HDAC7 knockdown abolishes PML sumoylation and PML NB formation. (A) HUVECs were treated with HDAC7 siRNA. Forty-eight hours posttransfection, cells were subjected to immunostaining using anti-PML (α-PML) and anti-HDAC7 antibodies, followed by confocal microscopy. Note that cells 1 and 2 express different levels of HDAC7 due to different efficiencies of knockdown by HDAC7 siRNA. They also display different intensities of PML or PML NBs. (B) HUVECs were transfected with PML siRNA (siPML). Forty-eight hours posttransfection, cells were subjected to immunostaining with anti-PML and anti-SUMO1 antibodies, followed by confocal microscopy. Cells transfected with PML siRNA are marked with arrows. siControl, control siRNA. (C) HUVECs were treated with siRNA against HDAC7. Whole-cell lysates were immunoprecipitated with anti-PML antibodies. Immunopellets and whole-cell lysates were probed with anti-PML, antiactin, anti-HDAC7, or anti-SUMO1 as indicated. A quantitative analysis revealed that HDAC7 knockdown led to a 60% loss of PML sumoylation and a loss of 58% of total PML protein levels. IB, immunoblotting; IP, immunoprecipitation; +, present; −, absent.

HDAC7 promotes PML sumoylation in mammalian cells. (A) HeLa cells were transfected with HA-PML, FLAG-SUMO1, and YFP-HDAC7 expression plasmids. Immunopellets precipitated with anti-FLAG (α-FLAG) and anti-HA antibodies were immunoblotted with anti-HA and anti-FLAG antibodies as indicated. The band migrating at 80 kDa is likely to be nonsumoylated HA-PML that is coprecipitated with sumoylated PML (FLAG-SUMO1-tagged HA-PML) due to the ability of PML to form dimers. It is marked with an asterisk. IB, immunoblotting; IP, immunoprecipitation; +, present; −, absent. (B) HeLa cells were transfected with HA-PML and YFP-HDAC7 expression constructs. Whole-cell lysates or immunopellets precipitated with anti-HA antibodies were immunoblotted with anti-HA and anti-SUMO1 antibodies as indicated. Note that not only was the intensity of sumoylated PML increased in cells cotransfected with an HDAC7 construct, but also slower-migrating sumoylated PML species were detected. (C) Cells were cotransfected with plasmids expressing increasing concentrations of YFP-HDAC7 (lanes 3 to 5), FLAG-SUMO1, and HA-PML. Whole-cell extracts (WCE) were immunoprecipitated with anti-FLAG antibodies (lanes 6 to 10), and the immunoprecipitates were analyzed by immunoblotting with anti-HA antibody. Nonsumoylated PML is marked with an asterisk. The expression levels of endogenous YFP-HDAC7 (lower band in lower panel, marked with an arrowhead) and YFP-HDAC7 introduced by transfection (upper band in lower panel) are shown.

The C terminus of HDAC7 stimulates PML sumoylation in mammalian cells and in vitro. (A) Schematic representation of HDAC7 functional domains. The structure of the HDAC7 C terminus has recently been resolved and shows that a CCHC zinc-binding motif is present at the N terminus of the HDAC enzymatic domain. WT, wild type. (B) HeLa cells were cotransfected with HA-PML and FLAG-SUMO1 expression plasmids and a plasmid expressing full-length HA-HDAC7, HDAC7 (2-254), or HDAC7 (500-938). Whole-cell lysates and immunoprecipitates were immunoblotted with anti-HA (α-HA) or anti-FLAG antibodies. IB, immunoblotting; IP, immunoprecipitation; +, present; −, absent. (C) HeLa cells were cotransfected with HA-PML and FLAG-SUMO1 expression plasmids and a wild-type HA-HDAC7 or mutant (Mut) HA-HDAC7 expression plasmid. Immunoprecipitations were carried out with anti-FLAG or anti-HA antibodies, and immunoprecipitates were immunoblotted with anti-HA or anti-FLAG antibodies as indicated. (D) HeLa cells were cotransfected with expression plasmids for FLAG-PML and HA-HDAC7 truncations. Whole-cell lysates were immunoprecipitated with anti-FLAG antibodies. The resulting immunopellets, along with the whole-cell lysates, were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies as indicated. WCE, whole-cell extracts. (E) HeLa whole-cell lysates expressing HA-PML were used in GST pulldowns with either GST alone or GST fused to the indicated truncations of HDAC7. The precipitated fractions were analyzed by immunoblotting with anti-HA antibodies. 500-ter, HDAC7 (500-938). (F) Recombinant His-tagged PML proteins were subjected to in vitro sumoylation assays with GST or GST-HDAC7 (500-938) fusion protein as described in Materials and Methods. The arrows and solid circles mark SUMO1-modified PML (sumo), and the asterisk marks nonsumoylated PML (non-sumo). Note that the amount of sumoylated PML compared to non sumoylated PML, as well as the relative sumoylation level, was increased when GST-HDAC7 (500-938) was added. It is estimated that five- to sixfold stimulation of PML sumoylation was observed when 100 ng of GST-HDAC7 (500-938) was used. For lanes 7 to 12, the intensity of sumoylated PML in lane 8 was used for normalization and was set at 1. The ratio of the intensity compared to that in lane 8 is shown and represents the increase (n-fold) in sumoylation stimulation (lanes 11 and 12).

Other class IIa HDACs also potentiate PML sumoylation. HA-PML, FLAG-SUMO1, and HA-HDAC4, HA-HDAC5, or HA-HDAC7 expression plasmids were singly expressed or coexpressed in HeLa cells. Whole-cell extracts were prepared, and immunoprecipitations were performed as described in the legend to Fig. 2A. Asterisks indicate the expression of HA-HDAC4, HA-HDAC5, and HA-HDAC7 in lanes 2, 3, and 4, respectively. The arrow marks HA-PML. We did not observe the sumoylation of HDAC4, HDAC5, or HDAC7 under our assay conditions (data not shown). IB, immunoblotting; IP, immunoprecipitation; +, present; −, absent; α-HA, anti-HA antibody.

HDAC7 associates with Ubc9. (A) HeLa cells were cotransfected with expression plasmids for HA-HDAC7 and FLAG-Ubc9, whole-cell extracts (WCE) were prepared and immunoprecipitated with anti-HA antibodies (α-HA), and the immunoprecipitates were analyzed by immunoblotting with anti-HA and anti-FLAG antibodies as indicated. IP, immunoprecipitation; +, present; −, absent. (B) HeLa cells were cotransfected with FLAG-Ubc9 and HA-HDAC7 full-length or truncation expression plasmids. Whole-cell lysates and immunopellets from immunoprecipitations with anti-FLAG antibodies were analyzed by immunoblotting with anti-FLAG and anti-HA antibodies as indicated. WT, wild type; 500-ter, HDAC7 (500-938). (C) Immobilized GST-HDAC7 fusion proteins were incubated with extracts from cells expressing HA-Ubc9 and analyzed by immunoblotting with anti-HA antibodies.

TNF-α promotes PML sumoylation and PML NB formation in HUVECs in an HDAC7-dependent manner. (A) HUVECs were treated with or without TNF-α. Twenty hours posttreatment, immunostaining with anti-PML (α-PML), SUMO1, or Daxx was carried out, followed by confocal microscopy. The laser used for panel b was seven times (7×) more intense than that used for panel g. Similarly, the laser used for panel l was three times more intense than that used for panel q. (B) HUVECs were treated with (+) or without (−) TNF-α. Twenty hours posttreatment, immunoprecipitation (IP) with anti-PML antibodies was carried out, followed by immunoblotting (IB) with anti-SUMO1. WCE, whole-cell extracts; Mr(K), molecular size in kilodaltons. (C) HUVECs were transfected with control siRNA (siControl) or the HDAC7 siRNA SMART pool (siHDAC7). Forty-eight hours posttransfection, cells were treated with TNF-α as described in Materials and Methods and whole-cell extracts were prepared and subjected to immunoprecipitation with PML antibodies and Western blotting with anti-SUMO1 antibodies. (D) HUVECs were treated with TNF-α and analyzed by immunostaining and confocal microscopy as described in the legend to panel A. HDAC7 staining was significantly decreased in four cells marked with asterisks or the number 2. Cells numbered 1 are those in which HDAC7 was moderately knocked down or in which HDAC7 was not knocked down.