Ralph Imhof

Acetylation of poly(ADP-ribose) polymerase-1 by p300/CREB-binding protein regulates coactivation of NF-kappaB-dependent transcription

Poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear factor κB (NF-κB) have both been demonstrated to play a pathophysiological role in a number of inflammatory disorders. We recently presented evidence that PARP-1 can act as a promoter-specific coactivator of NF-κB in vivo independent of its enzymatic activity. PARP-1 directly interacts with p300 and both subunits of NF-κB (p65 and p50) and synergistically coactivates NF-κB-dependent transcription. Here we show that PARP-1 is acetylated in vivo at specific lysine residues by p300/CREB-binding protein upon stimulation. Furthermore, acetylation of PARP-1 at these residues is required for the interaction of PARP-1 with p50 and synergistic coactivation of NF-κB by p300 and the Mediator complex in response to inflammatory stimuli. PARP-1 physically interacts with the Mediator. Interestingly, PARP-1 interacts in vivo with histone deacetylases (HDACs) 1-3 but not with HDACs 4-6 and might be deacetylated in vivo by HDACs 1-3. Thus, acetylation of PARP-1 by p300/CREB-binding protein plays an important regulatory role in NF-κB-dependent gene activation by enhancing its functional interaction with p300 and the Mediator complex.

Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-κB-dependent gene expression

Nuclear factor kappaB (NF‐κB) plays an important role in the transcriptional regulation of genes involved in inflammation and cell survival. Here, we show that coactivator‐associated arginine methyltransferase CARM1/PRMT4 is a novel transcriptional coactivator of NF‐κB and functions as a promoter‐specific regulator of NF‐κB recruitment to chromatin. Carm1 knockout cells showed impaired expression of a subset of NF‐κB‐dependent genes upon TNFα or LPS stimulation. CARM1 forms a complex with p300 and NF‐κB in vivo and interacts directly with the NF‐κB subunit p65 in vitro. CARM1 seems to act in a gene‐specific manner mainly by enhancing NF‐κB recruitment to cognate sites. Moreover, CARM1 synergistically coactivates NF‐κB‐mediated transactivation, in concert with the transcriptional coactivators p300/CREB‐binding protein and the p160 family of steroid receptor coactivators. For at least a subset of CARM1‐dependent NF‐κB target genes, the enzymatic activities of both CARM1 and p300 are necessary for the observed synergy between CARM1 and p300. Our results suggest that the cooperative action between protein arginine methyltransferases and protein lysine acetyltransferases regulates NF‐κB‐dependent gene activation in vivo.

Transcription coactivator p300 binds PCNA and may have a role in DNA repair synthesis.

The transcriptional coactivator p300 interacts with many transcription factors that participate in a broad spectrum of biological activities, such as cellular differentiation, homeostasis and growth control. Mouse embryos lacking both p300 alleles die around mid-gestation, with pleiotropic defects in morphogenesis, in cell differentiation and, unexpectedly, in cell proliferation because of reduced DNA synthesis. Here we show that p300 may have a role in DNA repair synthesis through its interaction with proliferating cell nuclear antigen (PCNA). We show that in vitro and in vivo p300 forms a complex with PCNA that does not depend on the S phase of cell cycle. A large fraction of both p300 and PCNA colocalize to speckled structures in the nucleus. Furthermore, the endogenous p300–PCNA complex stimulates DNA synthesis in vitro. Chromatin immunoprecipitation experiments indicate that p300 is associated with freshly synthesized DNA after ultraviolet irradiation. Our results suggest that p300 may participate in chromatin remodelling at DNA lesion sites to facilitate PCNA function in DNA repair synthesis.

Macrodomain-containing proteins are new mono-ADP-ribosylhydrolases

ADP-ribosylation is an important post-translational protein modification (PTM) that regulates diverse biological processes. ADP-ribosyltransferase diphtheria toxin-like 10 (ARTD10, also known as PARP10) mono-ADP-ribosylates acidic side chains and is one of eighteen ADP-ribosyltransferases that catalyze mono- or poly-ADP-ribosylation of target proteins. Currently, no enzyme is known that reverses ARTD10-catalyzed mono-ADP-ribosylation. Here we report that ARTD10-modified targets are substrates for the macrodomain proteins MacroD1, MacroD2 and C6orf130 from Homo sapiens as well as for the macrodomain protein Af1521 from archaebacteria. Structural modeling and mutagenesis of MacroD1 and MacroD2 revealed a common core structure with Asp102 and His106 of MacroD2 implicated in the hydrolytic reaction. Notably, MacroD2 reversed the ARTD10-catalyzed, mono-ADP-ribose–mediated inhibition of glycogen synthase kinase 3β (GSK3β) in vitro and in cells, thus underlining the physiological and regulatory importance of mono-ADP-ribosylhydrolase activity. Our results establish macrodomain-containing proteins as mono-ADP-ribosylhydrolases and define a class of enzymes that renders mono-ADP-ribosylation a reversible modification.

Regulation of Human Flap Endonuclease-1 Activity by Acetylation through the Transcriptional Coactivator p300

We describe a role for the transcriptional coactivator p300 in DNA metabolism. p300 formed a complex with flap endonuclease-1 (Fen1) and acetylated Fen1 in vitro. Furthermore, Fen1 acetylation was observed in vivo and was enhanced upon UV treatment of human cells. Remarkably, acetylation of the Fen1 C terminus by p300 significantly reduced Fen1s DNA binding and nuclease activity. Proliferating cell nuclear antigen (PCNA) was able to stimulate both acetylated and unacetylated Fen1 activity to the same extent. Our results identify acetylation as a novel regulatory modification of Fen1 and implicate that p300 is not only a component of the chromatin remodeling machinery but might also play a critical role in regulating DNA metabolic events.

Acetylation Regulates the DNA End-Trimming Activity of DNA Polymerase β

We describe a novel regulatory mechanism for DNA polymerase beta (Polbeta), a protein involved in DNA base excision repair (BER). Polbeta colocalized in vivo and formed a complex with the transcriptional coactivator p300. p300 interacted with Polbeta through distinct domains and acetylated Polbeta in vitro. Polbeta acetylation was furthermore observed in vivo. Lysine 72 of Polbeta was identified as the main target for acetylation by p300. Interestingly, acetylated Polbeta showed a severely reduced ability to participate in a reconstituted BER assay. This was due to an impairment of the dRP-lyase activity of Polbeta. Acetylation of Polbeta thus acts as an intranuclear regulatory mechanism and implies that p300 plays a critical regulatory role in BER.

Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle

We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse, and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.

Identification of lysines 36 and 37 of PARP-2 as targets for acetylation and auto-ADP-ribosylation

Poly-ADP-ribose polymerase-2 (PARP-2) was described to regulate cellular functions comprising DNA surveillance, inflammation and cell differentiation by co-regulating different transcription factors. Using an in vitro and in vivo approach, we identified PARP-2 as a new substrate for the histone acetyltransferases PCAF and GCN5L. Site directed mutagenesis indicated that lysines 36 and 37, located in the nuclear localization signal of PARP-2, are the main targets for PCAF and GCN5L activity in vitro. Interestingly, acetylation of the same two PARP-2 residues reduces the DNA binding and enzymatic activity of PARP-2. Finally, PARP-2 with mutated lysines 36 and 37 showed reduced auto-mono-ADP-ribosylation when compared to wild type PARP-2. Together, our results provide evidence that acetylation of PARP-2 is a key post-translational modification that may regulate DNA binding and consequently also the enzymatic activity of PARP-2.

DNA polymerase δ-interacting protein 2 is a processivity factor for DNA polymerase λ during 8-oxo-7,8-dihydroguanine bypass

Significance Macromolecules (DNA, proteins, and lipids) in all cells are constantly damaged by reactive oxygen species (ROS). In particular, ROS cause 1,000–7,000 DNA damages per day. Due to its lowest redox potential, the base guanine is mostly affected, resulting in the formation of 8-oxo-7,8-dihydroguanine. This modified base instructs incorporation of adenosine, instead of cytidine, by replicative DNA polymerases, potentially leading to GC→TA transversion mutations. DNA polymerase λ is the most efficient enzyme in performing accurate translesion synthesis over 8-oxo-7,8-dihydroguanine, since it preferentially incorporates the correct cytidine. In this paper we found that the protein called “DNA polymerase δ interacting protein 2” supports DNA polymerase λ in its important task and can protect cells from ROS DNA damage. The bypass of DNA lesions by the replication fork requires a switch between the replicative DNA polymerase (Pol) and a more specialized translesion synthesis (TLS) Pol to overcome the obstacle. DNA Pol δ-interacting protein 2 (PolDIP2) has been found to physically interact with Pol η, Pol ζ, and Rev1, suggesting a possible role of PolDIP2 in the TLS reaction. However, the consequences of PolDIP2 interaction on the properties of TLS Pols remain unknown. Here, we analyzed the effects of PolDIP2 on normal and TLS by five different human specialized Pols from three families: Pol δ (family B), Pol η and Pol ι (family Y), and Pol λ and Pol β (family X). Our results show that PolDIP2 also physically interacts with Pol λ, which is involved in the correct bypass of 8-oxo-7,8-dihydroguanine (8-oxo-G) lesions. This interaction increases both the processivity and catalytic efficiency of the error-free bypass of a 8-oxo-G lesion by both Pols η and λ, but not by Pols β or ι. Additionally, we provide evidence that PolDIP2 stimulates Pol δ without affecting its fidelity, facilitating the switch from Pol δ to Pol λ during 8-oxo-G TLS. PolDIP2 stimulates Pols λ and η mediated bypass of other common DNA lesions, such as abasic sites and cyclobutane thymine dimers. Finally, PolDIP2 silencing increases cell sensitivity to oxidative stress and its effect is further potentiated in a Pol λ deficient background, suggesting that PolDIP2 is an important mediator for TLS.

Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin

Summary The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1’s target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR.