Single-Cell Transcriptome Profiling of the Kidney Glomerulus Identifies Key Cell Types and Reactions to Injury.

BACKGROUND: The glomerulus is a specialized capillary bed that is involved in urine production and BP control. Glomerular injury is a major cause of CKD, which is epidemic and without therapeutic options. Single-cell transcriptomics has radically improved our ability to characterize complex organs, such as the kidney. Cells of the glomerulus, however, have been largely underrepresented in previous single-cell kidney studies due to their paucity and intractability. METHODS: Single-cell RNA sequencing comprehensively characterized the types of cells in the glomerulus from healthy mice and from four different disease models (nephrotoxic serum nephritis, diabetes, doxorubicin toxicity, and CD2AP deficiency). RESULTS: All cell types in the glomerulus were identified using unsupervised clustering analysis. Novel marker genes and gene signatures of mesangial cells, vascular smooth muscle cells of the afferent and efferent arterioles, parietal epithelial cells, and three types of endothelial cells were identified. Analysis of the disease models revealed cell type-specific and injury type-specific responses in the glomerulus, including acute activation of the Hippo pathway in podocytes after nephrotoxic immune injury. Conditional deletion of YAP or TAZ resulted in more severe and prolonged proteinuria in response to injury, as well as worse glomerulosclerosis. CONCLUSIONS: Generation of comprehensive high-resolution, single-cell transcriptomic profiles of the glomerulus from healthy and injured mice provides resources to identify novel disease-related genes and pathways.

Dna2 initiates resection at clean DNA double-strand breaks.

Nucleolytic resection of DNA double-strand breaks (DSBs) is essential for both checkpoint activation and homology-mediated repair; however, the precise mechanism of resection, especially the initiation step, remains incompletely understood. Resection of blocked ends with protein or chemical adducts is believed to be initiated by the MRN complex in conjunction with CtIP through internal cleavage of the 5' strand DNA. However, it is not clear whether resection of clean DSBs with free ends is also initiated by the same mechanism. Using the Xenopus nuclear extract system, here we show that the Dna2 nuclease directly initiates the resection of clean DSBs by cleaving the 5' strand DNA ∼10-20 nucleotides away from the ends. In the absence of Dna2, MRN together with CtIP mediate an alternative resection initiation pathway where the nuclease activity of MRN apparently directly cleaves the 5' strand DNA at more distal sites. MRN also facilitates resection initiation by promoting the recruitment of Dna2 and CtIP to the DNA substrate. The ssDNA-binding protein RPA promotes both Dna2- and CtIP-MRN-dependent resection initiation, but a RPA mutant can distinguish between these pathways. Our results strongly suggest that resection of blocked and clean DSBs is initiated via distinct mechanisms.

14-3-3 proteins restrain the Exo1 nuclease to prevent overresection.

The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment.

Sharpening the ends for repair: mechanisms and regulation of DNA resection.

DNA end resection is a key process in the cellular response to DNA double-strand break damage that is essential for genome maintenance and cell survival. Resection involves selective processing of 5' ends of broken DNA to generate ssDNA overhangs, which in turn control both DNA repair and checkpoint signaling. DNA resection is the first step in homologous recombination-mediated repair and a prerequisite for the activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent checkpoint that coordinates repair with cell cycle progression and other cellular processes. Resection occurs in a cell cycle-dependent manner and is regulated by multiple factors to ensure an optimal amount of ssDNA required for proper repair and genome stability. Here, we review the latest findings on the molecular mechanisms and regulation of the DNA end resection process and their implications for cancer formation and treatment.

PCNA promotes processive DNA end resection by Exo1.

Exo1-mediated resection of DNA double-strand break ends generates 3' single-stranded DNA overhangs required for homology-based DNA repair and activation of the ATR-dependent checkpoint. Despite its critical importance in inducing the overall DNA damage response, the mechanisms and regulation of the Exo1 resection pathway remain incompletely understood. Here, we identify the ring-shaped DNA clamp PCNA as a new factor in the Exo1 resection pathway. Using mammalian cells, Xenopus nuclear extracts and purified proteins, we show that after DNA damage, PCNA loads onto double-strand breaks and promotes Exo1 damage association through direct interaction with Exo1. By tethering Exo1 to the DNA substrate, PCNA confers processivity to Exo1 in resection. This role of PCNA in DNA resection is analogous to its function in DNA replication where PCNA serves as a processivity co-factor for DNA polymerases.

CHFR is important for the first wave of ubiquitination at DNA damage sites.

Protein ubiquitination plays an important role in activating the DNA damage response and maintaining genomic stability. In response to DNA double-strand breaks (DSBs), a ubiquitination cascade occurs at DNA lesions. Here, we show that checkpoint with Forkhead-associated (FHA) and RING finger domain protein (CHFR), an E3 ubiquitin ligase, is recruited to DSBs by poly(ADP-ribose) (PAR). At DSBs, CHFR regulates the first wave of protein ubiquitination. Moreover, CHFR ubiquitinates PAR polymerase 1 (PARP1) and regulates chromatin-associated PARP1 in vivo. Thus, these results demonstrate that CHFR is an important E3 ligase in the early stage of the DNA damage response, which mediates the crosstalk between ubiquitination and poly-ADP-ribosylation.

Regulators of tubulin polyglutamylation control nuclear shape and cilium disassembly by balancing microtubule and actin assembly.

Cytoskeletal networks play an important role in regulating nuclear morphology and ciliogenesis. However, the role of microtubule (MT) post-translational modifications in nuclear shape regulation and cilium disassembly has not been explored. Here we identified a novel regulator of the tubulin polyglutamylase complex (TPGC), C11ORF49/CSTPP1, that regulates cytoskeletal organization, nuclear shape, and cilium disassembly. Mechanistically, loss of C11ORF49/CSTPP1 impacts the assembly and stability of the TPGC, which modulates long-chain polyglutamylation levels on microtubules (MTs) and thereby balances the binding of MT-associated proteins and actin nucleators. As a result, loss of TPGC leads to aberrant, enhanced assembly of MTs that penetrate the nucleus, which in turn leads to defects in nuclear shape, and disorganization of cytoplasmic actin that disrupts the YAP/TAZ pathway and cilium disassembly. Further, we showed that C11ORF49/CSTPP1-TPGC plays mechanistically distinct roles in the regulation of nuclear shape and cilium disassembly. Remarkably, disruption of C11ORF49/CSTPP1-TPGC also leads to developmental defects in vivo. Our findings point to an unanticipated nexus that links tubulin polyglutamylation with nuclear shape and ciliogenesis.

Poly(ADP-ribose)-binding promotes Exo1 damage recruitment and suppresses its nuclease activities.

Exonuclease 1 (Exo1) has important roles in DNA metabolic transactions that are essential for genome maintenance, telomere regulation and cancer suppression. However, the mechanisms for regulating Exo1 activity in these processes remain incompletely understood. Here, we report that Exo1 activity is regulated by a direct interaction with poly(ADP-ribose) (PAR), a prominent posttranslational modification at the sites of DNA damage. This PAR-binding activity promotes the early recruitment of Exo1 to sites of DNA damage, where it is retained through an interaction with PCNA, which interacts with the C-terminus of Exo1. The effects of both PAR and PCNA on Exo1 damage association are antagonized by the 14-3-3 adaptor proteins, which interact with the central domain of Exo1. Although PAR binding inhibits both the exonuclease activity and the 5' flap endonuclease activity of purified Exo1, the pharmacological blockade of PAR synthesis does not overtly affect DNA double-strand break end resection in a cell free Xenopus egg extract. Thus, the counteracting effects of PAR on Exo1 recruitment and enzymatic activity may enable appropriate resection of DNA ends while preventing unscheduled or improper processing of DNA breaks in cells.