HDAC6 regulates DNA damage response via deacetylating MLH1

Abstract

MutL homolog 1 (MLH1) is a key DNA mismatch repair protein, which plays an important role in maintenance of genomic stability and the DNA damage response. Here, we report that MLH1 is a novel substrate of histone deacetylase 6 (HDAC6). HDAC6 interacts with and deacetylates MLH1 both in vitro and in vivo Interestingly, deacetylation of MLH1 blocks the assembly of the MutSα-MutLα complex. Moreover, we have identified four novel acetylation sites in MLH1 by MS analysis. The deacetylation mimetic mutant, but not the WT and the acetylation mimetic mutant, of MLH1 confers resistance to 6-thioguanine. Overall, our findings suggest that the MutSα-MutLα complex serves as a sensor for DNA damage response and that HDAC6 disrupts the MutSα-MutLα complex by deacetylation of MLH1, leading to the tolerance of DNA damage.

Keywords: DNA damage response; DNA mismatch repair; DNA repair; HDAC6; MLH1; acetylation; drug resistance; histone deacetylase 6 (HDAC6).

Figure 1.

HDAC6 interacts with MLH1. A and B, endogenous interaction of HDAC6 and MLH1 in A549 cells. A, immunoprecipitation (IP) was performed with either anti-IgG or anti-HDAC6 antibody in A549 cells, followed by anti-MLH1 and anti-HDAC6 Western blotting analyses as shown in the upper and lower panel, respectively. The input of MLH1 and HDAC6 is also shown. B, reciprocal immunoprecipitation of A was performed. C, physical interaction between HDAC6 and MLH1. GST pulldown assays were performed with the bacterially-expressed His–MLH1 and GST–HDAC6 proteins, followed by anti-His Western blot analysis (upper panel). Purified GST and GST–HDAC6 proteins were analyzed by Coomassie Blue staining (lower panel). GSH-agarose was used for pulldown as a negative control. D, schematic representation of HDAC6 full-length protein and its deletion mutants. The strength of interaction between HDAC6 and MLH1 was shown as follows: ++, very strong binding; +, strong binding; and −, no binding. E, MLH1 binds to HDAC6 deacetylase domain 1 (DAC1) and 2 (DAC2). Empty vector, FLAG-tagged full-length HDAC6 (1–1215), DAC1 (1–503), DAC2 (488–834), or ZnF–UBP (835–1215) was co-transfected with Myc-MLH1 in 293T cells. Anti-FLAG immunoprecipitation was performed, followed by Western blotting using anti-Myc and anti-FLAG antibodies (upper and middle panels). The input of Myc-MLH1 is shown in the anti-Myc Western blot analysis in the lower panel. F, schematic representation of MLH1 full-length protein and its deletion mutants. The strength of interaction between MLH1 and HDAC6 was shown as follows: +, strong binding, +/−, weak binding, and −, no binding. G, HDAC6 binds to MLH1's N-terminal (1–320) and C-terminal (491–756) regions. Empty vector, FLAG-tagged full-length MLH1(1–756), MLH1(1–147), MLH1(147–320), MLH1(320–491), or MLH1(491–756) was co-transfected with HA–HDAC6 in 293T cells. Anti-FLAG immunoprecipitation was performed and followed by Western blottings using anti-HA and anti-FLAG antibodies (upper and middle panels). The input of HA–HDAC6 is shown in the anti-HA Western blotting (lower panel). IB, immunoblot.

Figure 2.

HDAC6 interacts with MLH1 in the nucleus. A, HDAC6 interacts with MLH1 in the T29 nuclear extracts. Immunoprecipitation (IP) was performed with either anti-IgG or anti-HDAC6 antibody in T29 nuclear extracts, followed by anti-MLH1 and anti-HDAC6 Western blotting analyses as shown in the upper and lower panels, respectively. The input of MLH1 and HDAC6 is also shown. B and C, HDAC6 is co-localized with MLH1 in H1299 cells upon etoposide treatment. Representative images of immunofluorescence staining of HDAC6, MLH1, and DAPI as well as merged images in vehicle-treated H1299 cells (B) and 10 μm etoposide-treated H1299 cells are shown. D, nuclear (N)/cytoplasmic (C) expression of HDAC6 in four nonsmall cell lung cancer cell lines. The nuclear and cytoplasmic fractions were prepared as described under “Experimental procedures.” The Western blotting (IB) analyses were performed with the indicated antibodies.

Figure 3.

HDAC6 is an MLH1 deacetylase. A, level of acetylated MLH1 is increased upon TSA treatment. 293T cells were treated with vehicle or 200 μm of TSA for 12 h. Thirty six hours after transfection, cells were lysed, and anti-MLH1–agarose beads were used to immunoprecipitate (IP) MLH1. The resulting immunocomplexes were resolved on SDS-PAGE, followed by anti-acetylated lysine (anti-AcK) Western blot analysis (upper panel). The membrane was stripped and reprobed with the anti-MLH1 antibody (lower panel). B, level of acetylated MLH1 is increased upon tubastatin A treatment. 293T cells were treated with vehicle or 10 μm tubastatin A for 12 h. Thirty six hours after transfection, cells were lysed and anti-MLH1–agarose beads were used to immunoprecipitate MLH1. The resulting immunocomplexes were resolved on SDS-PAGE, followed by anti-acetylated lysine (anti-AcK) Western blot analysis (upper panel). The membrane was stripped and reprobed with the anti-MLH1 antibody (lower panel). C, p300 acetylates MLH1. 293T cells were transfected with an empty vector, F-MLH1, or F-MLH1 with HA-p300. Cell lysates were immunoprecipitated with anti-FLAG–M2–agarose beads, and an anti-Ac-K antibody was used to detect the Ac-MLH1 levels by Western blot analysis (upper panel). The membrane was then stripped and reprobed with the anti-FLAG antibody (lower panel). D, HDAC6 deacetylates MLH1 in 293T cells. Myc-MLH1 was transfected with either empty vector or FLAG–HDAC6 in 293T cells. The cell lysates were immunoprecipitated with anti-Myc–agarose beads, followed by anti-AcK Western blot analysis (upper panel). The membrane was stripped and reprobed with anti-Myc antibody (middle panel). The expression of FLAG–HDAC6 was detected by anti-FLAG Western blot analysis (lower panel). E, HDAC6 deacetylates MLH1 in vitro. FLAG–HDAC6 was purified from 293T cells. To obtain acetylated Myc-MLH1, 293T cells were transiently transfected with Myc-MLH1, followed by TSA treatment. Acetylated Myc-MLH1 was then purified and incubated with either buffer or purified FLAG–HDAC6. Reactions were stopped by adding SDS-PAGE loading buffer directly and were then subjected to Western blots with anti-AcK, and anti-Myc antibodies. The lower panel shows purified FLAG–HDAC6 by Coomassie blue staining. For all the panels, the acetylated MLH1 bands were quantified and the fold-changes were shown below the those bands. IB, immunoblot.

Figure 4.

Lysines 33, 241, 361, and 377 are acetylated in MLH1. A, lysine 33 is acetylated in MLH1. The peptide was detected with a m/z of 625.3074, which represents an error of 6.1 ppm. The tandem mass spectrum matched the following sequence, PANAIKEMIENCLDAK, indicating that the first lysine was acetylated. B, lysine 241 is acetylated in MLH1. The peptide was detected with a m/z of 1040.0105, which represents an error of 4.2 ppm. The tandem mass spectrum matched the following sequence, TLAFKMNGYISNANYSVK, indicating that the first lysine was acetylated. C, lysine 361 is acetylated in MLH1. The peptide was detected with an m/z of 1237.5928, which represents an error of 1.4 ppm. The tandem mass spectrum matched the following sequence, MYFTQTLLPGLAGPSGEMVKSTTSLTSSSTSGSSDK, indicating that the first lysine was acetylated. D, lysine 377 is acetylated in MLH1. The peptide was detected with an m/z of 858.7390, which represents an error of 4.3 ppm. The tandem mass spectrum matched the following sequence, STTSLTSSSTSGSSDKVYAHQMVR, indicating that the first lysine was acetylated.

Figure 5.

Conservation of four acetylated lysines in MLH1. A, stretch of MLH1 amino acids shows the conservation of lysine 33 among different species. B, stretches of MLH1 amino acids show the conservation of lysines 241, 361, and 377 among different species. Consensus amino acids are indicated as *. C, diagram of MLH1's domain structure showing the locations of four acetylated lysines.

Figure 6.

Acetylation/deacetylation of MLH1 regulates MLH1's binding to MutSα. A, Lys-33, Lys-241, Lys-361, and Lys-377 sites are the major acetylation sites in MLH1. MLH1-deficient SKOV3 cells were stably transfected with FLAG-tagged control vector, MLH1–PMS2, MLH1(4KR)–PMS2, or MLH1(4KQ)–PMS2. The cell lysates were immunoprecipitated (IP) with anti-FLAG–M2–agarose beads followed by Western blot analysis with anti-AcK. The membrane was stripped and reblotted with the anti-FLAG antibody. The input is shown by anti-FLAG, anti-PMS2, and anti-β-actin antibodies. B, MLH1–4KR mutant exhibits reduced binding affinity to MSH2 and MSH6. 293T cells were stably transfected with FLAG-tagged control vector, MLH1–PMS2, MLH1(4KR)–PMS2, or and MLH1(4KQ)–PMS2. The cell lysates were immunoprecipitated with anti-FLAG–M2–agarose beads followed by Western blottings with the indicated antibodies. The expression of F-MLH1, PMS2, MSH2, and MSH6 in transfected HEK293T cells was shown by the indicated Western blots. C, all MLH1 mutants (K33R, K241R, K361R, and K377R) display the same affinity of binding to MSH2 and MSH6 as WT MLH1. F-MLH1, F-MLH1-K33R, F-MLH1-K241R, F-MLH1-K361R, or F-MLH1-K377R was transiently co-transfected with HA–PMS2 into HEK293T cells. The cell lysates were immunoprecipitated with anti-FLAG–M2–agarose beads followed by Western blot analysis with the indicated antibodies. The immunoprecipitated F-MLH1 (WT and mutants) and HA–PMS2 are indicated by Ponceau staining. The expression of MSH2, MSH6, input of F-MLH1 (WT and mutants), and HA–PMS2 is shown by anti-MSH2, anti-MSH6, anti-FLAG, and anti-PMS2 Western blotting analyses. D and E, knockdown of HDAC6 enhances the formation of the MutLα–MutSα complex in U2OS and H292 cells. U2OS (D), or H292 (E) Tet-On–inducible HDAC6 knockdown cells were cultured with the Tet-free medium (lane 1) or doxycycline-containing medium (0.5 μg/ml) (lane 2). The cell lysates were immunoprecipitated with an anti-MLH1 antibody, followed by Western blotting with the indicated antibodies. The inputs of D and E were shown by direct Western blottings with indicated antibodies. IB, immunoblot.

Figure 7.

Deacetylation mimetic mutant of MLH1 confers 6-TG tolerance in HEK293T cells. A, reintroduction of WT MLH1 and PMS2 in HEK293T cells sensitizes cells to 6-TG. F-MLH1–PMS2 stably transfected 293T cells were treated with 50 μm 6-TG for the indicated time intervals. Anti-PARP1 and anti-β-actin Western blotting analyses were performed. B, deacetylation mimetic mutant of MLH1 cannot sensitize cells to 6-TG as does WT MLH1. The FLAG-tagged MLH1–PMS2, 4KR-PMS2 or 4KQ-PMS2 was stably transfected into 293T cells, followed by 50 μm 6-TG treatment for indicated time intervals. Anti-PARP1 and anti-β-actin Western blotting analyses were performed. C and D, deacetylation mimetic mutant of MLH1 confers 6-TG tolerance in 293T cells, as assessed via MTT and colony formation assays. The FLAG-tagged empty vector, MLH1–PMS2, 4KR-PMS2, or 4KQ-PMS2 was stably transfected into 293T cells. MTT assays were performed with indicated 6-TG concentrations for 3 days (C). Colony formation assays were performed with vehicle or 0.5 μm 6-TG for 10 days. Cells were stained with crystal violet and survival colonies were counted, as shown in D. C and D, Student's t test was performed with *, p < 0.05; ns, not significant. Error bars, S.D. E, expression of F-MLH1–WT, F-MLH1–4KR, F-MLH1–4KQ, and PMS2 in 293T stable cell lines. Lysates from the aforementioned cell lines were subjected to anti-FLAG, anti-PMS2, and anti-β-actin Western blotting analyses.

Figure 8.

MLH1 structure represented by molecular surface. Surface coloring is according to the electrostatic potential: red, white, and blue corresponding to negative, neutral, and positive potential, respectively. Two acetylated lysines, Lys-33 and Lys-241, are indicated. The potential MSH2-binding site is indicated in the region encircled by the dotted line. The structures of the sites of Lys-33 and Lys-241 are also enlarged.

Figure 9.

Working model of how HDAC6 regulates MutSα's recruiting MutLα via deacetylation of MLH1. Upon 6-TG treatment, 6-thioguanine (^sG) and S⁶-methylthioguanine (S⁶mG) can be inserted into the DNA, so that ^sG:T and S⁶mG:T mispairs can be formed. These mispairs would be recognized by the MSH2–MSH6 heterodimer (MutSα), and MutSα would then recruit the MLH1–PMS2 heterodimer (MutLα) and additional components to initiate futile mismatch repair, leading to apoptosis. However, HDAC6 can deacetylate MLH1 and prevent MutLα from being recruited to MutSα, leading to 6-TG tolerance.