As a critical developmental process, epithelial–mesenchymal transition (EMT) involves complex transcriptional reprogramming and has been closely linked to malignant progression. Although various epigenetic modifications, such as histone deacetylation and H3K9 methylation, have been implicated in this process, how they are coordinated remains elusive. We recently revealed that MPP8 couples H3K9 methylation and DNA methylation for E‐cadherin gene silencing and promotes tumor cell migration, invasion, and EMT. Here, we show that MPP8 cooperates with the class III HDAC SIRT1 in this process through their physical interaction. SIRT1 antagonizes PCAF‐catalyzed MPP8‐K439 acetylation to protect MPP8 from ubiquitin‐proteasome‐mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl‐H3K9 on target promoters. Consequently, disabling either MPP8 methyl‐H3K9 binding or SIRT1 interaction de‐represses E‐cadherin and reduces EMT phenotypes, as does knockdown of MPP8 or SIRT1 in prostate cancer cells. These results illustrate how SIRT1 and MPP8 reciprocally promote each other's function and coordinate epithelial gene silencing and EMT.
This study shows that two repressive chromatin modifiers, MPP8 and SIRT1, reciprocally promote each other's function through their physical interaction to coordinate epithelial gene silencing and promote EMT.
SIRT1 counteracts MPP8‐K439 acetylation catalyzed by PCAF to protect MPP8 from ubiquitin‐proteasome‐mediated degradation.
MPP8 binds to methyl‐H3K9 on target promoters and recruits SIRT1 to preferentially deacetylate H4K16 for transcription repression.
MPP8 not only interacts with the EMT‐inducing transcription factor ZEB1 but also facilitates ZEB1‐SIRT1 protein interaction.
Epithelial–mesenchymal transition (EMT) was initially defined as a developmental process which enables polarized epithelial cells reversibly lose their epithelial characteristics and acquire mesenchymal properties , . It is now evident that EMT is also an essential mechanism to endow epithelial cancer cells with migratory and invasive capabilities associated with metastatic competence . One hallmark of EMT is the functional loss of a key epithelial adhesion molecule E‐cadherin, which is mainly achieved at the transcription level during malignant progression . Several transcription factors (EMT‐TF), such as ZEB1/2 and SNAI1/2, directly bind to E‐cadherin promoter for gene silencing, while the elevated expression of these EMT‐TFs correlates with reduced E‐cadherin expression and poor clinical prognosis in different epithelial cancer cells . It has been shown that various histone modifications are essential mediators of EMT‐TFs and also play a critical role in this process , . For example, inhibition of histone deacetylation or H3K4 demethylation de‐represses E‐cadherin expression and prevents EMT in several tumor cell lines , , , , . Similar effects can also be achieved by inhibiting methylation of H3K9, H3K27, or H4K20 using different approaches , , , .
Histone deacetylation and H3K9 methylation are two intimately connected repressive modifications that have been implicated in E‐cadherin silencing. For example, HDAC1/2 coexist with G9a/GLP in CtBP1 complex which coordinates the stepwise deacetylation and methylation of H3K9 on E‐cadherin promoter . A class III HDAC SIRT1 has been implicated in E‐cadherin silencing and EMT by cooperating with ZEB1 , . The fact that ZEB1 was also co‐purified in CtBP1 complex  suggests a functional link between SIRT1 and H3K9 methylation. However, the underlying mechanism is largely unknown.
Methylation of H3K9 creates binding site for a group of adaptor proteins that mediate downstream effects. We have previously established the role of MPP8, a methyl‐H3K9 binding protein in E‐cadherin silencing and EMT . Here, we demonstrate that MPP8 and SIRT1 reciprocally regulate each other's function at multiple molecular layers through their physical interaction. Disruption of MPP8‐SIRT1 interaction de‐represses E‐cadherin expression and reduces cell motility and invasiveness, suggesting that this interplay plays critical role in MPP8‐ and SIRT1‐mediated EMT.
Results and Discussion
MPP8 interacts with SIRT1
Given that MPP8 and SIRT1 displayed similar function in E‐cadherin silencing and EMT , , , we first tested whether they have overlapping function in E‐cadherin silencing. As indicated in Fig 1A, stable knockdown (KD) of SIRT1 or MPP8, or treatment of EX‐527, a specific SIRT1 inhibitor , up‐regulated E‐cadherin protein level in PC3 prostate cancer cells. However, EX‐527 treatment of MPP8‐KD cells or simultaneous knockdown of SIRT1 and MPP8 did not lead to an additive increase. Although E‐cadherin mRNA level increased ~three‐ to eightfold upon EX‐527 treatment or knockdown of MPP8 or SIRT1, an additional increase was not detected either when SIRT1 and MPP8 were inhibited simultaneously. Similar expression changes were also observed when we analyzed another SIRT1 target gene CRBP1  (Fig 1B). Thus, these results support the possibility that MPP8 and SIRT1 are involved in the same transcription repression pathway.
Different histone‐modifying enzymes often coexist in the same multifunctional complex and act in concert for transcription regulation . We thus tested whether MPP8 interacts with SIRT1 by immunoprecipitation (IP)‐Western analysis. As shown in Fig 1C, Flag‐MPP8 and Myc‐SIRT1 specifically pulled down each other when they were co‐expressed in 293T cells. This interaction was not affected by either K622R mutation on SIRT1 or the methyl‐lysine binding disabling mutation W80A , ,  on MPP8, suggesting it is not mediated by the newly identified SIRT1‐K622 methylation . Importantly, Western blot analysis detected the robust MPP8‐SIRT1 interaction in 293T‐wt cells but not in SIRT1‐KD or MPP8‐KD cells after endogenous IP using their direct antibodies (Fig 1D), confirming that MPP8 specifically interacts with SIRT1 in cells.
To map MPP8‐SIRT1 interaction, we generated a serial of Flag‐MPP8 deletions and co‐expressed them with Myc‐SIRT1‐wt in 293T cells. IP‐Western blot analysis reveals that the deletion of first 263aa on MPP8, but not first 111aa or last 260aa or both, is sufficient to abolish its interaction with SIRT1 (Fig 1E), suggesting that a small region (112–263aa) on MPP8 is critical. This result was further confirmed by a similar IP‐Western blot analysis using an MPP8 internal deletion mutant (Δ112–225aa) (Fig 1F). We also co‐expressed Flag‐MPP8 with different GFP‐SIRT1 deletions for similar analysis and uncovered that the catalytic core of SIRT1 (254–489aa) is necessary and sufficient to interact with MPP8 (Fig 1G).
PCAF and SIRT1 dynamically regulate MPP8‐K439 acetylation
The finding that MPP8 interacts with the catalytic domain of SIRT1 prompted us to test whether MPP8 is a SIRT1 substrate. First, we expressed Flag‐MPP8 in MPP8‐KD 293T cells  followed by HDAC inhibitor treatment to test whether MPP8 is acetylated in cells. Anti‐acetyl‐lysine Western blot analysis of Flag‐IPed MPP8 did not detect MPP8 acetylation when cells were treated with a class I/II HDAC inhibitor TSA. However, a robust acetylation signal was readily detected from cells treated with a class III HDAC inhibitor nicotinamide (NIA) and this signal did not increase additionally when cells were treated with both inhibitors. These results suggest that MPP8 is subjected to acetylation in cells and its acetylation is regulated by Sir2 family HDACs.
Next, we co‐expressed Myc‐MPP8 with six different HATs in MPP8‐KD 293T cells. Anti‐myc IP‐coupled Western blot analysis detected the robust MPP8 acetylation only from cells co‐expressing PCAF (Fig 2B), suggesting that MPP8 is acetylated by PCAF. This possibility was further confirmed by observations that MPP8 acetylation level was in PCAF dosage‐dependent manner, while a catalytically inactive mutation in PCAF (Δ579–608) eliminated MPP8 acetylation (Fig 2C). We next purified PCAF‐acetylated MPP8 for LC–mass spectrometry and uncovered that the acetylation occurs at K439 (Fig 2D). Substitution of K439 to arginine eliminated MPP8 acetylation, while K‐R mutation at an acetylation consensus motif (K214KPKK) or an adjacent lysine (K433) did not (Fig 2E), demonstrating that PCAF specifically acetylates MPP8 at K439.
Finally, we co‐expressed MPP8 and PCAF together with four nuclear‐localized Sirtuins (SIRT‐1, 3, 6, 7)  in MPP8‐KD 293T cells and examined MPP8 acetylation by IP‐Western blot analysis. As shown in Fig 2F, MPP8 acetylation was only abrogated in cells co‐expressing SIRT1. Co‐expression of a catalytically inactive mutant SIRT1‐H363Y did not affect MPP8 acetylation (Fig 2G), indicating that SIRT1 enzymatic activity is required. We further purified PCAF‐acetylated MPP8 and incubated it with recombinant GST‐SIRT1 for an in vitro deacetylation assay. As indicated in Fig 2H, MPP8 acetylation was eliminated when GST‐SIRT1 and cofactor NAD+ were added into the reaction and this MPP8 deacetylation was effectively inhibited by nicotinamide but not TSA. These results together demonstrate that PCAF and SIRT1 dynamically regulate MPP8 acetylation at K439.
SIRT1 modulates MPP8 protein stability
The finding of MPP8‐K439 acetylation prompted us to test whether it affects MPP8 methyl‐H3K9 binding. This possibility was ruled out as MPP8‐wt, K439Q (acetylation‐mimic), and K439R (acetylation‐disabled) mutant displayed the similar binding profile to H3 peptides with different methyl‐K9 states (Supplementary Fig S1A). However, we noticed that MPP8 protein level, but not mRNA level, reduced in cells where SIRT1 was inhibited by EX‐527 or knockdown (Figs 1A and 3A), suggesting that SIRT1 affects MPP8 protein stability. We thus blocked protein synthesis in control and SIRT1‐KD PC3 cells using cycloheximide (CHX) and followed endogenous MPP8 degradation by Western blot (Fig 3B). In control cells, MPP8 degradation was detected only after 24‐h CHX treatment. In SIRT1‐KD cells, however, MPP8 protein level reduced significantly after 6‐h CHX treatment and became undetectable after 24 h. We further co‐expressed MPP8 (wt or K439R) and PCAF together with SIRT1 (wt or H363Y) in MPP8‐KD 293T cells and carried out similar analyses. As shown in Fig 3C, MPP8 protein remained stable in cells co‐expressing PCAF‐wt and SIRT1‐wt. However, it degraded much faster when SIRT1‐wt was replaced by SIRT1‐H363Y mutant. On the contrary, MPP8‐K439R protein remained stable when it was co‐expressed with PCAF‐wt and SIRT1‐H363Y. Together, we conclude that SIRT1 deacetylates MPP8 at K439 to increase its protein stability.
We next treated SIRT1‐KD PC3 cells with CHX and MG132 together. Following Western blot analysis reveals that the degraded MPP8 protein can be restored by MG132 in a dosage‐dependent manner (Fig 3D), suggesting that MPP8 is degraded through proteasome pathway. As most eukaryotes proteins destined for proteasomal degradation are initially polyubiquitinated , we co‐expressed Flag‐MPP8 and HA‐ubiquitin in MPP8‐KD 293T cells to test whether MPP8 is polyubiquitinated. Western blot analysis of Flag‐IPed MPP8 detected a strong high‐molecular‐mass smear signal (anti‐HA) indicating polyubiquitination. MPP8 polyubiquitination reduced significantly when SIRT1‐wt was co‐expressed but was not affected by SIRT1‐H363Y. A similar reduction in MPP8 polyubiquitination was also observed when we applied MPP8‐K439R mutant and co‐expression of SIRT1 did not lead to any additional reduction (Fig 3E). These results not only demonstrate that MPP8‐K439 deacetylation by SIRT1 protects MPP8 from ubiquitin‐proteasome‐mediated degradation, but also suggest that MPP8 polyubiquitination could be triggered by K439 acetylation. Although the underlying mechanism is still being investigated, we suspect that K439 acetylation could either recruit an E3 ligase or facilitate MPP8‐E3 interaction, according to several recent studies , , . PCAF might also contribute to MPP8 ubiquitination as it possesses an E3 ligase activity , .
MPP8‐SIRT1 interaction is important to maintain mesenchymal cell properties
To understand the role of MPP8‐SIRT1 interaction in cell function, we stably rescued MPP8‐KD PC3 cells with MPP8‐wt, Δ112–225 (deficient in SIRT1 binding, Fig 1D), or W80A mutant. Western blot analysis of EMT markers (Fig 4A) reveals that rescue expression of MPP8‐wt not only re‐repressed E‐cadherin, but also restored fibronectin expression. Because of the importance of MPP8 methyl‐H3K9 binding , such EMT marker changes were not observed in cells rescued by MPP8‐W80A mutant. However, E‐cadherin expression was only slightly repressed in MPP8 Δ112–225 mutant‐rescued cells, while fibronectin still remained at low level, suggesting that MPP8‐SIRT1 interaction is also important for the EMT marker rearrangement.
We next examined possible morphology changes in these cells. Immunofluorescence staining results indicate that de‐repressed E‐cadherin in SIRT1‐KD and MPP8‐KD PC3 cells localized on cell membrane to re‐establish cell–cell contacts. Although it completely disappeared in MPP8‐wt rescued cells, the membrane‐localized E‐cadherin remained in MPP8 Δ112–225 or W80A mutant‐rescued cells (Fig 4B). Consistently, differential interference contrast microscopy images reveal that both SIRT1‐KD and MPP8‐KD PC3 cells changed their shape to a cuboidal form. While rescue expression of MPP8‐wt impaired this morphologic change, the expression of either MPP8 Δ112–225 or W80A mutant failed to do so (Fig 4C). Furthermore, we evaluated cell migration and invasion abilities using Boyden chamber assay. As shown in Fig 4D, knockdown of SIRT1 or MPP8 leads to a severe reduction in migration (50–75%) and invasiveness (> 80%) of PC3 cells. Although this reduction in MPP8‐KD cells was abrogated by rescue expression of MPP8‐wt, MPP8 Δ112–225 or W80A mutant only displayed moderate effects. Given that MPP8 Δ112–225 mutant contains an intact chromodomain which is sufficient for methyl‐H3K9 binding ,  and SIRT1 binds to MPP8‐wt and W80A similarly, these results demonstrate that MPP8‐SIRT1 interaction as well as MPP8 methyl‐H3K9 binding is necessary to maintain E‐cadherin silencing and mesenchymal characteristics of PC3 cells.
MPP8 contributes to SIRT1 promoter targeting
MPP8 and SIRT1 have been previously shown on E‐cadherin promoter in different cancer cells , , and we thus determined whether MPP8‐SIRT1 interaction plays a role in their promoter targeting. Chromatin IP‐coupled qPCR (ChIP–qPCR) analyses (Fig 5A) indicate that MPP8 and SIRT1 indeed target E‐cadherin promoter in control PC3 cells. We also carried out ChIP‐reChIP analysis, and the results indicate that MPP8‐bound DNA was also occupied by SIRT1 (Fig 5B), suggesting that they co‐localize on E‐cadherin promoter through their physical interaction. In SIRT1‐KD PC3 cells, SIRT1 localization was abolished, while MPP8 localization only decreased slightly. However, MPP8 knockdown severely reduced the occupancy of SIRT1 in addition to MPP8, suggesting that the promoter localization of MPP8 is necessary for SIRT1 targeting. This possibility is also supported by results that rescue‐expressed MPP8‐wt restored promoter localization of both MPP8 and SIRT1. Although MPP8 Δ112–225 mutant displayed an intact promoter targeting, it failed to restore SIRT1 localization, whereas MPP8‐W80A mutant did not restore either MPP8 or SIRT1 localization. We further generated the same set of stable knockdown and rescue MDA‐MB‐231 cells, while Western blot and ChIP–qPCR analyses detected similar changes on E‐cadherin expression and promoter localization of MPP8 and SIRT1 (Supplementary Fig S2). Therefore, these results illustrate a scenario that MPP8 occupies E‐cadherin promoter by binding to methyl‐H3K9 and in turn recruits SIRT1 through their physical interaction. Although fibronectin expression was also effected in these knockdown and rescue cells, it is likely an indirect effect as MPP8 does not target FN1 promoter .
It has been shown that SIRT1 is recruited to E‐cadherin promoter by ZEB1 through their interaction . We next wondered whether MPP8 contributes to SIRT1 recruitment by ZEB1 by regulating SIRT1‐ZEB1 interaction. To test this possibility, we co‐expressed Flag‐ZEB1 and myc‐SIRT1 in 293T cells with or without HA‐MPP8. IP‐Western blot analysis not only confirmed ZEB1‐SIRT1 interaction, but also detected a robust ZEB1‐MPP8 interaction when they were co‐expressed. Intriguingly, ZEB1‐SIRT1 interaction increased significantly in the presence of MPP8 (Fig 5C). We next carried out endogenous IP using ZEB1 antibody and cell extracts derived from control, MPP8‐KD, and SIRT1‐KD PC3 cells. Western blot analysis reveals that endogenous MPP8 and SIRT1 also interacted with ZEB1 specifically. While knockdown of SIRT1 did not affect ZEB1‐MPP8 interaction, knockdown of MPP8 severely reduced endogenous ZEB1‐SIRT1 interaction (Fig 5D). These results suggest that ZEB1 could recruit both SIRT1 and MPP8 to target promoters through their physical interactions; importantly, MPP8 also bridges or stabilizes ZEB1‐SIRT1 interaction to facilitate SIRT1 recruitment by ZEB1.
MPP8 recruits SIRT1 to preferentially deacetylate H4K16
As histone deacetylation is one of the major functions of SIRT1 , we examined E‐cadherin promoter acetylation at two SIRT1 direct target sites, H3K9 and H4K16 , , , , , , in different knockdown and rescue PC3 cells. As indicated in ChIP–qPCR results (Fig 5E and Supplementary Fig S3), knockdown of SIRT1 significantly increased H4K16 acetylation on E‐cadherin promoter but only slightly affected H3K9 acetylation. As expected, we observed similar changes in MPP8‐KD PC3 cells. Although rescue expression of MPP8‐wt completely abrogated the increased H4K16 acetylation, MPP8 Δ112–225 or W80A mutant only showed modest effects. We thus conclude that MPP8 recruits SIRT1 for H4K16 deacetylation and E‐cadherin silencing. In addition, ChIP–qPCR analysis detected similar changes on H3K9 and H4K16 acetylation as well as the occupancy of MPP8 and SIRT1 on CRBP1 promoter (Fig 5F and Supplementary S4A–F), but not on the control GAPDH promoter (Supplementary Fig S4G), suggesting that the recruitment of SIRT1 and promoter H4K16 deacetylation could be a general mechanism for MPP8‐mediated transcription repression.
While MPP8 and SIRT1 preferentially affect H4K16 acetylation, promoter H3K9 acetylation has also been associated with E‐cadherin expression , , , . We thus treated control and SIRT1‐KD PC3 cells with TSA, a class I/II HDAC inhibitor that has being widely used to restore H3K9 acetylation to test whether they are independent mechanisms in E‐cadherin silencing. Although E‐cadherin expression is de‐repressed in SIRT1‐KD cells, TSA treatment increased E‐cadherin protein and mRNA levels in both control and SIRT1‐KD cells in a dosage‐dependent manner (Supplementary Fig S5A and B). When normalized to control treatment, E‐cadherin mRNA displayed similar up‐regulation pattern in control and SIRT1‐KD PC3 cells (Supplementary Fig S5C), indicating that TSA and SIRT1 knockdown induce E‐cadherin expression in a non‐overlapping manner. Upon TSA treatment, ChIP–qPCR analysis detected a significant increase in H3K9 acetylation on E‐cadherin promoter in both control and SIRT1‐KD PC3 cells (Supplementary Fig S5D). However, we only observed modest increases on H4K16 acetylation (Supplementary Fig S5E). These results suggest that H3K9 deacetylation and SIRT‐mediated H4K16 deacetylation both play important roles in E‐cadherin silencing but contribute to different repression pathways.
Collectively, our results uncovered a novel molecular mechanism by which two repressive chromatin modifiers MPP8 and SIRT1 positively regulate each other's function through protein interaction. While SIRT1 counteracts MPP8‐K439 acetylation catalyzed by PCAF to increase MPP8 protein stability, MPP8 recruits SIRT1 to deacetylate H4K16 on target promoters for gene silencing. Disruption of MPP8‐SIRT1 interaction reduces cellular motility, invasiveness, and other mesenchymal properties, indicating that this interaction plays an important role in MPP8‐ and SIRT1‐mediated EMT. The finding that MPP8 interacts with ZEB1 and greatly facilitates SIRT1‐ZEB1 interaction (Fig 5C and D) suggests that MPP8 not only is targeted by ZEB1 to promoters, but also bridges SIRT1 recruitment by this key EMT‐TF (Fig 5G). Since ZEB1 was co‐purified with G9a/GLP in CtBP1 complex , we suspect it also facilitates the retention of MPP8 on target promoters by recruiting G9a/GLP for H3K9 methylation. Disabling MPP8 methyl‐H3K9 binding does not affect MPP8‐SIRT1 interaction but impairs SIRT1 promoter targeting and E‐cadherin silencing (Figs 1 and 5, Supplementary Fig S2), suggesting that the stable binding of MPP8 is critical for assembly of ZEB1‐MPP8‐SIRT1 complex on target promoters.
Consistent with the fact that SIRT1 preferentially deacetylates H4K16 at the physiological concentration , our results did not detect significant changes on promoter H3K9 acetylation in SIRT1‐KD or MPP8‐KD cells. However, we demonstrate that H3K9 deacetylation plays a pivotal role in E‐cadherin silencing in a SIRT1‐independent manner (Supplementary Fig S5). H3K9 could be deacetylated by HDAC1/2‐containing Sin3A and NuRD complexes as they are recruited to E‐cadherin promoter by Snail and Twist, respectively , . HDAC1/2 also exist in ZEB1‐CtBP1 complex  while knockdown of ZEB1 decreases HDAC1/2 binding  and increases H3K9 acetylation on E‐cadherin promoter , suggesting that ZEB1 recruit HDAC1/2 to deacetylate H3K9 as well. As H3K9 deacetylation is a prerequisite for the stepwise H3K9 methylation, we predict it occurs prior to promoter targeting of MPP8 and SIRT1. In addition, SIRT1 might also indirectly facilitate H3K9 methylation and MPP8 binding as a positive feedback as SIRT1 deacetylates SUV39H1 at K266 to increase its H3K9 MTase activity .
MPP8‐wt and Δ112–225 mutant displayed similar promoter enrichment (Fig 5A and F) suggesting that MPP8 interaction with SIRT1 does not affect its binding to methyl‐H3K9 or DNMT3A‐K47me . We thus postulate that MPP8 recruits DNMT3A and SIRT1 independently. It has been shown that CpG methylation prevents promoter binding by RNA Pol II, while H4K16 acetylation promotes the release of Pol II from promoter‐proximal pausing and the transition into active elongation , MPP8‐directed promoter DNA methylation and H4K16 deacetylation may introduce multiple barriers for transcription. SIRT1 interacts with DNMT1  and positively regulates its enzymatic activity , indicating that SIRT1 may also recruit DNMT1 to facilitate DNA methylation. Moreover, the facts that H4K16 acetylation directly de‐compacts chromatin in vitro , , ,  and SIRT1 promotes facultative heterochromatin formation by deacetylating H1  suggest that MPP8‐SIRT1 pathway might also compact local chromatin directly for transcription repression.
Materials and Methods
Knockdown and rescue
Knockdown and shRNA sequence targeting MPP8 was described previously , while shRNA targeting SIRT1 are “GTATTGCTGAACAGATGGA” and “GGAAATATATCCTGGACAA.” For rescue, lentiviral vector expressing shRNA‐resistant (1102‐AACCAG‐1107 of NM_017520) Flag‐MPP8‐wt, Δ112‐225, and W80A was transfected into 293T cells with helper vectors for virus production. MPP8‐KD cells were then infected with lentiviruses and selected by both blasticidin (10 μg/ml) and puromycin (1 μg/ml).
In vitro deacetylation assay
To generate substrate for in vitro deacetylation assays, HA‐MPP8 (FL) and Flag‐PCAF were co‐expressed in MPP8‐KD 293T cells. K439‐acetylated MPP8‐FL protein was next purified by HA‐IP. To generate enzyme, GST‐SIRT1  was expressed and purified from E. coli BL21 (DE3) Codon‐plus (Stratagene) using standard protocol. To set up the deacetylation reaction, purified HA‐MPP8‐K439ac on HA‐7 agarose was incubated with purified 1 μg GST‐SIRT1 at 30°C for 10 min with rotation in a 40 μl reaction containing 50 mM Tris–Cl (pH 8.0), 0.8 mM MgCl2, 1 mM NAD+. Control, nicotinamide (NIA, 10 mM), or trichostatin A (TSA, 2 μM) was added in different reactions. The reactions were then stopped and analyzed by Western blot.
ChIP and ReChIP assays
ChIP assays were carried out as previously described  with some modifications. Briefly, formaldehyde‐cross‐linked cells were sonicated using a Bioruptor (Diagenode), while sheared chromatin was incubated with different antibodies. ChIPed complexes were captured on G‐protein‐conjugated magnetic beads (Cell Signaling) and then reverse‐cross‐linked. For ReChIP assays, chromatin bound to MPP8 antibody was eluted with reChIP elution buffer (50 mM Tris–Cl, 10 mM DTT, 1% SDS, pH 8.0) and diluted tenfold with reChIP dilution buffer (50 mM Tris–Cl, 167 mM NaCl, 2.2 mM EDTA, 1.1% Triton X‐100, pH 8.0). SIRT1 antibody was next added for 2nd IP followed by the same process. Elution was purified using PCR Cleanup kit (Invitrogen) and assayed by real‐time qPCR using primer pairs listed in the Supplementary Experimental Procedures.
LS designed and performed the experiments and analyzed the data; KK participated in MPP8 protein stability analysis; VI and JMK performed peptide acetylation analysis using the LC‐MS/MS approach; ES and JC provided important intellectual input and reagents; JF conceived the study, designed the experiments, analyzed the data, and wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary Experimental Procedures and Figures [embr201439792-sup-0001-SupInfo.pdf]
We thank Drs. Srikumar Chellappan and John Cleveland for their criticisms, suggestions, and discussions. This work was supported by NIH grant (R01CA172774) and Florida Department of Health grant (3KN02) to J.F., NIH grant (R01CA169210) to E.S., and NIH grants (CA141244 and CA109636) and Florida Department of Health grant (4BB14) to J.C.
FundingNIH R01CA172774 R01CA169210 CA141244 CA109636
- © 2015 The Authors