Transcriptional Activity of Sp1 Is Regulated by Molecular Interactions between the Zinc Finger DNA Binding Domain and the Inhibitory Domain with Corepressors, and This Interaction Is Modulated by MEK

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

Download Full-text

Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6858-6865.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6858-6865

Author(s):

M W Russo ◽

C Matheny ◽

J Milbrandt

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activity ◽

Zinc Fingers ◽

Fold Increase ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domains ◽

Cellular Factor ◽

Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

Download Full-text

A position-dependent transcription-activating domain in TFIIIA.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7496 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7496-7506 ◽

Cited By ~ 15

Author(s):

X Mao ◽

M K Darby

Keyword(s):

Amino Acids ◽

Dna Binding ◽

Rna Polymerase ◽

Zinc Finger ◽

Transcriptional Activation ◽

Transcriptional Activity ◽

Rna Polymerase Iii ◽

Binding Domain ◽

Dna Binding Domain ◽

5S Rna

Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain.

Download Full-text

Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6858 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6858-6865 ◽

Cited By ~ 65

Author(s):

M W Russo ◽

C Matheny ◽

J Milbrandt

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activity ◽

Zinc Fingers ◽

Fold Increase ◽

Binding Domain ◽

Dna Binding Domain ◽

Activation Domains ◽

Cellular Factor ◽

Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

Download Full-text

The C6 zinc finger and adjacent amino acids determine DNA-binding specificity and affinity in the yeast activator proteins LAC9 and PPR1.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5128 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5128-5137 ◽

Cited By ~ 10

Author(s):

M M Witte ◽

R C Dickson

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Dna Binding ◽

Zinc Finger ◽

Binding Specificity ◽

Binding Domain ◽

Dna Binding Domain ◽

Transcription Activator ◽

Activator Proteins ◽

Dna Binding Specificity

LAC9 is a DNA-binding protein that regulates transcription of the lactose-galactose regulon in Kluyveromyces lactis. The DNA-binding domain is composed of a zinc finger and nearby amino acids (M. M. Witte and R. C. Dickson, Mol. Cell. Biol. 8:3726-3733, 1988). The single zinc finger appears to be structurally related to the zinc finger of many other fungal transcription activator proteins that contain positively charged residues and six conserved cysteines with the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-9-Cys-Xaa2-Cys-Xaa 6-Cys, where Xaan indicates a stretch of the indicated number of any amino acids (R. M. Evans and S. M. Hollenberg, Cell 52:1-3, 1988). The function(s) of the zinc finger and other amino acids in DNA-binding remains unclear. To determine which portion of the LAC9 DNA-binding domain mediates sequence recognition, we replaced the C6 zinc finger, amino acids adjacent to the carboxyl side of the zinc finger, or both with the analogous region from the Saccharomyces cerevisiae PPR1 or LEU3 protein. A chimeric LAC9 protein, LAC9(PPR1 34-61), carrying only the PPR1 zinc finger, retained the DNA-binding specificity of LAC9. However, LAC9(PPR1 34-75), carrying the PPR1 zinc finger and 14 amino acids on the carboxyl side of the zinc finger, gained the DNA-binding specificity of PPR1, indicating that these 14 amino acids are necessary for specific DNA binding. Our data show that C6 fingers can substitute for each other and allow DNA binding, but binding affinity is reduced. Thus, in a qualitative sense C6 fingers perform a similar function(s). However, the high-affinity binding required by natural C6 finger proteins demands a unique C6 finger with a specific amino acid sequence. This requirement may reflect conformational constraints, including interactions between the C6 finger and the carboxyl-adjacent amino acids; alternatively or in addition, it may indicate that unique, nonconserved amino acid residues in zinc fingers make sequence-specifying or stabilizing contacts with DNA.

Download Full-text

The Dof domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain

The Plant Journal ◽

10.1111/j.1365-313x.2003.01997.x ◽

2004 ◽

Vol 37 (5) ◽

pp. 741-749 ◽

Cited By ~ 49

Author(s):

Yoshimi Umemura ◽

Tomoko Ishiduka ◽

Rie Yamamoto ◽

Muneharu Esaka

Keyword(s):

Dna Binding ◽

Zinc Finger ◽

Higher Plants ◽

Binding Domain ◽

Dna Binding Domain

Download Full-text

A New Coactivator Function for Zac1's C2H2 Zinc Finger DNA-Binding Domain in Selectively Controlling PCAF Activity

Molecular and Cellular Biology ◽

10.1128/mcb.00842-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 6078-6093 ◽

Cited By ~ 16

Author(s):

Anke Hoffmann ◽

Dietmar Spengler

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Zinc Finger ◽

Transcriptional Control ◽

Zinc Finger Protein ◽

Embryonic Stem ◽

Binding Domain ◽

Dna Binding Domain ◽

Histone H4 ◽

Histone H4 Acetylation

ABSTRACT The generally accepted paradigm of transcription by regulated recruitment defines sequence-specific transcription factors and coactivators as separate categories that are distinguished by their abilities to bind DNA autonomously. The C2H2 zinc finger protein Zac1, with an established role in canonical DNA binding, also acts as a coactivator. Commensurate with this function, p73, which is related to p53, is here shown to recruit Zac1, together with the coactivators p300 and PCAF, to the p21Cip1 promoter during the differentiation of embryonic stem cells into neurons. In the absence of autonomous DNA binding, Zac1's zinc fingers stabilize the association of PCAF with p300, suggesting its scaffolding function. Furthermore, Zac1 regulates the affinities of PCAF substrates as well as the catalytic activities of PCAF to induce a selective switch in favor of histone H4 acetylation and thereby the efficient transcription of p21Cip1. These results are consistent with an authentic coactivator function of Zac1's C2H2 zinc finger DNA-binding domain and suggest coactivation by sequence-specific transcription factors as a new facet of transcriptional control.

Download Full-text