Regulating the Regulators: The Post-Translational Code of Class I HDAC1 and HDAC2

Class I histone deacetylases (HDACs) are cellular enzymes expressed in many tissues and play crucial roles in differentiation, proliferation, and cancer. HDAC1 and HDAC2 in particular are highly homologous proteins that show redundant or specific roles in different cell types or in response to different stimuli and signaling pathways. The molecular details of this dual regulation are largely unknown. HDAC1 and HDAC2 are not only protein modifiers, but are in turn regulated by post-translational modifications (PTMs): phosphorylation, acetylation, ubiquitination, SUMOylation, nitrosylation, and carbonylation. Some of these PTMs occur and crosstalk specifically on HDAC1 or HDAC2, creating a rational “code” for a differential, context-related regulation. The global comprehension of this PTM code is central for dissecting the role of single HDAC1 and HDAC2 in physiology and pathology

Recent developments in research on catalytic reaction networks

Over the last years, analyses performed on a stochastic model of catalytic reaction networks have provided some indications about the reasons why wet-lab experiments hardly ever comply with the phase transition typically predicted by theoretical models with regard to the emergence of collectively self-replicating sets of molecule (also defined as autocatalytic sets, ACSs), a phenomenon that is often observed in nature and that is supposed to have played a major role in the emergence of the primitive forms of life. The model at issue has allowed to reveal that the emerging ACSs are characterized by a general dynamical fragility, which might explain the difficulty to observe them in lab experiments. In this work, the main results of the various analyses are reviewed, with particular regard to the factors able to affect the generic properties of catalytic reactions network, for what concerns, not only the probability of ACSs to be observed, but also the overall activity of the system, in terms of production of new species, reactions and matter

Sumoylation of UL44 in mammalian cells.

<p>(<b>A</b>) Phoenix cells were transfected to express the indicated proteins and analyzed by western blotting with anti-FLAG, anti-HA, anti-Ubc9, and anti-GAPDH antibodies. (<b>B</b>) Cell lysates were incubated with the anti-FLAG antibody and the input proteins (left panels) or the immunoprecipitated samples (right panels) were analyzed by western blot with the indicated antibodies. (<b>C</b>) Sumoylation of a UL44 mutant lacking the NLS was analyzed as in (A). For all panels, the arrowhead indicates the unmodified form of UL44 or free SUMO-1 and the asterisks indicate the sumoylated forms.</p

Sumoylation of UL44 <i>in vitro</i>.

<p>(<b>A</b>) To analyze UL44 sumoylation <i>in vitro</i>, purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either wild-type SUMO-1 (SUMO-1 wt) or a mutant form of SUMO-1 (SUMO-1 mut) which cannot be covalently linked to substrates. The reaction products were analyzed by western blotting with anti-UL44 and anti-SUMO-1 antibodies. As a positive control, <i>in vitro</i> sumoylation of p53 was also analyzed. (<b>B</b>) Purified 6His-UL44 was incubated in the absence or the presence of sumoylation enzymes and either SUMO-2 or SUMO-3 and analyzed by western blotting with anti-UL44 and anti-SUMO-2/−3 antibodies. For all panels, the arrowhead indicates the unmodified form of UL44 or p53 and the asterisks indicate the respective sumoylated forms.</p

UL44 interacts with human Ubc9 in yeast two-hybrid assays.

1<p>UL44, UL54, and Ubc9 proteins were fused to the C-terminus of LexA protein and/or of GAL4 activation domain (GAD). Fusion proteins were then assayed for interaction by qualitative β-galactosidase (β-gal) filter assays and by quantitative β-gal liquid assays.</p>2<p>β-gal expression was scored as follows +, strong blue color detected within 2 h of incubation; ±, blue color detected after more than 2 h of incubation; −, no signal detected after 16–24 h of incubation. Values within parentheses represent β-gal units ± standard deviation (SD) of 3–4 yeast colonies from at least three independent transformations.</p

Sumoylation of UL44 in HCMV-infected cells.

<p>(<b>A</b>) HFFs were either mock-infected or infected with HCMV for the indicated times. Cell lysates were analyzed by western blotting with an anti-UL44 antibody. (<b>B</b>) Blots were analyzed by densitometry and the percentage of sumoylated UL44 bands relative to that of unmodified UL44 at each time p.i. was plotted versus the p.i. time point. Data represent the means ± standard deviations (error bars) of values from three independent experiments such as that shown in (A). (<b>C</b>) Lysates from either mock-infected or HCMV-infected HFF cells were prepared at 120 h p.i. and immunoprecipitated with an anti-UL44 antibody. Immunoprecipitates were analyzed by western blotting with anti-SUMO-1 (left panel) and anti-UL44 (right panel) antibodies. For all panels, the arrowhead indicates the unmodified form of UL44, the arrow indicates the immunoglobulin G heavy chain (IgG hc) and the asterisks indicate the sumoylated UL44 forms.</p

UL44 sumoylation is stimulated by DNA.

<p>(<b>A</b>) Purified 6His-UL44 protein was incubated with sumoylation proteins in the absence or presence of dsDNA (left panel) or ssDNA (right panel). Samples were analyzed by western blotting with an anti-UL44 antibody. (<b>B, C</b>) Phoenix cells were transfected to express wild-type UL44 or the FLAG-UL44Δloop or FLAG-UL44L86A/L87A mutant, which are defective for DNA binding. At 48 h post-transfection, cell lysates were analyzed by western blotting with anti-FLAG, anti-HA, anti-Ubc9, and anti-GAPDH antibodies. (<b>D</b>) Phoenix cells were transfected to express the indicated proteins. At 48 h post-transfection, cell lysates were analyzed by western blotting with anti-UL44, anti-Ubc9, and anti-vinculin antibodies (left panel). Cell lysates were incubated with anti-FLAG-M2-Agarose beads and the immunoprecipitated samples were analyzed by western blotting with anti-UL44 and anti-Ubc9 antibodies (right panel). (<b>E</b>) The sumoylation <i>in vitro</i> of wild-type 6His-UL44 and mutant 6His-UL44Δloop and 6His-UL44L86A/L87A proteins was carried out as in (A) and analyzed by western blotting with an anti-UL44 antibody. (<b>F</b>) The sumoylation <i>in vitro</i> of a UL44 mutant bearing the K167R substitution in the flexible loop of UL44 involved in DNA binding was carried out in the presence of DNA and compared to that of wild-type UL44. For all panels, the arrowhead indicates the unmodified form of UL44 or free SUMO-1 and the asterisks indicate the sumoylated forms.</p

Overexpression of SUMO-1 modifies the subnuclear localization of UL44 and promotes virus replication in HCMV-infected cells.

<p>(A) U373 cells that constitutively overexpress SUMO-1 (U373-SUMO-1), control U373-Neo cells, and U373-SUMO-1 cells transduced with lentiviral particles expressing either a <i>Ubc9</i>-silencing shRNA (U373-SUMO-1 shUbc9) or a non-silencing shRNA sequence (U373-SUMO-1 NS) were mock-infected or infected with HCMV at an MOI of 5 or 1 PFU/cell. At 72 h p.i., cells were fixed and stained with a primary antibody against UL44 (upper panels) or against UL57 (lower panels), and successively with a secondary fluorescein-conjugated antibody (green) which contained Evans Blue to counterstain cells (red). Cell samples were then analyzed by CLSM. (B) Western blot analysis of Ubc9 expression in Ubc9-knocked-down and control cells. Cell lysates were prepared from the indicated cell lines and subjected to SDS-PAGE. Filter was then probed with anti-Ubc9 and anti-α-tubulin MAbs. (C) The indicated cell lines were infected with HCMV at an MOI of 1 and viral DNA levels were measured at 72 h p.i. by quantitative real-time PCR. (D) The indicated cell lines were infected with HCMV at an MOI of 1 and the titers of infectious virus progeny produced at 120 h p.i. were determined by plaque assays. In both (C) and (D), the data shown represent the means ± standard deviations (error bars) of two independent experiments performed in duplicate or triplicate. The asterisks denote a statistically significant difference (<i>P<</i>0.05) between the values relative to U373-SUMO-1 and U373-SUMO-1 NS and the values relative to control U373-Neo cells.</p