Androgen signaling uses a writer and a reader of ADP-ribosylation to regulate protein complex assembly.

Androgen signaling through the androgen receptor (AR) directs gene expression in both normal and prostate cancer cells. Androgen regulates multiple aspects of the AR life cycle, including its localization and post-translational modification, but understanding how modifications are read and integrated with AR activity has been difficult. Here, we show that ADP-ribosylation regulates AR through a nuclear pathway mediated by Parp7. We show that Parp7 mono-ADP-ribosylates agonist-bound AR, and that ADP-ribosyl-cysteines within the N-terminal domain mediate recruitment of the E3 ligase Dtx3L/Parp9. Molecular recognition of ADP-ribosyl-cysteine is provided by tandem macrodomains in Parp9, and Dtx3L/Parp9 modulates expression of a subset of AR-regulated genes. Parp7, ADP-ribosylation of AR, and AR-Dtx3L/Parp9 complex assembly are inhibited by Olaparib, a compound used clinically to inhibit poly-ADP-ribosyltransferases Parp1/2. Our study reveals the components of an androgen signaling axis that uses a writer and reader of ADP-ribosylation to regulate protein-protein interactions and AR activity.

A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break repair.

DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.

The Barrier Molecules Junction Plakoglobin, Filaggrin, and Dystonin Play Roles in Melanoma Growth and Angiogenesis.

OBJECTIVE: To understand role of barrier molecules in melanomas. BACKGROUND: We have reported poor patient survival and low immune infiltration of melanomas that overexpress a set of genes that include filaggrin (FLG), dystonin (DST), junction plakoglobin (JUP), and plakophilin-3 (PKP3), and are involved in cell-cell adhesions. We hypothesized that these associations are causal, either by interfering with immune cell infiltration or by enhancing melanoma cell growth. METHODS: FLG and DST were knocked out by CRISPR/Cas9 in human DM93 and murine B16-F1 melanoma cells. PKP3 and JUP were overexpressed in murine B16-AAD and human VMM39 melanoma cells by lentiviral transduction. These cell lines were evaluated in vitro for cell proliferation and in vivo for tumor burden, immune composition, cytokine expression, and vascularity. RESULTS: Immune infiltrates were not altered by these genes. FLG/DST knockout reduced proliferation of human DM93 melanoma in vitro, and decreased B16-F1 tumor burden in vivo. Overexpression of JUP, but not PKP3, in B16-AAD significantly increased tumor burden, increased VEGF-A, reduced IL-33, and enhanced vascularity. CONCLUSIONS: FLG and DST support melanoma cell growth in vitro and in vivo. Growth effects of JUP were only evident in vivo, and may be mediated, in part, by enhancing angiogenesis. In addition, growth-promoting effects of FLG and DST in vitro suggest that these genes may also support melanoma cell proliferation through angiogenesis-independent pathways. These findings identify FLG, DST, and JUP as novel therapeutic targets whose down-regulation may provide clinical benefit to patients with melanoma.