The transcription corepressor LEUNIG interacts with the histone deacetylase HDA19 and mediator components MED14 (SWP) and CDK8 (HEN3) to repress transcription - PubMed (original) (raw)

The transcription corepressor LEUNIG interacts with the histone deacetylase HDA19 and mediator components MED14 (SWP) and CDK8 (HEN3) to repress transcription

Deyarina Gonzalez et al. Mol Cell Biol. 2007 Aug.

Abstract

Transcription corepressors are general regulators controlling the expression of genes involved in multiple signaling pathways and developmental programs. Repression is mediated through mechanisms including the stabilization of a repressive chromatin structure over control regions and regulation of Mediator function inhibiting RNA polymerase II activity. Using whole-genome arrays we show that the Arabidopsis thaliana corepressor LEUNIG, a member of the GroTLE transcription corepressor family, regulates the expression of multiple targets in vivo. LEUNIG has a role in the regulation of genes involved in a number of different physiological processes including disease resistance, DNA damage response, and cell signaling. We demonstrate that repression of in vivo LEUNIG targets is achieved through histone deacetylase (HDAC)-dependent and -independent mechanisms. HDAC-dependent mechanisms involve direct interaction with HDA19, a class 1 HDAC, whereas an HDAC-independent repression activity involves interactions with the putative Arabidopsis Mediator components AtMED14/SWP and AtCDK8/HEN3. We suggest that changes in chromatin structure coupled with regulation of Mediator function are likely to be utilized by LEUNIG in the repression of gene transcription.

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Figures

FIG. 1.

FIG. 1.

HDAC-dependent and -independent regulation of in vivo LUG targets. TSA treatment reveals HDAC-dependent (A) and HDAC-independent (B) LUG target genes. Shown are results of real-time PCR analysis of reverse-transcribed mRNA from Ler and lug-3 mutant seedlings 6 h post-TSA treatment. Relative quantification of gene expression data was carried out using CT values for each sample normalized to ACTIN2 expression levels. Normalized values for lug-3 expression levels are given as change (_n_-fold) relative to values for Ler seedlings.

FIG. 2.

FIG. 2.

LUG interacts functionally and physically with HDACs to repress transcription. (A) LUG repression activity in plant protoplasts is abolished by TSA treatment. Arabidopsis leaf protoplasts were transfected with GAL4-LUG, GAL4-LUG derivatives, or GAL4-only effector plasmids plus reporter plasmid pJC1 and treated with 20 μM TSA (dark bars) or ethanol (white bars) for 6 h before fluorescence levels were determined relative to untransformed controls (blank). (B) LUG repression activity in yeast is abolished by TSA treatment. Yeast strain FT5 was transformed with the reporter vector pJK1621 (16) together with the indicated LexA-LUG derivatives (27) or LexA only. Individual transformants were treated with 20 μM TSA (dark bars) or ethanol (white bars) and grown overnight in liquid medium to early log phase (OD600 of <1) before β-galactosidase activity was determined. (C) LUG copurifies with an HDAC activity. Nuclear proteins isolated from Ler and lug-3 mutant leaves, flowers, and seedlings (with or without 6 h of TSA treatment) were incubated with immobilized anti-LUG antibody to immunopurify LUG-associated proteins, and the immunoprecipitate was either analyzed for HDAC activity relative to the negative bead-only control (top) or immunoblotted and probed with anti-LUG antibody (bottom). (D) LUG requires RPD3 to repress transcription. Yeast strains FT5 and FT5::_rpd3_Δ were transformed with the reporter vector pJK1621 (16) together with full-length LexA-LUG (27) or LexA only. Individual transformants were assayed as described for panel B. (E) LUG interacts with HDA19 in vitro. LexA-LUG was immunoprecipitated from yeast whole-cell extracts and incubated with [35S]methionine-labeled HDA19 or Luciferase (Luc). Input (I) and bound (B) 35S-labeled proteins were visualized using a phosphorimager. LUG was detected using an anti-LexA antibody (W).

FIG. 3.

FIG. 3.

The Arabidopsis Mediator components AtMED14/SWP and AtCDK8/HEN3 are involved in LUG-mediated repression. (A) LUG requires yeast y_MED14_ and y_CDK8_ for repression function. Yeast strains FT5 (white bars), FT5::med14Δ (dark bars; viable partial deletion), and FT5::cdk8Δ (light gray bars) were transformed with the reporter vector pJK1621 (4× LexA operator) or the control vector pLG312S (0× LexA operator) together with the indicated LexA-LUG derivatives (27) or LexA only. Individual transformants were grown in liquid medium to an early log phase (OD600 of <1), and β-galactosidase activity was determined. Error bars show standard deviations. (B) LUG interacts with AtMED14 and AtCDK8 in vivo. Interactions were determined using yeast two-hybrid assays. FT5 was transformed with the reporter plasmid JK103 (4× LexA operator sequences-CYC1 minimal promoter-LacZ) plus LexA-AtMED14 or LexA-AtCDK8 together with either AD (AD only; white bars), LUG-AD (dark bars), or SEU-AD (light gray bars) (27). LexA-SEU was used as a positive control for LUG interaction. Error bars show standard deviations. Unpaired t tests comparing bait vector alone with bait plus prey vectors were used to test significance (**, P < 0.01). (C and E) LUG interacts with AtMED14 (C) and AtCDK8 (E) in vitro. LexA-MED14 or LexA-AtCDK8 was immunoprecipitated from whole-cell extracts and incubated with [35S]methionine-labeled LUG, LUFS+Q (L+Q), or luciferase (Luc) proteins. 35S-labeled proteins were visualized using a phosphorimager. LUG was detected using an anti-LexA antibody (W). (D and F) Like panels C and E but using LexA-LUG immunoprecipitated from yeast whole-cell extracts incubated with [35S]methionine-labeled AtMED14 (conserved N-terminal domain) (D) and AtCDK8 (F) or luciferase (Luc). I, input; B, bound. (G) AtMED14 and AtCDK8 display inherent repression activity. Repression activity of LexA-AtMED14 or LexA-AtCDK8 was determined by comparing LacZ activity in FT5 transformed with pJK1621 and activity in FT5 transformed with pLG312S, and significance was determined using unpaired t tests between pJK1621 and pLG312S values (**, P < 0.01). Error bars show standard deviations.

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