PRAMEL7 and CUL2 decrease NuRD stability to establish ground-state pluripotency.

Pluripotency is established in E4.5 preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of pluripotency, however, their gene expression signature only partially resembles that of developmental ground-state. Induced PRAMEL7 expression, a protein highly expressed in the ICM but lowly expressed in ESCs, reprograms developmentally advanced ESC+serum into ground-state pluripotency by inducing a gene expression signature close to developmental ground-state. However, how PRAMEL7 reprograms gene expression remains elusive. Here we show that PRAMEL7 associates with Cullin2 (CUL2) and this interaction is required to establish ground-state gene expression. PRAMEL7 recruits CUL2 to chromatin and targets regulators of repressive chromatin, including the NuRD complex, for proteasomal degradation. PRAMEL7 antagonizes NuRD-mediated repression of genes implicated in pluripotency by decreasing NuRD stability and promoter association in a CUL2-dependent manner. Our data link proteasome degradation pathways to ground-state gene expression, offering insights to generate in vitro models to reproduce the in vivo ground-state pluripotency.

PARP7-mediated ADP-ribosylation of FRA1 promotes cancer cell growth by repressing IRF1- and IRF3-dependent apoptosis.

PARP7 was reported to promote tumor growth in a cell-autonomous manner and by repressing the antitumor immune response. Nevertheless, the molecular mechanism of how PARP7-mediated ADP-ribosylation exerts these effects in cancer cells remains elusive. Here, we identified PARP7 as a nuclear and cysteine-specific mono-ADP-ribosyltransferase that modifies targets critical for regulating transcription, including the AP-1 transcription factor FRA1. Loss of FRA1 ADP-ribosylation via PARP7 inhibition by RBN-2397 or mutation of the ADP-ribosylation site C97 increased FRA1 degradation by the proteasome via PSMC3. The reduction in FRA1 protein levels promoted IRF1- and IRF3-dependent cytokine as well as proapoptotic gene expression, culminating in CASP8-mediated apoptosis. Furthermore, high PARP7 expression was indicative of the PARP7 inhibitor response in FRA1-positive lung and breast cancer cells. Collectively, our findings highlight the connected roles of PARP7 and FRA1 and emphasize the clinical potential of PARP7 inhibitors for FRA1-driven cancers.

Combinatorial Treatment with PARP and MAPK Inhibitors Overcomes Phenotype Switch-Driven Drug Resistance in Advanced Melanoma.

Metastatic melanoma is either intrinsically resistant or rapidly acquires resistance to targeted therapy treatments, such as MAPK inhibitors (MAPKi). A leading cause of resistance to targeted therapy is a dynamic transition of melanoma cells from a proliferative to a highly invasive state, a phenomenon called phenotype switching. Mechanisms regulating phenotype switching represent potential targets for improving treatment of patients with melanoma. Using a drug screen targeting chromatin regulators in patient-derived three-dimensional MAPKi-resistant melanoma cell cultures, we discovered that PARP inhibitors (PARPi) restore sensitivity to MAPKis, independent of DNA damage repair pathways. Integrated transcriptomic, proteomic, and epigenomic analyses demonstrated that PARPis induce lysosomal autophagic cell death, accompanied by enhanced mitochondrial lipid metabolism that ultimately increases antigen presentation and sensitivity to T-cell cytotoxicity. Moreover, transcriptomic and epigenetic rearrangements induced by PARP inhibition reversed epithelial-mesenchymal transition-like phenotype switching, which redirected melanoma cells toward a proliferative and MAPKi-sensitive state. The combination of PARP and MAPKis synergistically induced cancer cell death both in vitro and in vivo in patient-derived xenograft models. Therefore, this study provides a scientific rationale for treating patients with melanoma with PARPis in combination with MAPKis to abrogate acquired therapy resistance. SIGNIFICANCE: PARP inhibitors can overcome resistance to MAPK inhibitors by activating autophagic cell death and reversing phenotype switching, suggesting that this synergistic combination could help improve the prognosis of patients with melanoma.

Absence of mitochondrial SLC25A51 enhances PARP1-dependent DNA repair by increasing nuclear NAD+ levels.

Though the effect of the recently identified mitochondrial NAD+ transporter SLC25A51 on glucose metabolism has been described, its contribution to other NAD+-dependent processes throughout the cell such as ADP-ribosylation remains elusive. Here, we report that absence of SLC25A51 leads to increased NAD+ concentration not only in the cytoplasm and but also in the nucleus. The increase is not associated with upregulation of the salvage pathway, implying an accumulation of constitutively synthesized NAD+ in the cytoplasm and nucleus. This results in an increase of PARP1-mediated nuclear ADP-ribosylation, as well as faster repair of DNA lesions induced by different single-strand DNA damaging agents. Lastly, absence of SLC25A51 reduces both MMS/Olaparib induced PARP1 chromatin retention and the sensitivity of different breast cancer cells to PARP1 inhibition. Together these results provide evidence that SLC25A51 might be a novel target to improve PARP1 inhibitor based therapies by changing subcellular NAD+ redistribution.

Tankyrase-mediated ADP-ribosylation is a regulator of TNF-induced death.

Tumor necrosis factor (TNF) is a key component of the innate immune response. Upon binding to its receptor, TNFR1, it promotes production of other cytokines via a membrane-bound complex 1 or induces cell death via a cytosolic complex 2. To understand how TNF-induced cell death is regulated, we performed mass spectrometry of complex 2 and identified tankyrase-1 as a native component that, upon a death stimulus, mediates complex 2 poly-ADP-ribosylation (PARylation). PARylation promotes recruitment of the E3 ligase RNF146, resulting in proteasomal degradation of complex 2, thereby limiting cell death. Expression of the ADP-ribose-binding/hydrolyzing severe acute respiratory syndrome coronavirus 2 macrodomain sensitizes cells to TNF-induced death via abolishing complex 2 PARylation. This suggests that disruption of ADP-ribosylation during an infection can prime a cell to retaliate with an inflammatory cell death.

Establishment of a Mass-Spectrometry-Based Method for the Identification of the In Vivo Whole Blood and Plasma ADP-Ribosylomes.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Engineering Af1521 improves ADP-ribose binding and identification of ADP-ribosylated proteins.

Protein ADP-ribosylation is a reversible post-translational modification that regulates important cellular functions. The identification of modified proteins has proven challenging and has mainly been achieved via enrichment methodologies. Random mutagenesis was used here to develop an engineered Af1521 ADP-ribose binding macro domain protein with 1000-fold increased affinity towards ADP-ribose. The crystal structure reveals that two point mutations K35E and Y145R form a salt bridge within the ADP-ribose binding domain. This forces the proximal ribose to rotate within the binding pocket and, as a consequence, improves engineered Af1521 ADPr-binding affinity. Its use in our proteomic ADP-ribosylome workflow increases the ADP-ribosylated protein identification rates and yields greater ADP-ribosylome coverage. Furthermore, generation of an engineered Af1521 Fc fusion protein confirms the improved detection of cellular ADP-ribosylation by immunoblot and immunofluorescence. Thus, this engineered isoform of Af1521 can also serve as a valuable tool for the analysis of cellular ADP-ribosylation under in vivo conditions.

Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies.

Purpose: The purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases. Methods: Corneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12. Results: The Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2). Conclusions: This novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.

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.

Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy.

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

Keratin 9 is required for the structural integrity and terminal differentiation of the palmoplantar epidermis.

Keratin 9 (K9) is a type I intermediate filament protein whose expression is confined to the suprabasal layers of the palmoplantar epidermis. Although mutations in the K9 gene are known to cause epidermolytic palmoplantar keratoderma, a rare dominant-negative skin disorder, its functional significance is poorly understood. To gain insight into the physical requirement and importance of K9, we generated K9-deficient (Krt9(-/-)) mice. Here, we report that adult Krt9(-/-)mice develop calluses marked by hyperpigmentation that are exclusively localized to the stress-bearing footpads. Histological, immunohistochemical, and immunoblot analyses of these regions revealed hyperproliferation, impaired terminal differentiation, and abnormal expression of keratins K5, K14, and K2. Furthermore, the absence of K9 induces the stress-activated keratins K6 and K16. Importantly, mice heterozygous for the K9-null allele (Krt9(+/-)) show neither an overt nor histological phenotype, demonstrating that one Krt9 allele is sufficient for the developing normal palmoplantar epidermis. Together, our data demonstrate that complete ablation of K9 is not tolerable in vivo and that K9 is required for terminal differentiation and maintaining the mechanical integrity of palmoplantar epidermis.

Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder.

Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.