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Comparative Study
. 1996 Nov 12;93(23):12845-50.
doi: 10.1073/pnas.93.23.12845.

Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3

W M Yang  1 , C Inouye, Y Zeng, D Bearss, E Seto
Affiliations
Comparative Study

Transcriptional repression by YY1 is mediated by interaction with a mammalian homolog of the yeast global regulator RPD3

W M Yang et al. Proc Natl Acad Sci U S A. .

Abstract

YY1 is a mammalian zinc-finger transcription factor with unusual structural and functional features. It has been implicated as a positive and a negative regulatory factor that binds to the CCATNTT consensus DNA element located in promoters of many cellular and viral genes. A mammalian cDNA that encodes a YY1-binding protein and possesses sequence homology with the yeast transcriptional factor RPD3 has been identified. A Gal4 DNA binding domain-mammalian RPD3 fusion protein strongly represses transcription from a promoter containing Gal4 binding sites. Association between YY1 and mammalian RPD3 requires a glycine-rich region on YY1. Mutations in this region abolish the interaction with mammalian RPD3 and eliminate transcriptional repression by YY1. These data suggest that YY1 negatively regulates transcription by tethering RPD3 to DNA as a cofactor and that this transcriptional mechanism is highly conserved from yeast to human.

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Figures

Figure 1
Figure 1
Predicted amino acid sequences of mouse and human RPD3 and their homology to RPD3 from other species. The deduced amino acid sequence of mRPD3 and human RPD3 (hRPD3) are aligned with the sequences of the yeast RPD3 [yRPD3; National Center for Biotechnology Information S22284 (ref. 17)], the Xenopus laevis RPD3 (xRPD3; gi:576995; unpublished sequence), and the Caenorhabditis elegans RPD3 (cRPD3; X78454; unpublished sequence). Amino acid positions are boxed where there is identity among all five species.
Figure 2
Figure 2
Identification of a glycine-rich mRPD3-interacting domain in YY1. (A) Schematic drawing of YY1 and GST–YY1 fusion constructs. The glycine-rich region (mRPD3 binding domain) is shaded in gray. The location of activation, repression, and DNA binding domains on YY1 is from refs. , , and –. The ability of each GST–YY1 fusion protein to bind mRPD3 is indicated (+ or −). (B) Representative autoradiogram of in vitro translated mRPD3 protein captured by GST–YY1 fusion proteins. Three experiments yielded consistent results. The input lane (lane 16) was loaded with one-third the amount of mRPD3 used in the binding reactions. The sizes of molecular weight markers are indicated to the left.
Figure 3
Figure 3
Gal4–mRPD3 fusion protein represses transcription from promoters with Gal4-binding sites. (A) Schematic drawing of plasmids used in transfections. (B) Representative CAT assays showing that mRPD3 can repress transcription. Seven experiments yielded consistent results. Plasmids included in each transfection are indicated (+).
Figure 4
Figure 4
mRPD3 protein increases Gal4–YY1-induced transcriptional repression through a glycine-rich domain in YY1. (A) Schematic drawing of plasmids used in transfections. (B, C, and E–G) Representative CAT assays showing that mRPD3 can further repress transcription through a Gal4–YY1 glycine-rich domain. Each CAT assay shown is only a representative of at least three experiments. Plasmids included in each transfection are indicated (+). Reporter plasmid is pCAT-Control for C and pGal4-tkCAT for E–G. (D) Mouse RPD3 has no effect on Gal4–YY1 as determined by EMSA. No protein was added in reaction presented in lane 1. Arrow indicates position of Gal4–YY1–DNA complex.
Figure 5
Figure 5
Nonfusion mRPD3 protein represses transcription from promoters with YY1-binding sites. Representative CAT assays showing that mRPD3 can further repress transcription through YY1. Each CAT assay shown is a representative of at least three experiments. All relative CAT activities are normalized with control β-galactosidase expressions. Plasmids included in each transfection are indicated (+). Effector plasmid is pCMV-YY1 and pCMV-mRPD3. Reporter plasmid is pSVECAT for no YY1-binding site, pP5-60SVECAT for wild-type YY1-binding sites, and pP5-60(mt2)SVECAT for mutated YY1-binding sites.
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