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Kaposi sarcoma is the most common cancer in human immunodeficiency virus–positive individuals and is caused by Kaposi sarcoma–associated herpesvirus (KSHV). It is believed that a small number of latently infected Kaposi sarcoma tumor cells undergo spontaneous lytic reactivation to produce viral progeny for infection of new cells. Here, we use matched donor-derived human dermal blood and lymphatic endothelial cells (BEC and LEC, respectively) to show that KSHV-infected BECs progressively lose viral genome as they proliferate. In sharp contrast, KSHV-infected LECs predominantly entered lytic replication, underwent cell lysis, and released new virus. Continuous lytic cell lysis and de novo infection allowed LEC culture to remain infected for a prolonged time. Because of the strong propensity of LECs toward lytic replication, LECs maintained virus as a population, despite the death of individual host cells from lytic lysis. The master regulator of lymphatic development, Prox1, bound the promoter of the RTA gene to upregulate its expression and physically interacted with RTA protein to coregulate lytic genes. Thus, LECs may serve as a proficient viral reservoir that provides viral progeny for continuous de novo infection of tumor origin cells, and potentially BECs and mesenchymal stem cells, which give rise to Kaposi sarcoma tumors. Our study reveals drastically different host cell behaviors between BEC and LEC and defines the underlying mechanisms of the lymphatic cell environment supporting persistent infection in Kaposi sarcoma tumors. Significance: This study defines the mechanism by which Kaposi's sarcoma could be maintained by virus constantly produced by lymphatic cells in HIV-positive individuals.

Glaucoma surgeries, such as trabeculectomy, are performed to lower intraocular pressure to reduce risk of vision loss. These surgeries create a new passage in the eye that reroutes the aqueous humor outflow to the subconjunctival space, where the fluid is presumably absorbed by the conjunctival lymphatics. Here, we characterized the development and function of the ocular lymphatics using transgenic lymphatic reporter mice and rats. We found that the limbal and conjunctival lymphatic networks are progressively formed from a primary lymphatic vessel that grows from the nasal-side medial canthus region at birth. This primary lymphatic vessel immediately branches out, invades the limbus and conjunctiva, and bidirectionally encircles the cornea. As a result, the distribution of the ocular lymphatics is significantly polarized toward the nasal side, and the limbal lymphatics are directly connected to the conjunctival lymphatics. New lymphatic sprouts are produced mainly from the nasal-side limbal lymphatics, posing the nasal side of the eye as more responsive to fluid drainage and inflammatory stimuli. Consistent with this polarized distribution of the ocular lymphatics, a higher drainage efficiency was observed in the nasal side than the temporal side of the eye when injected with a fluorescent tracer. In contrast, blood vessels are evenly distributed at the anterior surface of the eyes. Also, we found that these distinct vascular distribution patterns were conserved in human eyes. Together, our study demonstrated that the ocular surface lymphatics are more densely present in the nasal side and uncovered the potential clinical benefits in selecting the nasal side as a glaucoma surgery site to improve fluid drainage.

Objective Mutations in receptor expression enhancing protein 1 (REEP1) are associated with hereditary spastic paraplegias (HSPs). Although axonal degeneration is thought to be a predominant feature in HSP, the role of REEP1 mutations in degeneration is largely unknown. Previous studies have implicated a role for REEP1 in the endoplasmic reticulum (ER), whereas others localized REEP1 with mitochondria. We sought to resolve the cellular localization of REEP1 and further elucidate the pathobiology underlying REEP1 mutations in patients. Methods A combination of cellular imaging and biochemical approaches was used to refine the cellular localization of REEP1. Next, Reep1 mutations associated with HSP were functionally tested in neuritic growth and degeneration assays using mouse cortical culture. Finally, a novel assay was developed and used with wild-type and mutant Reep1s to measure the interactions between the ER and mitochondria. Results We found that REEP1 is present at the ER-mitochondria interface, and it contains subdomains for mitochondrial as well as ER localization. Knockdown of Reep1 and expression of pathological Reep1 mutations resulted in neuritic growth defects and degeneration. Finally, using our novel split-RLuc8 assay, we show that REEP1 facilitates ER-mitochondria interactions, a function diminished by disease-associated mutations. Interpretation Our data potentially reconcile the current conflicting reports regarding REEP1 being either an ER or a mitochondrial protein. Furthermore, our results connect, for the first time, the disrupted ER-mitochondria interactions to a failure in maintaining health of long axons in HSPs. Finally, the split-RLuc8 assay offers a new tool to identify potential drugs for multiple neurodegenerative diseases with ER-mitochondria interaction defects. Ann Neurol 2015;78:Ann Neurol 2015;78:679–696

Osteoclast maturation and function primarily depend on receptor activator of NF-κB ligand (RANKL)-mediated induction of nuclear factor of activated T cells c1 (NFATc1), which is further activated via increased intracellular calcium ([Ca(2+)](i)) oscillation. However, the coordination mechanism that mediates Ca(2+) oscillation during osteoclastogenesis remains ill defined. Here, we identified transmembrane protein 64 (Tmem64) as a regulator of Ca(2+) oscillation during osteoclastogenesis. We found that Tmem64-deficient mice exhibit increased bone mass due in part to impaired osteoclast formation. Using in vitro osteoclast culture systems, we show here that Tmem64 interacts with sarcoplasmic endoplasmic reticulum Ca(2+) ATPase 2 (SERCA2) and modulates its activity. Consequently, Tmem64 deficiency significantly diminishes RANKL-induced [Ca(2+)](i) oscillation, which results in reduced Ca(2+)/calmodulin-dependent protein kinases (CaMK) IV and mitochondrial ROS, both of which contribute to achieving the CREB activity necessary for osteoclast formation. These data demonstrate that Tmem64 is a positive modulator of osteoclast differentiation via SERCA2-dependent Ca(2+) signaling.

The Cockayne syndrome complementation group B (CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome—a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP–dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP–dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB–binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP–dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome., Author Summary Cockayne syndrome is a devastating inherited disease; the average life span of those afflicted is 12 years. Cockayne syndrome patients have features of premature aging, are highly sensitive to sunlight, and suffer from numerous developmental and neurological disorders. The majority of Cockayne syndrome patients have mutations in the CSB protein; however, how these mutations can lead to Cockayne syndrome is largely unknown. CSB is essential for transcription-coupled DNA repair—a process that preferentially removes bulky DNA lesions that stall transcription, such as those created by ultraviolet light. In eukaryotes, DNA is packaged into nucleosomes, which consists of DNA wrapped around a set of core histone proteins, and nucleosomes can create barriers to the DNA repair process. In this study, we found that CSB can slide histones along DNA. We also found that histone chaperones, proteins that accept and donate histones, greatly facilitate this process. Importantly, we show that CSB derivatives that are unable to move nucleosomes or collaborate with histone chaperones cannot repair UV-induced DNA lesions. Our study reveals that nucleosome remodeling by CSB is important for transcription-coupled DNA repair and suggests that an inability to efficiently mobilize nucleosomes might contribute to the underlying mechanism of Cockayne syndrome.

DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC).We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells.Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P.001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P.001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities.These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Colorectal cancer (CRC) is the second most common fatal cancer in the U.S., with ∼ 30% cases familial (FCRC). Genetic predispositions have only been defined for 25% of FCRC (including Lynch syndrome, Familial Adenomatous Polyposis (FAP), and MutYH polyposis). For the genetically undefined group (uFCRC), understanding of disease pathogenesis is limited, and the absence of defined defects in relevant pathways that could support molecular diagnosis results in under- and over-screening. We hypothesized that uFCRC would arise distinctly from moderately penetrant germline defects in repair of DNA double-strand breaks (DSBs). Such defects in DSB repair would lead to a moderate level of constitutional genomic instability (CGI) that could be assayed from patient peripheral blood lymphocytes (PBLs). We further hypothesized that such DNA lesions would often be heterozygous, associated with haploinsufficiency or dominant-negative effects, and would impair protein interactions important for DNA repair. We combined exome sequencing with biological assays to screen a rich resource of uFCRC clinical samples from the Fox Chase Cancer Center Risk Assessment Program. Exome sequencing of PBLs from patients (N = 25) identified a mean of 1.4 rare variants/patient that were 1) strongly predicted to damage protein function using multiple functional predictors (SIFT, Polyphen-2, Provean, and MutationAssessor), 2) involved in pathways that suppress DSBs, and 3) have not been previously implicated in CRC. Further, many of the variant genes were commonly mutated in CRCs and other cancers in TCGA database. Comparison of PBLs from patients and age- and sex- matched controls revealed increased DSB levels (basal and with DNA damaging agents) in 18/25 patients vs. 1/25 normal controls (P = 0.001, AUC = 0.85). Next, knockdown of variant genes in HCT116 CRC cells increased DSB formation with or without damaging agents. As a proof-of-concept, we studied one patient in detail who had candidate disease-causing variants in 2 DNA repair genes, ERCC6 and WRN. Both variants were dysfunctional in biochemical tests. We established patient-derived EBV-lines and observed, at baseline and with several DNA damaging agents, elevated levels of γH2AX foci and larger nuclear comets, indicative of DSBs. This phenotype could be rescued by expression of wild type WRN or ERCC6 and conversely induced by knockdown in CRC cells. Further, the patient derived lines exhibited reduced expression of WRN and ERCC6, implicating haploinsufficiency of these genes in the phenotype observed. Our data support the hypothesis that defects in DSB repair genes cause moderate-level CGI that commonly presents as uFCRC. Our studies identify novel genetic subsets of FCRC, and could potentially predict familial risk and improve screening and diagnosis. Citation Format: sanjeevani arora, Hong Yan, Iltaeg Cho, Hua-Ying Fan, Biao Luo, xiaowu gai, dale bodian, joe vockley, yan zhou, elizabeth handorf, mark andrake, emmanuelle nicolas, Ilya Serebriiskii, tim yen, michael hall, greg enders, erica golemis. Genetic predisposition to DNA double strand break repair defect defines a new class of familial colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4739. doi:10.1158/1538-7445.AM2015-4739