Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression - PubMed (original) (raw)

. 2000 Jan 1;14(1):55-66.

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Isolation of a novel histone deacetylase reveals that class I and class II deacetylases promote SMRT-mediated repression

H Y Kao et al. Genes Dev. 2000.

Abstract

The transcriptional corepressor SMRT functions by mediating the repressive effect of transcription factors involved in diverse signaling pathways. The mechanism by which SMRT represses basal transcription has been proposed to involve the indirect recruitment of histone deacetylase HDAC1 via the adaptor mSin3A. In contrast to this model, a two-hybrid screen on SMRT-interacting proteins resulted in the isolation of the recently described HDAC5 and a new family member termed HDAC7. Molecular and biochemical results indicate that this interaction is direct and in vivo evidence colocalizes SMRT, mHDAC5, and mHDAC7 to a distinct nuclear compartment. Surprisingly, HDAC7 can interact with mSin3A in yeast and in mammalian cells, suggesting association of multiple repression complexes. Taken together, our results provide the first evidence that SMRT-mediated repression is promoted by class I and class II histone deacetylases and that SMRT can recruit class II histone deacetylases in a mSin3A-independent fashion.

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Figures

Figure 1

Figure 1

SMRT repression domains III and IV interact with deacetylase domain of the newly described deacetylases. (A) Quantitation of yeast two-hybrid assays between SMRT and HDAC5 and HDAC7. Yeast cells were cotransformed with the indicated plasmids. Two viable transformants were picked for liquid β-galactosidase activity. (Open bars) AD; (solid bars) AD–HDAC. The number represents average of duplicates from two colonies. (B) SMRT interaction requires amino acid carboxy-terminal to the deacetylase domain of mHDAC5. Yeast method as described in A. (Open bars) pGBT9 alone; (solid bars) pGBT9–SMRT. (C) SMRT RD III and RD IV do not interact with HDAC1 or mHDAC6. The bait construct pGBT9–SMRT (RD III + RD IV) was cotransformed with GAL AD fusion to various HDAC constructs into yeast strain Y190. After 3 days, two isolated colonies were picked, resuspended in water, and 103 cells were dropped on both −Trp–Leu and −Trp–Leu–His + 40 m

m

3-AT plates. Pictures were taken after 3 days.

Figure 1

Figure 1

SMRT repression domains III and IV interact with deacetylase domain of the newly described deacetylases. (A) Quantitation of yeast two-hybrid assays between SMRT and HDAC5 and HDAC7. Yeast cells were cotransformed with the indicated plasmids. Two viable transformants were picked for liquid β-galactosidase activity. (Open bars) AD; (solid bars) AD–HDAC. The number represents average of duplicates from two colonies. (B) SMRT interaction requires amino acid carboxy-terminal to the deacetylase domain of mHDAC5. Yeast method as described in A. (Open bars) pGBT9 alone; (solid bars) pGBT9–SMRT. (C) SMRT RD III and RD IV do not interact with HDAC1 or mHDAC6. The bait construct pGBT9–SMRT (RD III + RD IV) was cotransformed with GAL AD fusion to various HDAC constructs into yeast strain Y190. After 3 days, two isolated colonies were picked, resuspended in water, and 103 cells were dropped on both −Trp–Leu and −Trp–Leu–His + 40 m

m

3-AT plates. Pictures were taken after 3 days.

Figure 1

Figure 1

SMRT repression domains III and IV interact with deacetylase domain of the newly described deacetylases. (A) Quantitation of yeast two-hybrid assays between SMRT and HDAC5 and HDAC7. Yeast cells were cotransformed with the indicated plasmids. Two viable transformants were picked for liquid β-galactosidase activity. (Open bars) AD; (solid bars) AD–HDAC. The number represents average of duplicates from two colonies. (B) SMRT interaction requires amino acid carboxy-terminal to the deacetylase domain of mHDAC5. Yeast method as described in A. (Open bars) pGBT9 alone; (solid bars) pGBT9–SMRT. (C) SMRT RD III and RD IV do not interact with HDAC1 or mHDAC6. The bait construct pGBT9–SMRT (RD III + RD IV) was cotransformed with GAL AD fusion to various HDAC constructs into yeast strain Y190. After 3 days, two isolated colonies were picked, resuspended in water, and 103 cells were dropped on both −Trp–Leu and −Trp–Leu–His + 40 m

m

3-AT plates. Pictures were taken after 3 days.

Figure 2

Figure 2

Putative amino acid sequence and tissue distribution of mHDAC7. (A) The deduced amino acid sequence of mHDAC7 and sequence alignment of human HDAC4 and mouse HDAC5 and HDAC7. Sequence alignment of HDAC4, HDAC5, and HDAC7 were performed according to the Jotun Hein method with the DNA STAR program. (Arrow) Beginning of the histone deacetylase domain. (B) Schematic representation of the novel histone deacetylase family. The percentage of homology in each domain is determined by blasting against the amino acid sequence of mHDAC7. (Open rectangle) Histone deacetyalse domain. (Solid ovals) Position of the glutamate-rich (E-rich) regions. (Solid rectangle) Three copies of the zinc finger motif. (C) Expression pattern of mHDAC7 in mouse. Multiple mouse tissue Northern blots were probed with mHDAC7 cDNA. The size marker is indicated. (D) mHDAC5 and mHDAC7 associate with histone deacetylase activity. Whole-cell extracts prepared from cells expressing vector alone, mHDAC5 (lanes 2,3), and mHDAC7 (lanes 4,5) were immunoprecipitated with anti-HA antibodies conjugated agarose beads. Immunoprecipitates were resuspended in deacetylase assay buffer for histone deacetylase assays in the presence (lanes 3,5) or absence (lanes 2,4) of 100 n

m

trichostatin A. (E) SMRT and N-CoR associate with histone deacetylase activity.

Figure 2

Figure 2

Putative amino acid sequence and tissue distribution of mHDAC7. (A) The deduced amino acid sequence of mHDAC7 and sequence alignment of human HDAC4 and mouse HDAC5 and HDAC7. Sequence alignment of HDAC4, HDAC5, and HDAC7 were performed according to the Jotun Hein method with the DNA STAR program. (Arrow) Beginning of the histone deacetylase domain. (B) Schematic representation of the novel histone deacetylase family. The percentage of homology in each domain is determined by blasting against the amino acid sequence of mHDAC7. (Open rectangle) Histone deacetyalse domain. (Solid ovals) Position of the glutamate-rich (E-rich) regions. (Solid rectangle) Three copies of the zinc finger motif. (C) Expression pattern of mHDAC7 in mouse. Multiple mouse tissue Northern blots were probed with mHDAC7 cDNA. The size marker is indicated. (D) mHDAC5 and mHDAC7 associate with histone deacetylase activity. Whole-cell extracts prepared from cells expressing vector alone, mHDAC5 (lanes 2,3), and mHDAC7 (lanes 4,5) were immunoprecipitated with anti-HA antibodies conjugated agarose beads. Immunoprecipitates were resuspended in deacetylase assay buffer for histone deacetylase assays in the presence (lanes 3,5) or absence (lanes 2,4) of 100 n

m

trichostatin A. (E) SMRT and N-CoR associate with histone deacetylase activity.

Figure 3

Figure 3

HDAC5 and HDAC7 repress basal transcription. (A) HDAC5 and HDAC7 repress basal transcription in transient transfection assays. CV-1 cells were transfected with reporter constructs pCMX–β–GAL and pMH100–TK–Luc as well as increasing amounts of plasmids expressing GAL–mHADC5 (lanes 2_–_4), GAL–mHADC7 (lanes 5_–_7), and GAL–HDAC1 (lanes 8_–_10). (Lane 1) Control. Fold repression activity is shown at top of each bar. (B) mHDAC7 possesses three repression domains. The GAL DBD1–147 was fused to the amino-terminal of a series of truncation constructs of mHDAC7, and 0.1 μg of the fusion construct was tested in transient transfection assays for repression activity. Fold repression is shown at top of each bar. Fold repression was determined relative to the basal transcription activity of the reporter in the presence of GAL4 DBD. (C) mHDAC5 contains two repression domains. The assays were carried out the same as described above except that the GAL DBD–mHDAC5 constructs were used.

Figure 3

Figure 3

HDAC5 and HDAC7 repress basal transcription. (A) HDAC5 and HDAC7 repress basal transcription in transient transfection assays. CV-1 cells were transfected with reporter constructs pCMX–β–GAL and pMH100–TK–Luc as well as increasing amounts of plasmids expressing GAL–mHADC5 (lanes 2_–_4), GAL–mHADC7 (lanes 5_–_7), and GAL–HDAC1 (lanes 8_–_10). (Lane 1) Control. Fold repression activity is shown at top of each bar. (B) mHDAC7 possesses three repression domains. The GAL DBD1–147 was fused to the amino-terminal of a series of truncation constructs of mHDAC7, and 0.1 μg of the fusion construct was tested in transient transfection assays for repression activity. Fold repression is shown at top of each bar. Fold repression was determined relative to the basal transcription activity of the reporter in the presence of GAL4 DBD. (C) mHDAC5 contains two repression domains. The assays were carried out the same as described above except that the GAL DBD–mHDAC5 constructs were used.

Figure 4

Figure 4

SMRT interacts with mHDAC5 and mHDAC7 in vitro and in vivo. (A) Summary of yeast two-hybrid assays. SMRT contains four repression domains denoted as RD I, RD II, RD III, and RD IV. RD III and RD IV were renamed from SRD I and SRD II, respectively. Note that the HDAC5-interacting domain was mapped to amino acids 1281–1785. Quantitation of the yeast two-hybrid assays are indicated following each construct shown. (Open bars) AD alone; (solid bars) AD–mHDAC. (B) HDAC5 interacts with SMRT in vitro. (Lane 1) 12.5% of input, (lane 2) GST alone, (lane 3) GST–HDAC5 (590–1122). (D) SMRT interacts with HDAC5 and HDAC7 in mammalian cells. Anti-Flag antibodies were incubated with whole cell extracts prepared from cells expressing mHDAC5–HA (lane 1), mHDAC7–HA (lane 2), mHDAC5–HA and SMRT–Flag (lane 3), and mHDAC7–HA and SMRT–Flag (lane 4). Immunoprecipitates were separated onto a SDS-polyacrylamide gel and Western analysis was carried out with anti-HA antibodies as a probe. The expression level of mHDAC5–HA and mHDAC7–HA is equivalent in all extracts as determined by Western blots (not shown).

Figure 5

Figure 5

mHDAC5 colocalizes with HDAC7 and SMRT. (A) mHDAC7 predominantly localizes in nucleus with dot-like subnuclear structure. Note that mHDAC7 and DAPI staining are not overlapped. (B) mHDAC5 and mHDAC7 colocalize in the nucleus. (C) mHDAC5 colocalizes with SMRT, but not CBP in CV-1 cells. Note that CBP also gives speckle staining, which represents PML nuclear bodies.

Figure 5

Figure 5

mHDAC5 colocalizes with HDAC7 and SMRT. (A) mHDAC7 predominantly localizes in nucleus with dot-like subnuclear structure. Note that mHDAC7 and DAPI staining are not overlapped. (B) mHDAC5 and mHDAC7 colocalize in the nucleus. (C) mHDAC5 colocalizes with SMRT, but not CBP in CV-1 cells. Note that CBP also gives speckle staining, which represents PML nuclear bodies.

Figure 5

Figure 5

mHDAC5 colocalizes with HDAC7 and SMRT. (A) mHDAC7 predominantly localizes in nucleus with dot-like subnuclear structure. Note that mHDAC7 and DAPI staining are not overlapped. (B) mHDAC5 and mHDAC7 colocalize in the nucleus. (C) mHDAC5 colocalizes with SMRT, but not CBP in CV-1 cells. Note that CBP also gives speckle staining, which represents PML nuclear bodies.

Figure 6

Figure 6

mHDAC7 complexes with mSin3A in yeast and in mammalian cells. (A) mHDAC7 complexes with mSin3A in 293 cells. Whole-cell extracts prepared from 293 cells with (lanes 2,4,6,8) or without mHDAC7–HA expression (lanes 1,3,5,7) were incubated with anti-HA antibodies conjugated with agarose beads. Immunoprecipitates were subjected to Western blot analysis and probed with anti-HA antibodies (lanes 3,4), anti-mSin3A antibodies (lanes 5,6), and anti-SMRT antibodies (lanes 7,8). (Lanes 1,2) Whole cell extracts probed with anti-HA antibodies. (B) mHDAC7 interacts with PAH1 of mSin3A in yeast. Plasmids pGBT9–HDAC7 (500–938) and pACTII–mSin3A were cotransformed into yeast Y190 strain and β-galactosidase lifting assays were performed. (C) The carboxy-terminal region (amino acids 864–938) of HDAC7 is required for mSin3A interaction. Plasmids pGBT9–HDAC and pACTII–mSin3A (1–386) were cotransformed into yeast strain Y190 in this experiment.

Figure 7

Figure 7

SMRT represses transcription by recruiting both class I and class II histone deacetylase. Repression by a sequence-specific transcription factor such as nuclear receptor heterodimer retinoid X receptor (RXR) and retinoid acid receptor (RAR) is mediated by a SMRT corepressor complex that represses transcription by recruiting both class I and class II histone deacetylases. Models for mechanism of SMRT repression including: (1) SMRT recruits class I deacetylase HDAC1/HDAC2 through direct interaction with mSin3A. (B) SMRT represses transcription by direct interaction with class II deacetylases HDAC5 and HDAC7 (and possibly HDAC4), which also binds to mSin3A through a region different from where HDAC1 binds. (C) SMRT recruits both class I and class II histone deacetylases and mSin3A.

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