the expression of these T-cellspecific genes is a window on the process of T-cell development.The CD4 glycoprotein is one of the surface molecules that is important as a receptor in the development and function of T cells (reviewed in references 39 and 46). In mature antigenspecific T cells, CD4 is expressed only on cells that have a T-cell receptor (TCR) specific for antigen associated with class II MHC molecules, while CD8, a cell surface protein functionally similar to CD4, is expressed only on T cells that recognize antigen bound to MHC class I molecules (56). The mutually exclusive expression of CD4 and CD8 on mature T cells also corresponds to T-cell function; CD4 is expressed primarily on helper T cells, while CD8 is expressed on cytotoxic T cells. CD4 participates in antigen recognition by binding to nonpolymorphic regions of class II MHC molecules, while the T-cell antigen receptor recognizes a specific peptide antigen that is bound by the MHC molecule (7). The CD4-MHC interaction serves to increase the avidity of the T cell to the antigenpresenting cell but also induces an intracellular signaling event through the CD4-...

show abstract

Section: Resultsmentioning

confidence: 99%

Elf-1 binds to a critical element in a second CD4 enhancer.

Wurster¹,

Siu²,

Leiden³

et al. 1994

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…Selection against G was also seen for the second zinc finger of Sp1, which has the same base-contacting residues as zinc fingers 3 and 1 of the NGFI-A family (14). Additionally, there was diminished binding by Sp1 to an oligonucleotide with G at the position expected to interact with the Glu of its second zinc finger (13), and other experiments demonstrated that substitution of G for C at the same position reduced binding 30-fold (34). The nature of this interaction was not resolved by the crystal structure, but it was later suggested that a contributory influence might be an electrostatic repulsion between the O6 and N7 groups of G and the nearby Glu carboxylate oxygens, following the loss of their shell of (42).…”

Section: Discussionmentioning

confidence: 84%

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

Swirnoff

Milbrandt

1995

Molecular and Cellular Biology

299

287

View full text Add to dashboard Cite

NGFI-A is the prototypic member of a family of immediate-early gene-encoded transcription factors which includes NGFI-C, Egr3, and Krox20. These proteins possess highly homologous DNA-binding domains, composed of three Cys 2 -His 2 zinc fingers, and all bind to and activate transcription from the sequence GCGGGGGCG. We used a PCR-mediated random site selection protocol to determine whether other sites could be bound by these proteins and the extent to which their binding site preferences are similar or different. The high-affinity consensus sites generated from the selection data are similar, and the combined consensus sequence is T -G-C-G-T/g-G/A-G-G-C/a/t-G-G/T (lowercase letters indicate bases selected less frequently).Using gel shift assays, we found that sequences that diverge from the consensus were bound by NGFI-A, confirming that there is greater variability in binding sites than has generally been acknowledged. We also provide evidence that protein-DNA interactions not noted, or whose importance was not apparent from the X-ray cocrystal structure of the NGFI-A zinc fingers complexed with DNA, contribute significantly to the binding energy of these proteins and confirm that an optimal site is at least 10 instead of 9 nucleotides in length. In contrast to the similarities in binding specificity among these proteins, we found that while NGFI-A, Egr3, and Krox20 have comparable DNA binding affinities and kinetics of dissociation, the affinity of NGFI-C is more than threefold lower. This could result in differential regulation of target genes in cells where NGFI-C and the other proteins are coexpressed. Furthermore, we show that this affinity difference is a property not of the zinc fingers themselves but rather of the protein context of the DNA-binding domain.

show abstract

“…The results of these experiments show that all three footprinted sequences are required for activation of a linked ␤-globin gene. Sp1 family members recognize a 9-bp sequence GGG GT/CG GGG (Letovsky and Dynan 1989) and thus it could be predicted that fp 2 contains two overlapping binding sites, in addition to potential weaker binding sites (Fig. 1B, wild type).…”

Section: Experimental Strategymentioning

confidence: 99%

“…Thus, we conclude that each G box contributes to the activity of fp 1-3. Because the 3Ј sequence has the best match with the Sp1 consensus binding site (Letovsky and Dynan 1989), we concentrated on this motif. For reasons of clarity, we will refer to this permutation of fp 2 as control (ct in Fig.…”

Section: Experimental Strategymentioning

confidence: 99%

Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo

Gillemans

Tewari²,

Lindeboom³

et al. 1998

Genes Dev.

View full text Add to dashboard Cite

The locus control region of the ␤-globin cluster contains five DNase I hypersensitive sites (5 HS1-5) required for locus activation. 5 HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5 HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5 HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5 HS3.

show abstract

Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence

Cited by 228 publications

References 23 publications

Elf-1 binds to a critical element in a second CD4 enhancer.

Elf-1 binds to a critical element in a second CD4 enhancer.

DNA-Binding Specificity of NGFI-A and Related Zinc Finger Transcription Factors

Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo

Contact Info

Product

Resources

About