KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing

R F Ryan, D C Schultz, K Ayyanathan, P B Singh, J R Friedman, W J Fredericks, F J Rauscher

Research output: Contribution to journalArticle

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Abstract

Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

Original languageEnglish
Pages (from-to)4366-78
Number of pages13
JournalMolecular and Cellular Biology
Volume19
Issue number6
Publication statusPublished - Jun 1999

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Co-Repressor Proteins
Heterochromatin
Zinc Fingers
Gene Silencing
Genes
Proteins
Euchromatin
Interphase
Amino Acid Substitution
Epigenomics
Drosophila
Genome
In Vitro Techniques

Keywords

  • 3T3 Cells
  • Animals
  • Blotting, Western
  • COS Cells
  • Cell Nucleus
  • Chromatin
  • Chromatography, Liquid
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Electrophoresis, Polyacrylamide Gel
  • Euchromatin
  • Fluorescent Antibody Technique, Indirect
  • Heterochromatin
  • Luciferases
  • Mice
  • Models, Biological
  • Models, Genetic
  • Mutagenesis
  • Nuclear Proteins
  • Protein Binding
  • Protein Conformation
  • Recombinant Fusion Proteins
  • Repressor Proteins
  • Transcription Factors
  • Transfection
  • Tripartite Motif-Containing Protein 28
  • Zinc Fingers
  • Journal Article
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

Cite this

KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins : a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. / Ryan, R F; Schultz, D C; Ayyanathan, K; Singh, P B; Friedman, J R; Fredericks, W J; Rauscher, F J.

In: Molecular and Cellular Biology, Vol. 19, No. 6, 06.1999, p. 4366-78.

Research output: Contribution to journalArticle

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T2 - a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing

AU - Ryan, R F

AU - Schultz, D C

AU - Ayyanathan, K

AU - Singh, P B

AU - Friedman, J R

AU - Fredericks, W J

AU - Rauscher, F J

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N2 - Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

AB - Krüppel-associated box (KRAB) domains are present in approximately one-third of all human zinc finger proteins (ZFPs) and are potent transcriptional repression modules. We have previously cloned a corepressor for the KRAB domain, KAP-1, which is required for KRAB-mediated repression in vivo. To characterize the repression mechanism utilized by KAP-1, we have analyzed the ability of KAP-1 to interact with murine (M31 and M32) and human (HP1alpha and HP1gamma) homologues of the HP1 protein family, a class of nonhistone heterochromatin-associated proteins with a well-established epigenetic gene silencing function in Drosophila. In vitro studies confirmed that KAP-1 is capable of directly interacting with M31 and hHP1alpha, which are normally found in centromeric heterochromatin, as well as M32 and hHP1gamma, both of which are found in euchromatin. Mapping of the region in KAP-1 required for HP1 interaction showed that amino acid substitutions which abolish HP1 binding in vitro reduce KAP-1 mediated repression in vivo. We observed colocalization of KAP-1 with M31 and M32 in interphase nuclei, lending support to the biochemical evidence that M31 and M32 directly interact with KAP-1. The colocalization of KAP-1 with M31 is sometimes found in subnuclear territories of potential pericentromeric heterochromatin, whereas colocalization of KAP-1 and M32 occurs in punctate euchromatic domains throughout the nucleus. This work suggests a mechanism for the recruitment of HP1-like gene products by the KRAB-ZFP-KAP-1 complex to specific loci within the genome through formation of heterochromatin-like complexes that silence gene activity. We speculate that gene-specific repression may be a consequence of the formation of such complexes, ultimately leading to silenced genes in newly formed heterochromatic chromosomal environments.

KW - 3T3 Cells

KW - Animals

KW - Blotting, Western

KW - COS Cells

KW - Cell Nucleus

KW - Chromatin

KW - Chromatography, Liquid

KW - Chromosomal Proteins, Non-Histone

KW - DNA-Binding Proteins

KW - Electrophoresis, Polyacrylamide Gel

KW - Euchromatin

KW - Fluorescent Antibody Technique, Indirect

KW - Heterochromatin

KW - Luciferases

KW - Mice

KW - Models, Biological

KW - Models, Genetic

KW - Mutagenesis

KW - Nuclear Proteins

KW - Protein Binding

KW - Protein Conformation

KW - Recombinant Fusion Proteins

KW - Repressor Proteins

KW - Transcription Factors

KW - Transfection

KW - Tripartite Motif-Containing Protein 28

KW - Zinc Fingers

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

KW - Research Support, U.S. Gov't, P.H.S.

M3 - Article

C2 - 10330177

VL - 19

SP - 4366

EP - 4378

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

SN - 0270-7306

IS - 6

ER -