Two mechanisms in the action of repressor deltaEF1

binding site competition with an activator and active repression

R Sekido, K Murai, Y Kamachi, H Kondoh

Research output: Contribution to journalArticle

52 Citations (Scopus)

Abstract

BACKGROUND: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers.

RESULTS: The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 were separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters.

CONCLUSION: Two mechanisms are involved in the repressor action of deltaEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.

Original languageEnglish
Pages (from-to)771-83
Number of pages13
JournalGenes to Cells
Volume2
Issue number12
Publication statusPublished - Dec 1997

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Zinc Fingers
Binding Sites
DNA
Crystallins
Repressor Proteins
Competitive Binding
Cell Count

Keywords

  • Adenovirus E2 Proteins
  • Animals
  • Base Sequence
  • Binding Sites
  • Binding, Competitive
  • COS Cells
  • Consensus Sequence
  • Crystallins
  • DNA
  • DNA-Binding Proteins
  • Enhancer Elements, Genetic
  • Gene Expression Regulation
  • Genes, Reporter
  • Homeodomain Proteins
  • Molecular Sequence Data
  • Nuclear Proteins
  • Plasmids
  • Promoter Regions, Genetic
  • Protein Binding
  • Repressor Proteins
  • Transcription Factors
  • Transcriptional Activation
  • Zinc Fingers

Cite this

Two mechanisms in the action of repressor deltaEF1 : binding site competition with an activator and active repression. / Sekido, R; Murai, K; Kamachi, Y; Kondoh, H.

In: Genes to Cells, Vol. 2, No. 12, 12.1997, p. 771-83.

Research output: Contribution to journalArticle

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TY - JOUR

T1 - Two mechanisms in the action of repressor deltaEF1

T2 - binding site competition with an activator and active repression

AU - Sekido, R

AU - Murai, K

AU - Kamachi, Y

AU - Kondoh, H

PY - 1997/12

Y1 - 1997/12

N2 - BACKGROUND: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers.RESULTS: The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 were separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters.CONCLUSION: Two mechanisms are involved in the repressor action of deltaEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.

AB - BACKGROUND: Counteraction between activators and repressors is crucial for the regulation of a number of cell-specific enhancers, where an activator and a repressor are mutually competitive in binding to the same site. DeltaEF1 is a repressor protein of delta1-crystallin minimal enhancer DC5 binding at the CACCT site, and inhibits activator deltaEF3 from binding to the overlapped site. It has two zinc finger clusters N-fin and C-fin, close to N- and C-termini, respectively, and a homeodomain in the middle. deltaEF1 also binds to the E2-box sequence CACCTG, and represses E2-box-dependent enhancers.RESULTS: The mechanism of the repressor action of deltaEF1 was investigated by examining various deletion mutants of deltaEF1 for their activity to repress delta1-crystallin enhancer fragment HN which contained DC5 sequence and an additional activator site. Both zinc finger clusters were found to be essential for DNA binding and repression, but the homeodomain was not. In addition, the NR domain close to the N-terminus was required for full repression. The NR domain showed active repression when fused to the Gal4 DNA binding domain. Active repression by deltaEF1, dependent on the NR domain, was also demonstrated in a situation where the binding sites of deltaEF1 and deltaEF3 were separated. N-fin and C-fin in their isolated forms bind the 5'-(T/C)ACCTG-3' and 5'-(t/C)ACCT-3' sequences, respectively, while the homeodomain showed no DNA binding activity. An analysis of DNA binding of the delta(Int)F form, having both N-fin and C-fin, indicated that a single DNA binding domain is assembled from two zinc finger clusters.CONCLUSION: Two mechanisms are involved in the repressor action of deltaEF1. First, a binding site competition with an activator which depends on the integrity of both zinc finger clusters, and second, an active repression to silence an enhancer which is attributed to the NR domain.

KW - Adenovirus E2 Proteins

KW - Animals

KW - Base Sequence

KW - Binding Sites

KW - Binding, Competitive

KW - COS Cells

KW - Consensus Sequence

KW - Crystallins

KW - DNA

KW - DNA-Binding Proteins

KW - Enhancer Elements, Genetic

KW - Gene Expression Regulation

KW - Genes, Reporter

KW - Homeodomain Proteins

KW - Molecular Sequence Data

KW - Nuclear Proteins

KW - Plasmids

KW - Promoter Regions, Genetic

KW - Protein Binding

KW - Repressor Proteins

KW - Transcription Factors

KW - Transcriptional Activation

KW - Zinc Fingers

M3 - Article

VL - 2

SP - 771

EP - 783

JO - Genes to Cells

JF - Genes to Cells

SN - 1356-9597

IS - 12

ER -