Fig 1.
SRID mutants of HDAC4c and -5c obtained by Partitioned OPTHiS.
(A) Schematic depiction of gap plasmids and targeted regions of HDAC4c and -5c for the SRID mutant screening by Partitioned OPTHiS. The gap plasmids G4N and G4T were used for the screening of SRID mutants targeted for the HDAC4cN (amino acids 651 to 865) and HDAC4cT (amino acids 865 to 1055) regions of HDAC4c, respectively. For HDAC5c, the gap plasmids G5N and G5M were used to isolate SRID alleles present in the HDAC5cN (amino acids 680 to 831) and HDAC5cM (amino acids 829 to 1010) regions of HDAC5c, respectively. The linearized gap plasmids and mutagenic PCR products were prepared with the use of the indicated restriction enzymes (top) and the denoted primer sets (arrow), respectively. (B) The list of SRID alleles of HDAC4c (upper) or HDAC5c (lower). The residues in gray boxes were commonly found between SRID mutants of HDAC4c and -5c based on sequence alignment. The residues existing on the surface of HDAC4c are presented in bold, and the class IIa HDAC-specific residues are shown in red.
Fig 2.
Defective interactions of SRD3c with the isolated HDAC4c and -5c mutants in GST pull-down assays.
GST-SRD3c protein was purified and tested for interactions with the wild-type (WT) and mutant versions of 35S-labeled HDAC4c (A) or -5c (B) proteins. GST protein was used as the negative control. Input indicates 10% of the in vitro translated HDAC proteins used in the pull-down analysis.
Fig 3.
Defective interactions of SRD3c with SRID mutants of HDAC4c and -5c in HEK293 cells.
(A) Confocal laser scanning microscope images illustrating the interactions between SRD3c and HDAC4c or -5c via BiFC assay. Fluorescent signals were mainly localized in the nucleus, and observed in the shape of speckles. Magnification: 180 X. (B),(C) Quantitative measurement of fluorescence signals in the BiFC assay, generated by the association of SRD3c with the indicated SRID mutants of HDAC4c (B) or -5c (C). The expression constructs for KGN-SRD3c (1 μg) and the indicated KGC-HDAC4c or -5c mutants (1 μg) were transiently cotransfected into HEK293 cells. After 48 hours of transfection, the fluorescent signals from whole cell lysates were measured with the use of a fluorescence spectrophotometer. E.V (empty vector) and N.T (no transfection) samples were used as negative controls. The p-values for all compared groups between wild-type and mutants are less than 0.01. RFU: relative fluorescence units.
Fig 4.
Positions of SRID mutations over the MSA of HDAC domains.
Structure-based MSA of the catalytic domains from HDAC1 to HDAC11 was built using the ESPript 3.0 program, and secondary structural elements are shown for the inhibitor-bound HDAC4c structure (PDB code 2VQJ). The residues in pink and blue shades represent the SRID alleles specifically found in HDAC4c and -5c mutants, respectively. The residues with yellow shade indicate the SRID alleles commonly found at the same positions between HDAC4c and -5c sequences on the basis of MSA. Red triangles: the residues of HDAC4c mutants located on the surface region of the HDAC4c structure. Blue Boxes indicate hot-spot regions for SRID mutations which are commonly found in all classes of the HDAC family. Red boxes indicate hot-spot regions for SRID mutations which correspond to class IIa HDAC-specific regions.
Fig 5.
Surface presentation of SRID mutations on HDAC4c structure.
(A) Ribbon diagram of HDAC4c structure (PDB code 2VQJ) [40]. The structural zinc-binding domain is shown by magenta. Two zinc ions and their chelating residues are drawn as spheres and sticks, respectively. (B) Positions of SRID mutations of HDAC4c are presented on the surface of the HDAC4c structure (PDB code 2VQJ). Among SRID mutants of HDAC4c, 21 residues located on the surface are indicated, and their surface positions are shown in orange. (C) Surface presentation of SRID mutations of HDAC5c at the corresponding positions of the HDAC4c structure. The positions of the SRID mutations of HDAC5c were changed to those of HDAC4c according to MSA data, and presented on the surface of HDAC4c structure in orange. The residues specific to class IIa HDACs are indicated in yellow color.
Fig 6.
Loss of in vitro HDAC activity of immuno-purified HDAC4c mutants due to inability to associate with endogenous SMRT-HDAC3 complex.
(A) In vitro HDAC assay and (B) co-immunoprecipitation assay for HDAC4c SRID mutant proteins immuno-purified from HEK293 cells. The HA-tagged versions of the indicated HDAC4c proteins were coexpressed with HDAC3 in HEK293 cells by transient transfection and purified from cell lysates (300 μg) with the use of agarose beads coupled with anti-HA-antibody. (A) The immune complex was subsequently subjected to in vitro HDAC assay using fluorescent-coupled Lys acetamide as a substrate. HeLa nuclear extract (1 μg) was used as the positive control. The p-values for all compared groups between wild-type and mutants are less than 0.0001. RFU: relative fluorescence units. (B) For co-immunoprecipitation assay, immunoprecipitates from 1 mg of whole cell lysate were prepared and analyzed for the presence of HA-HDAC4c and HDAC3 by immunoblot analysis.
Fig 7.
Transcriptional repressive activities of Gal4N-fused SRID mutants of HDAC4c.
(A) Mammalian one-hybrid assay of HDAC4c mutants. HEK293 cells were cotransfected with the Gal4-tk-luciferase reporter plasmid (200 ng) and wild-type (WT) or the indicated mutants of Gal4N-HDAC4c plasmids (50 and 100 ng), together with the pCMV-β-gal vector (100 ng). After 48 hours of transfection, the luciferase activities were measured and normalized, as described in the materials and methods section. Fold repression indicates the mean ± S. E. value obtained from at least two independent experiments performed in duplicate. The p-values for all compared groups between wild-type and mutants are less than 0.05. RLU: relative luciferase activity. (B) The expression levels of wild-type (WT) or indicated mutants of Gal4N-HDAC4c proteins were examined by immunoblot analysis of whole cell lysates (15 μg) using anti-Gal4N mouse monoclonal antibody (Santa Cruz, sc-510; 1:3,000 dilution).