Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling.

PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.

Serine ADP-Ribosylation Depends on HPF1.

ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.

Inhibitors of PARP: Number crunching and structure gazing.

SignificancePARP is an important target in the treatment of cancers, particularly in patients with breast, ovarian, or prostate cancer that have compromised homologous recombination repair (i.e., BRCA-/-). This review about inhibitors of PARP (PARPi) is for readers interested in the development of next-generation drugs for the treatment of cancer, providing insights into structure-activity relationships, in vitro vs. in vivo potency, PARP trapping, and synthetic lethality.

[Poly(ADP-Ribose) Polymerases 1 and 2: Classical Functions and Interaction with New Histone Poly(ADP-Ribosyl)ation Factor HPF1].

Poly(ADP-ribose) (PAR) is a negatively charged polymer, linear or branched, that consists of ADP-ribose monomers. PAR is synthesized by poly(ADP-ribose)polymerase (PARP) enzymes, which are activated upon DNA damage and use nicotinamide adenine dinucleotide (NAD^(+)) as a substrate. The best-studied members of the PARP family, PARP1 and PARP2, are the most important nuclear proteins involved in many cell processes, including the regulation of DNA repair. PARP1 and PARP2 catalyze PAR synthesis and transfer to amino acid residues of target proteins, including autoPARylation. PARP1 and PARP2 are promising targets for chemotherapy in view of their key role in regulating DNA repair. A novel histone PARylation factor (HPF1) was recently discovered to modulate PARP1/2 activity by forming a transient joint active site with PARP1/2. Histones are modified at serine residues in the presence of HPF1. The general mechanism of the interaction between HPF1 and PARP1/2 is a subject of intense research now. The review considers the discovery and classical mechanism of PARylation in higher eukaryotes and the role of HPF1 in the process.

Comprehensive ADP-ribosylome analysis identifies tyrosine as an ADP-ribose acceptor site.

Despite recent mass spectrometry (MS)-based breakthroughs, comprehensive ADP-ribose (ADPr)-acceptor amino acid identification and ADPr-site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP-ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1-dependent in vivo ADPr-acceptor amino acid. MS analyses of in vitro ADP-ribosylated proteins confirmed tyrosine as an ADPr-acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans-modification of RPS3A is dependent on HPF1. We provide an ADPr-site Localization Spectra Database (ADPr-LSD), which contains 288 high-quality ADPr-modified peptide spectra, to serve as ADPr spectral references for correct ADPr-site localizations.

Analysis of hpf1 expression and function in early embryonic development of zebrafish.

About 70% of zebrafish (Danio rerio) genes are orthologues of the human's, which are of great interests, but still largely unknown for their functions. Recently, a report on human histone PARylation factor 1 (HPF1/C4orf27) showed that it is involved in DNA damage response along with poly (ADP-ribose) polymerase 1 (PARP1). However, its function in living organism remains unclear. Given that zebrafish has showed its values in modeling human diseases and physiology, we characterized a zebrafish homolog of human HPF1 by sequence alignment. We also analyzed its expression pattern during early development and among adult tissues. Furthermore, knocking down hpf1 by morpholinos affected zebrafish early development. Our work provides a novel clue for the mechanism of genome stability and early embryogenesis.

ANP32B promotes colorectal cancer cell progression and reduces cell sensitivity to PRAP1 inhibitor through up-regulating HPF1.

ANP32B, a member of the acidic leucine-rich nuclear phosphoprotein 32 family member B, is aberrantly expressed in various cancers, including colorectal cancer. However, the function and mechanism of action of ANP32B in colorectal cancer remain unclear. The present study therefore analyzed the expression of ANP32B and its activity in colorectal cancer patient samples and colorectal cancer cell lines. ANP32B expression was found to be significantly upregulated in colorectal cancer patient samples and cell lines. Upregulation of ANP32B enhanced colorectal cancer cell proliferation and migration, whereas downregulation of ANP32B suppressed colorectal cancer cell proliferation. RNA sequencing analysis of differentially expressed genes in ANP32B silenced colorectal cancer cells showed that histone PARylation factor 1 (HPF1), which protects against DNA damage by interacting with the anti-tumor target PARP1, was significantly downregulated. Luciferase promoter assays testing the regulatory association between ANP32B and HPF1 showed that ANP32B interacted with the HPF1 promoter. Analysis of colorectal cancer samples from The Cancer Genome Atlas showed that ANP32B and HPF1 expression were positively correlated, and recovery assays showed that ANP32B promoted colorectal cancer progression by up-regulating HPF1. Overexpression of ANP32B also reduced the sensitivity of colorectal cancer cells to PARP1 inhibitor, consistent with the oncogenic role of ANP32B. ANP32B may alter the sensitivity of colorectal cancer cells to PARP1 inhibitor via a mechanism associated with the HPF1 gene. In summary, these findings showed that ANP32B acted as a tumor promoter, potentiating both colorectal cancer malignancy and drug resistance. Targeting the ANP32B/HPF1 axis may have benefit for patients with colorectal cancer.

HPF1 regulates tendon stem/progenitor cell senescence and tendon repair via PARP1-mediated poly-ADP ribosylation of HuR.

BACKGROUND: Tendon stem/progenitor cells (TSPCs) play a vital role in tendon repair, regeneration and homeostasis. However, the specific mechanism of TSPCs aging is still unclear. OBJECTIVE: This study aims to explore the role and molecular mechanism of HPF1 in the aging of TSPCs. METHODS: Young and aged TSPCs (Y-TSPCs and A-TSPCs) were acquired from 3 to 4 and 24-26-month-old Sprague-Dawley male rats, TSPCs (Y-TSPCs and A-TSPCs) were subjected to senescence-associated ß-galactosidase (SA-ß-Gal))staining and telomerase activity detection, p16, p21, Scx, Tnmd, Col1, Col3HPF1 and PAPR1 expression levels were detected by Western blot or Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), Reciprocal co-immunoprecipitation (co-IP) was used to explore the interaction between HPF1 and PARP1. Ribonucleoprotein immunoprecipitation (RNP-IP) was used to analyze the binding of HuR to the senescence marker gene mRNAs, IP was used to perform HPF1 to the PARylation of HuR, and the half-life of p16 and p21 were detected. Finally, we established an in vivo model, and the tendon tissue was used to perform hematoxylin and eosin (HE) and masson's trichrome staining, as well as the immunohistochemical analysis of Col I and TNMD. RESULTS: Compared with Y-TSPCs, A-TSPCs had significantly enhanced cell senescence and significantly reduced tendon differentiation ability, and significantly increased the expression of HPF1 and PARP1. In addition, HPF1 and PARP1 interacted and coordinated the senescence and differentiation of TSPCs, HPF1 could also regulate the expression of p21 and p21, the interaction of p16 or p21 with HuR, and the poly-ADP ribosylation of PARP1 to HuR. HPF1 overexpression and siHuR co-transfection significantly reduced the half-life of p16 and p21, and HPF1 and PARP1 regulated the mRNA levels of p16 and p21 through HuR. Finally, in vivo experiments have shown that HPF1 or PARP1 overexpression could both inhibit the ability of tendon differentiation and promote cell senescence. CONCLUSIONS: HPF1 promoted the senescence of TSPCs and inhibits the tendon differentiation of TSPCs through PARP1-mediated poly-ADP ribosylation of HuR.

PARticular MARks: Histone ADP-ribosylation and the DNA damage response.

Cellular and molecular responses to DNA damage are highly orchestrated and dynamic, acting to preserve the maintenance and integrity of the genome. Histone proteins bind DNA and organize the genome into chromatin. Post-translational modifications of histones have been shown to play an essential role in orchestrating the chromatin response to DNA damage by regulating the DNA damage response pathway. Among the histone modifications that contribute to this intricate network, histone ADP-ribosylation (ADPr) is emerging as a pivotal component of chromatin-based DNA damage response (DDR) pathways. In this review, we survey how histone ADPr is regulated to promote the DDR and how it impacts chromatin and other histone marks. Recent advancements have revealed histone ADPr effects on chromatin structure and the regulation of DNA repair factor recruitment to DNA lesions. Additionally, we highlight advancements in technology that have enabled the identification and functional validation of histone ADPr in cells and in response to DNA damage. Given the involvement of DNA damage and epigenetic regulation in human diseases including cancer, these findings have clinical implications for histone ADPr, which are also discussed. Overall, this review covers the involvement of histone ADPr in the DDR and highlights potential future investigations aimed at identifying mechanisms governed by histone ADPr that participate in the DDR, human diseases, and their treatments.

HTS discovery of PARP1-HPF1 complex inhibitors in cancer.

PARP1/2 inhibitors (PARPi) are effective clinically used drugs for the treatment of cancers with BRCA deficiencies. PARPi have had limited success and applicability beyond BRCA deficient cancers, and their effect is diminished by resistance mechanisms. The recent discovery of Histone PARylation Factor (HPF1) and the role it plays in the PARylation reaction by forming a shared active site with PARP1 raises the possibility that novel inhibitors that target the PARP1-HPF1 complex can be identified. Herein we describe a simple and cost-effective high-throughput screening (HTS) method aimed at discovering inhibitors of the PARP1-HPF1 complex. Upon HTS validation, we first applied this method to screen a small PARP-focused library of compounds and then scale up our approach using robotic automation to conduct a pilot screen of 10,000 compounds and validating >100 hits. This work demonstrates for the first time the capacity to discover potent inhibitors of the PARP1-HPF1 complex, which may have utility as probes to better understand the DNA damage response and as therapeutics for cancer.

The HPF1-dependent histone PARylation catalyzed by PARP2 is specifically stimulated by an incised AP site-containing BER DNA intermediate.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are DNA-dependent poly(ADP-ribose)transferases localized in nucleus. They have a significant homology in the C-terminal catalytic domain structure but differ in their N-terminal DNA-binding parts. The structural difference has an impact on the interaction of PARP1 and PARP2 with DNA and their DNA-dependent activation. Here, we compare the interaction of PARP1 and PARP2 with free 147 bp nucleosomal DNA and its nucleosome-associated variant (NCP) that contain in one strand a 1-nucleotide gap with 5'-dRP (imitating the intermediate of Base Excision Repair) or no specific damage. The affinity of PARP2 for the DNA strongly depends on the gap presence and to a lesser extent on the association with nucleosomes, while PARP1 interacts primarily with blunt ends of all DNAs and with a lower affinity with the single-strand break. The activities of PARP1 and PARP2 in the autoPARylation reaction and heteromodification of histones are distinctly stimulated by HPF1, depending on the gap presence in activating DNA. The most significant HPF1-induced stimulation of the histone modification in the presence of gapped NCP is a peculiar feature of PARP2. We propose a specific regulatory role of PARP2 in the process of DNA repair in the context of chromatin.

Progress and outlook in studying the substrate specificities of PARPs and related enzymes.

Despite decades of research on ADP-ribosyltransferases (ARTs) from the poly(ADP-ribose) polymerase (PARP) family, one key aspect of these enzymes - their substrate specificity - has remained unclear. Here, we briefly discuss the history of this area and, more extensively, the recent breakthroughs, including the identification of protein serine residues as a major substrate of PARP1 and PARP2 in human cells and of cysteine and tyrosine as potential targets of specific PARPs. On the molecular level, the modification of serine residues requires a composite active site formed by PARP1 or PARP2 together with a specificity-determining factor, HPF1; this represents a new paradigm not only for PARPs but generally for post-translational modification (PTM) catalysis. Additionally, we discuss the identification of DNA as a substrate of PARP1, PARP2 and PARP3, and some bacterial ARTs and the discovery of noncanonical RNA capping by several PARP family members. Together, these recent findings shed new light on PARP-mediated catalysis and caution to 'expect the unexpected' when it comes to further potential substrates.

Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling.

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here, we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at histone H2B serine 6 or histone H3 serine 10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

Interplay of Histone Marks with Serine ADP-Ribosylation.

Serine ADP-ribosylation (Ser-ADPr) is a recently discovered protein modification that is catalyzed by PARP1 and PARP2 when in complex with the eponymous histone PARylation factor 1 (HPF1). In addition to numerous other targets, core histone tails are primary acceptors of Ser-ADPr in the DNA damage response. Here, we show that specific canonical histone marks interfere with Ser-ADPr of neighboring residues and vice versa. Most notably, acetylation, but not methylation of H3K9, is mutually exclusive with ADPr of H3S10 in vitro and in vivo. We also broaden the O-linked ADPr spectrum by providing evidence for tyrosine ADPr on HPF1 and other proteins. Finally, we facilitate wider investigations into the interplay of histone marks with Ser-ADPr by introducing a simple approach for profiling posttranslationally modified peptides. Our findings implicate Ser-ADPr as a dynamic addition to the complex interplay of modifications that shape the histone code.