Nfu-type proteins are essential in the biogenesis of iron-sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here we report that recombinant AtNfu2 is able to assemble both [2Fe-2S] and [4Fe-4S] clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one [2Fe-2S] cluster/homotetramer and one [4Fe-4S] cluster/homodimer. The combination of UV-visible absorption and circular dichroism, resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging [2Fe-2S]2+ and [4Fe-4S]2+ clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for maturation of chloroplastic Fe-S proteins via intact, rapid and quantitative cluster transfer. [2Fe-2S] cluster-bound Nfu2 is shown to be an effective [2Fe-2S]2+ cluster donor for glutaredoxin S16, but not glutaredoxin S14. Moreover, [4Fe-4S] cluster-bound Nfu2 is shown to be a very rapid and efficient [4Fe-4S]2+ cluster donor for adenosine 5′-phosphosulfate reductase (APR1) and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2, but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.

Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe-S clusters. The best characterized examples are DHAD, which contains an oxygen-labile [FeS] cluster, and spinach DHAD, which contains an oxygen-resistant [FeS] cluster. Although the Fe-S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism and resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe-S center in DHAD from (). Our results indicated that DHAD can accommodate [FeS] and [FeS] clusters. However, only the [FeS] cluster-bound form is catalytically active. We found that the [FeS] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol. cluster transfer and reconstitution reactions revealed that [FeS] cluster-containing NFU2 protein is likely the physiological cluster donor for maturation ofDHAD. In summary, DHAD binds either one [FeS] or one [FeS] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [FeS] cluster donor for DHAD maturation. This work represents the first characterization of recombinantDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe-S center in a plant DHAD.

Poly (ADP-ribose) polymerase (PARP) inhibitors are one of the most exciting classes of targeted therapy agents for cancers with homologous recombination (HR) deficiency. However, many patients without apparent HR defects also respond well to PARP inhibitors/cisplatin. The biomarker responsible for this mechanism remains unclear. Here, we identified a set of ribosomal genes that predict response to PARP inhibitors/cisplatin in HR-proficient patients. PARP inhibitor/cisplatin selectively eliminates cells with high expression of the eight genes in the identified panel via DNA damage (ATM) signaling-induced pro-apoptotic ribosomal stress, which along with ATM signaling-induced pro-survival HR repair constitutes a new model to balance the cell fate in response to DNA damage. Therefore, the combined examination of the gene panel along with HR status would allow for more precise predictions of clinical response to PARP inhibitor/cisplatin. The gene panel as an independent biomarker was validated by multiple published clinical datasets, as well as by an ovarian cancer organoids library we established. More importantly, its predictive value was further verified in a cohort of PARP inhibitor-treated ovarian cancer patients with both RNA-seq and WGS data. Furthermore, we identified several marketed drugs capable of upregulating the expression of the genes in the panel without causing HR deficiency in PARP inhibitor/cisplatin-resistant cell lines. These drugs enhance PARP inhibitor/cisplatin sensitivity in both intrinsically resistant organoids and cell lines with acquired resistance. Together, our study identifies a marker gene panel for HR-proficient patients and reveals a broader application of PARP inhibitor/cisplatin in cancer therapy.

Selective serotonin reuptake inhibitors (SSRIs) are standard of care for major depressive disorder (MDD) pharmacotherapy, but only approximately half of these patients remit on SSRI therapy. Our previous genome-wide association study identified a single-nucleotide polymorphism (SNP) signal across the glutamate-rich 3 (ERICH3) gene that was nearly genome-wide significantly associated with plasma serotonin (5-HT) concentrations, which were themselves associated with SSRI response for MDD patients enrolled in the Mayo Clinic PGRN-AMPS SSRI trial. In this study, we performed a meta-analysis which demonstrated that those SNPs were significantly associated with SSRI treatment outcomes in four independent MDD trials. However, the function of ERICH3 and molecular mechanism(s) by which it might be associated with plasma 5-HT concentrations and SSRI clinical response remained unclear. Therefore, we characterized the human ERICH3 gene functionally and identified ERICH3 mRNA transcripts and protein isoforms that are highly expressed in central nervous system cells. Coimmunoprecipitation identified a series of ERICH3 interacting proteins including clathrin heavy chain which are known to play a role in vesicular function. Immunofluorescence showed ERICH3 colocalization with 5-HT in vesicle-like structures, and ERICH3 knock-out dramatically decreased 5-HT staining in SK-N-SH cells as well as 5-HT concentrations in the culture media and cell lysates without changing the expression of 5-HT synthesizing or metabolizing enzymes. Finally, immunofluorescence also showed ERICH3 colocalization with dopamine in human iPSC-derived neurons. These results suggest that ERICH3 may play a significant role in vesicular function in serotonergic and other neuronal cell types, which might help explain its association with antidepressant treatment response.

Understanding the function of non-coding genetic variants represents a formidable challenge for biomedicine. We previously identified genetic variants that influence gene expression only after exposure to a hormone or drug. Using glucocorticoid signaling as a model system, we have now demonstrated, in a genome-wide manner, that exposure to glucocorticoids triggered disease risk variants with previously unclear function to influence the expression of genes involved in autoimmunity, metabolic and mood disorders, osteoporosis and cancer. Integrating a series of genomic and epigenomic assays, we identified the cis-regulatory elements and 3-dimensional interactions underlying the ligand-dependent associations between those genetic variants and distant risk genes. These observations increase our understanding of mechanisms of non-coding genetic variant-chemical environment interactions and advance the fine-mapping of disease risk and pharmacogenomic loci.One Sentence SummaryGenomic and epigenomic fine-mapping of ligand-dependent genetic variants unmasks novel disease risk genes

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Characterization of the recurrent genetic alterations in PDAC will yield improved understanding and therapies for this disease. Here, we report that PDAC patients with elevated expression of METTL16, one of the "writers" of RNA N 6 -methyladenosine (m 6 A) modi cation, may bene ts from poly (ADP-ribose) polymerase (PARP) inhibitor treatment. Mechanistically, METTL16 interacts with MRE11 in an RNA-dependent manner; and, this interaction inhibits MRE11's exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 at Ser419 within its C terminus, resulting in METTL16 conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression levels show increased sensitivity to PARP inhibitors, especially when combined with gemcitabine. Thus, our ndings have revealed a role for METTL16 in homologous recombination repair and suggest that combination of PARP inhibitors with gemcitabine could be an effective treatment strategy for PDAC patients with high METTL16 expression.DNA end resection to generate short stretches of single-stranded DNA (ssDNA), which is a critical step in HR 24 . Subsequently, EXO1/DNA2 are recruited to DSBs sites to expand end resection. Therefore, MRE11 is the functional subunit of the MRN complex, which plays a key role in DNA repair regulation. Meanwhile, the function of MRE11 is strictly regulated in cells 25,26 . PLK1 phosphorylates MRE11 at Ser649 to inhibit its DNA binding activity and antagonize HR repair 27 . UBQLN4 promotes the ubiquitination and degradation of chromatin MRE11, thus affects DSBs repair choice. In addition to post translational modi cation, protein-protein interactions were also reported to affect MRE11 function 28 . C1QBP interacts with the GAR domain of MRE11 and inhibits the nuclease activity of MRE11 29 . DYNLL1 and Nej1 were also recently identi ed as MRE11 interacting proteins and reported to inhibit MRE11-mediated end resection 30 . N6-methyladenosine (m 6 A) methylation is one of the most common RNA modi cations 31,32 . m 6 A modi cations occur via the m 6 A methyltransferases called "writers", they are removed by demethylases termed "erasers", and they are recognized by m 6 A-binding proteins called "readers". Methyltransferaselike family members of METTL3, METTL14, and METTL16 have been shown acting as "writers" to initiate the m 6 A modi cation process 33 . Recently, Xiang et al. reported that m 6 A modi cation can help to recruit DNA polymerase k (Pol k) for nucleotide excision and transl-esion synthesis-mediated single-strand breaks in response to ultra-violet (UV) irradiation 34 . Furthermore, Zhang et al. revealed that METTL3 modulates accumulation of DNA-RNA hybrids at DSBs sites depending on its methyltransferase (MTase) activity, and then promote HR repair by assistin...

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