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PMID:16024659

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Citation

Horn, PJ, Bastie, JN and Peterson, CL (2005) A Rik1-associated, cullin-dependent E3 ubiquitin ligase is essential for heterochromatin formation. Genes Dev. 19:1705-14

Abstract

Heterochromatin is critical for proper centromere and telomere function, and it plays a key role in the transcriptional silencing of specific genomic loci. In fission yeast, the Rik1 protein functions with the Clr4 histone methyltransferase at an early step in heterochromatin formation. Here, we use mass spectrometry and tandem affinity purification of a Rik1-TAP fusion protein to identify Rik1-associated proteins. These studies identify two novel proteins, Raf1 and Raf2, which we find are required for H3-K9 methylation and for transcriptional silencing within centromeric heterochromatin. We also find that subunits of a cullin-dependent E3 ubiquitin ligase are associated with Rik1 and Clr4, and Rik1-TAP preparations exhibit robust E3 ubiquitin ligase activity. Furthermore, expression of a dominant-negative allele of the Pcu4 cullin subunit disrupts regulation of K4 methylation within heterochromatin. These studies provide evidence for a novel Rik1-associated E3 ubiquitin ligase that is required for heterochromatin formation.

Links

PubMed PMC1176008 Online version:10.1101/gad.1328005

Keywords

Cell Cycle Proteins/genetics; Cell Cycle Proteins/metabolism; Chromosomal Proteins, Non-Histone/genetics; Chromosomal Proteins, Non-Histone/metabolism; Cullin Proteins/genetics; Cullin Proteins/metabolism; Genes, Fungal; Heterochromatin/genetics; Heterochromatin/metabolism; Methyltransferases/genetics; Methyltransferases/metabolism; Multiprotein Complexes/chemistry; Multiprotein Complexes/genetics; Multiprotein Complexes/metabolism; Mutation; Protein Subunits; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/metabolism; Schizosaccharomyces/genetics; Schizosaccharomyces/metabolism; Schizosaccharomyces pombe Proteins/chemistry; Schizosaccharomyces pombe Proteins/genetics; Schizosaccharomyces pombe Proteins/metabolism; Ubiquitin-Protein Ligases/chemistry; Ubiquitin-Protein Ligases/genetics; Ubiquitin-Protein Ligases/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

SCHPO:RBX1

enables

GO:0061630: ubiquitin protein ligase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

SCHPO:CUL4

involved_in

GO:0031508: pericentric heterochromatin assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

SCHPO:CUL4

part_of

GO:0043494: CLRC complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

SCHPO:CUL4

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q10426

F

Seeded From UniProt

complete

SCHPO:CLR4

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q10426

F

Seeded From UniProt

complete

SCHPO:RAF2

involved_in

GO:0031508: pericentric heterochromatin assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

SCHPO:RAF2

is_active_in

GO:0005721: pericentric heterochromatin

ECO:0000305: curator inference used in manual assertion

GO:0043494

C

Seeded From UniProt

complete

SCHPO:RAF2

part_of

GO:0043494: CLRC complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

SCHPO:RAF1

involved_in

GO:0031508: pericentric heterochromatin assembly

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

SCHPO:RAF1

part_of

GO:0043494: CLRC complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

SCHPO:RAF1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q10426

F

Seeded From UniProt

complete

SCHPO:H2B1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q10426

F

Seeded From UniProt

complete

SCHPO:RIK1

part_of

GO:0043494: CLRC complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

SCHPO:RIK1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O14122

F

Seeded From UniProt

complete

SCHPO:RIK1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O60016

F

Seeded From UniProt

complete

SCHPO:RIK1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O74910

F

Seeded From UniProt

complete

SCHPO:RIK1

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P04913

F

Seeded From UniProt

complete


See also

References

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