Chromatin run-on sequencing analysis finds that ECM remodeling plays an important role in canine hemangiosarcoma pathogenesis.

BACKGROUND: Canine visceral hemangiosarcoma (HSA) is a highly aggressive cancer of endothelial origin that closely resembles visceral angiosarcoma in humans, both clinically and histopathologically. Currently there is an unmet need for new diagnostics and therapies for both forms of this disease. The goal of this study was to utilize Chromatin run-on sequencing (ChRO-seq) and immunohistochemistry (IHC) to identify gene and protein expression signatures that may be important drivers of HSA progression. RESULTS: ChRO-seq was performed on tissue isolated from 17 HSA samples and 4 normal splenic samples. Computational analysis was then used to identify differentially expressed genes and these factors were subjected to gene ontology analysis. ChRO-seq analysis revealed over a thousand differentially expressed genes in HSA tissue compared with normal splenic tissue (FDR < 0.005). Interestingly, the majority of genes overexpressed in HSA tumor tissue were associated with extracellular matrix (ECM) remodeling. This observation correlated well with our histological analysis, which found that HSA tumors contain a rich and complex collagen network. Additionally, we characterized the protein expression patterns of two highly overexpressed molecules identified in ChRO-seq analysis, podoplanin (PDPN) and laminin alpha 4 (LAMA4). We found that the expression of these two ECM-associated factors appeared to be largely limited to transformed endothelial cells within the HSA lesions. CONCLUSION: Outcomes from this study suggest that ECM remodeling plays an important role in HSA progression. Additionally, our study identified two potential novel biomarkers of HSA, PDPN and LAMA4. Interestingly, given that function-blocking anti-PDPN antibodies have shown anti-tumor effects in mouse models of canine melanoma, our studies raise the possibility that these types of therapeutic strategies could potentially be developed for treating canine HSA.

BarkBase: Epigenomic Annotation of Canine Genomes.

Dogs are an unparalleled natural model for investigating the genetics of health and disease, particularly for complex diseases like cancer. Comprehensive genomic annotation of regulatory elements active in healthy canine tissues is crucial both for identifying candidate causal variants and for designing functional studies needed to translate genetic associations into disease insight. Currently, canine geneticists rely primarily on annotations of the human or mouse genome that have been remapped to dog, an approach that misses dog-specific features. Here, we describe BarkBase, a canine epigenomic resource available at barkbase.org. BarkBase hosts data for 27 adult tissue types, with biological replicates, and for one sample of up to five tissues sampled at each of four carefully staged embryonic time points. RNA sequencing is complemented with whole genome sequencing and with assay for transposase-accessible chromatin using sequencing (ATAC-seq), which identifies open chromatin regions. By including replicates, we can more confidently discern tissue-specific transcripts and assess differential gene expression between tissues and timepoints. By offering data in easy-to-use file formats, through a visual browser modeled on similar genomic resources for human, BarkBase introduces a powerful new resource to support comparative studies in dogs and humans.

ER-positive breast cancer cells are poised for RET-mediated endocrine resistance.

The RET tyrosine kinase signaling pathway is involved in the development of endocrine resistant ER+ breast cancer. However, we know little about how ER+ cells activate RET signaling and initiate an endocrine resistant phenotype. Here we show that both ER+ endocrine resistant and sensitive breast cancers have a functional RET tyrosine kinase signaling pathway, but that endocrine sensitive breast cancer cells lack RET ligands that are necessary to drive endocrine resistance. Transcription of one RET ligand, GDNF, is necessary and sufficient to confer resistance in the ER+ MCF-7 cell line. Endogenous GDNF produced by endocrine resistant cells is translated, secreted into the media, and activates RET signaling in nearby cells. In patients, RET ligand expression predicts responsiveness to endocrine therapies and correlates with survival. Collectively, our findings show that ER+ tumor cells are "poised" for RET mediated endocrine resistance, expressing all components of the RET signaling pathway, but endocrine sensitive cells lack high expression of RET ligands that are necessary to initiate the resistance phenotype.

PAD2 overexpression in transgenic mice augments malignancy and tumor-associated inflammation in chemically initiated skin tumors.

We previously found that transgenic mice overexpressing MMTV-FLAG-hPAD2 (PAD2OE) developed spontaneous skin lesions, with a subset of these lesions progressing to invasive squamous cell carcinoma (SCC). The goal of this report was to better understand the potential mechanisms by which PAD2 overexpression promotes skin cancer. Here, PAD2OE mice were treated with the carcinogen, 9,10-dimethyl-1,2-benzanthracene and with O-tetradecanoylphorbol-13-acetate and then scored for papilloma formation. Additionally, tumor sections were evaluated for evidence of tumor cell invasion and inflammation. We found that the total number of papillomas was significantly increased in PAD2OE mice compared to controls. Histopathologic analysis of the lesions found that in PAD2OE skin tumors progressed to invasive SCC more frequently than controls. Additionally, we found that PAD2OE lesions were highly inflamed, with a dense inflammatory cell infiltrate and an associated increase in nuclear phospho-STAT3 (signal transducer and activator of transcription 3) in the transgenic tumors. These data suggest that overexpression of the hPAD2 transgene in the epidermis increases the malignant conversion rate of benign tumors by promoting an inflammatory microenvironment.

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration.

BACKGROUND: Penetration of the mammary gland basement membrane by cancer cells is a crucial first step in tumor invasion. Using a mouse model of ductal carcinoma in situ, we previously found that inhibition of peptidylarginine deiminase 2 (PAD2, aka PADI2) activity appears to maintain basement membrane integrity in xenograft tumors. The goal of this investigation was to gain insight into the mechanisms by which PAD2 mediates this process. METHODS: For our study, we modulated PAD2 activity in mammary ductal carcinoma cells by lentiviral shRNA-mediated depletion, lentiviral-mediated PAD2 overexpression, or PAD inhibition and explored the effects of these treatments on changes in cell migration and cell morphology. We also used these PAD2-modulated cells to test whether PAD2 may be required for EGF-induced cell migration. To determine how PAD2 might promote tumor cell migration in vivo, we tested the effects of PAD2 inhibition on the expression of several cell migration mediators in MCF10DCIS.com xenograft tumors. In addition, we tested the effect of PAD2 inhibition on EGF-induced ductal invasion and elongation in primary mouse mammary organoids. Lastly, using a transgenic mouse model, we investigated the effects of PAD2 overexpression on mammary gland development. RESULTS: Our results indicate that PAD2 depletion or inhibition suppresses cell migration and alters the morphology of MCF10DCIS.com cells. In addition, we found that PAD2 depletion suppresses the expression of the cytoskeletal regulatory proteins RhoA, Rac1, and Cdc42 and also promotes a mesenchymal to epithelial-like transition in tumor cells with an associated increase in the cell adhesion marker, E-cadherin. Our mammary gland organoid study found that inhibition of PAD2 activity suppresses EGF-induced ductal invasion. In vivo, we found that PAD2 overexpression causes hyperbranching in the developing mammary gland. CONCLUSION: Together, these results suggest that PAD2 plays a critical role in breast cancer cell migration. Our findings that EGF treatment increases protein citrullination and that PAD2 inhibition blocks EGF-induced cell migration suggest that PAD2 likely functions within the EGF signaling pathway to mediate cell migration.

Effects of Nanoparticle Size on Multilayer Formation and Kinetics of Tethered Enzymes.

Despite numerous applications, we lack fundamental understanding of how variables such as nanoparticle (NP) size influence the activity of tethered enzymes. Previously, we showed that biomimetic oriented immobilization yielded higher specific activities versus nonoriented adsorption or carboxyl-amine binding. Here, we standardize NP attachment strategy (oriented immobilization via hexahistidine tags) and composition (Ni-NTA coated gold NPs), to test the impact of NP size (⌀5, 10, 20, and 50 nm) on multilayer formation, activity, and kinetic parameters (kcat, KM, kcat/KM) of enzymes representing three different classes: glucose-6-phosphate isomerase (GPI), an isomerase; Glyceraldehyde-3-phosphate dehydrogenase S (GAPDHS), an oxidoreductase; and pyruvate kinase (PK), a transferase. Contrary to other reports, we observed no trend in kinetic parameters for individual enzymes when found in monolayers (<100% enzyme coverage), suggesting an advantage for oriented immobilization versus other attachment strategies. Saturating the NPs to maximize activity per NP resulted in enzyme multilayer formation. Under these conditions, total activity per NP increased with increasing NP size. Conversely, specific activity for all three enzymes was highest when tethered to the smallest NPs, retaining a remarkable 73-94% of the activity of free/untethered enzymes. Multilayer formations caused a clear trend of kcat decreasing with increasing NP size, yet negligible change in KM. Understanding the fundamental relationships between NP size and tethered enzyme activity enables optimized design of various applications, maximizing activity per NP or activity per enzyme molecule.

Design, synthesis, and biological evaluation of tetrazole analogs of Cl-amidine as protein arginine deiminase inhibitors.

Protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of arginine residues to form citrulline. This once obscure modification is now known to play a key role in the etiology of multiple autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, lupus, and ulcerative colitis) and in some forms of cancer. Among the five human PADs (PAD1, -2, -3, -4, and -6), it is unclear which isozyme contributes to disease pathogenesis. Toward the identification of potent, selective, and bioavailable PAD inhibitors that can be used to elucidate the specific roles of each isozyme, we describe tetrazole analogs as suitable backbone amide bond bioisosteres for the parent pan PAD inhibitor Cl-amidine. These tetrazole based analogs are highly potent and show selectivity toward particular isozymes. Importantly, one of the compounds, biphenyl tetrazole tert-butyl Cl-amidine (compound 13), exhibits enhanced cell killing in a PAD4 expressing osteosarcoma bone marrow (U2OS) cell line and can also block the formation of neutrophil extracellular traps. These bioisosteres represent an important step in our efforts to develop stable, bioavailable, and selective inhibitors for the PADs.

PAD2 overexpression in transgenic mice promotes spontaneous skin neoplasia.

Peptidylarginine deiminase 2 (PAD2/PADI2) has been implicated in various inflammatory diseases and, more recently, cancer. The goal of this study was to test the hypothesis that PAD2 promotes oncogenesis using a transgenic mouse model. We found that about 37% of transgenic mice overexpressing human FLAG-PAD2 downstream of the MMTV-LTR promoter develop spontaneous neoplastic skin lesions. Molecular and histopathologic analyses of the resulting lesions find that they contain increased levels of markers for invasion, inflammation, and epithelial-to-mesenchymal transition (EMT) and that a subset of the lesions progress to invasive squamous cell carcinoma (SCC). We then stably overexpressed FLAG-PAD2 in the human SCC cell line, A431, and found that the PAD2-overexpressing cells were more tumorigenic in vitro and also contained elevated levels of markers for inflammation and EMT. Collectively, these studies provide the first genetic evidence that PAD2 functions as an oncogene and suggest that PAD2 may promote tumor progression by enhancing inflammation within the tumor microenvironment.

Virus-mediated compartmentalization of the host translational machinery.

UNLABELLED: Viruses require the host translational apparatus to synthesize viral proteins. Host stress response mechanisms that suppress translation, therefore, represent a significant obstacle that viruses must overcome. Here, we report a strategy whereby the mammalian orthoreoviruses compartmentalize the translational machinery within virus-induced inclusions known as viral factories (VF). VF are the sites of reovirus replication and assembly but were thought not to contain ribosomes. It was assumed viral mRNAs exited the VF to undergo translation by the cellular machinery, and proteins reentered the factory to participate in assembly. Here, we used ribopuromycylation to visualize active translation in infected cells. These studies revealed that active translation occurs within VF and that ribosomal subunits and proteins required for translation initiation, elongation, termination, and recycling localize to the factory. Interestingly, we observed components of the 43S preinitiation complex (PIC) concentrating primarily at factory margins, suggesting a spatial and/or dynamic organization of translation within the VF. Similarly, the viral single-stranded RNA binding protein σNS localized to the factory margins and had a tubulovesicular staining pattern that extended a short distance from the margins of the factories and colocalized with endoplasmic reticulum (ER) markers. Consistent with these colocalization studies, σNS was found to associate with both eukaryotic translation initiation factor 3 subunit A (eIF3A) and the ribosomal subunit pS6R. Together, these findings indicate that σNS functions to recruit 43S PIC machinery to the primary site of viral translation within the viral factory. Pathogen-mediated compartmentalization of the translational apparatus provides a novel mechanism by which viruses might avoid host translational suppression. IMPORTANCE: Viruses lack biosynthetic capabilities and depend upon the host for protein synthesis. This dependence requires viruses to evolve mechanisms to coerce the host translational machinery into synthesizing viral proteins in the face of ongoing cellular stress responses that suppress global protein synthesis. Reoviruses replicate and assemble within cytoplasmic inclusions called viral factories. However, synthesis of viral proteins was thought to occur in the cytosol. To identify the site(s) of viral translation, we undertook a microscopy-based approach using ribopuromycylation to detect active translation. Here, we report that active translation occurs within viral factories and that translational factors are compartmentalized within factories. Furthermore, we find that the reovirus nonstructural protein σNS associates with 43S preinitiation complexes at the factory margins, suggesting a role for σNS in translation. Together, virus-induced compartmentalization of the host translational machinery represents a strategy for viruses to spatiotemporally couple viral protein synthesis with viral replication and assembly.

Targeted H3R26 deimination specifically facilitates estrogen receptor binding by modifying nucleosome structure.

Transcription factor binding to DNA in vivo causes the recruitment of chromatin modifiers that can cause changes in chromatin structure, including the modification of histone tails. We previously showed that estrogen receptor (ER) target gene activation is facilitated by peptidylarginine deiminase 2 (PAD2)-catalyzed histone H3R26 deimination (H3R26Cit). Here we report that the genomic distributions of ER and H3R26Cit in breast cancer cells are strikingly coincident, linearly correlated, and observed as early as 2 minutes following estradiol treatment. The H3R26Cit profile is unlike that of previously described histone modifications and is characterized by sharp, narrow peaks. Paired-end MNase ChIP-seq indicates that the charge-neutral H3R26Cit modification facilitates ER binding to DNA by altering the fine structure of the nucleosome. Clinically, we find that PAD2 and H3R26Cit levels correlate with ER expression in breast tumors and that high PAD2 expression is associated with increased survival in ER+ breast cancer patients. These findings provide insight into how transcription factors gain access to nucleosomal DNA and implicate PAD2 as a novel therapeutic target for ER+ breast cancer.

A FluoPol-ABPP PAD2 high-throughput screen identifies the first calcium site inhibitor targeting the PADs.

The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 µM), to preferentially bind the apoenzyme.

Preparation of mammalian oocytes for transmission electron microscopy.

The visualization of subcellular organelles and structures is a valuable tool for understanding cellular changes that occur in oocytes and early embryos as a result of genetic alterations, incubation conditions, drug treatments, and many other manipulations. Preparing oocytes for transmission electron microscopic analysis can be challenging as these cells cannot be visualized without a microscope and they are more susceptible to mechanical disruption during manipulation. Here we describe methods for immobilizing oocytes on either a solid surface or within a matrix and then document our embedding techniques which work well for preserving the ultrastructure of the mouse oocyte.

Identification of PADI2 as a potential breast cancer biomarker and therapeutic target.

BACKGROUND: We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects. METHODS: RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes. RESULTS: We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67. CONCLUSION: Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

Potential role for PAD2 in gene regulation in breast cancer cells.

The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2-8-17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells.

D-amino acid based protein arginine deiminase inhibitors: Synthesis, pharmacokinetics, and in cellulo efficacy.

The protein arginine deiminases (PADs) are known to play a crucial role in the onset and progression of multiple inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and cancer. However, it is not known how each of the five PAD isozymes contributes to disease pathogenesis. As such, potent, selective, and bioavailable PAD inhibitors will be useful chemical probes to elucidate the specific roles of each isozyme. Since D-amino amino acids often possess enhanced in cellulo stability, and perhaps unique selectivities, we synthesized a series of D-amino acid analogs of our pan-PAD inhibitor Cl-amidine, hypothesizing that this change would provide inhibitors with enhanced pharmacokinetic properties. Herein, we demonstrate that d-Cl-amidine and d-o-F-amidine are potent and highly selective inhibitors of PAD1. The pharmacokinetic properties of d-Cl-amidine were moderately improved over those of l-Cl-amidine, and this compound exhibited similar cell killing in a PAD1 expressing, triple-negative MDA-MB-231 breast cancer cell line. These inhibitors represent an important step in our efforts to develop stable, bioavailable, and highly selective inhibitors for all of the PAD isozymes.

Subcellular localization of cytoplasmic lattice-associated proteins is dependent upon fixation and processing procedures.

We and others have recently demonstrated by immuno-EM and mutation analysis that two oocyte-restricted maternal effect genes, PADI6 and MATER, localize, in part, to the oocyte cytoplasmic lattices (CPLs). During these ongoing studies, however, we found that the localization of these factors by confocal immunofluorescence (IF) analysis can vary dramatically depending upon how the oocytes and embryos are processed, with the localization pattern sometimes appearing more uniformly cytoplasmic while at other times appearing to be primarily cortical. We set out to better understand this differential staining pattern by testing a range of IF protocol parameters, changing mainly time and temperature conditions of the primary antibody solution incubation, as well as fixation methods. We found by confocal IF whole mount analysis that PADI6 and MATER localization in germinal vesicle stage oocytes is mainly cytoplasmic when the oocytes are fixed and then incubated with primary antibodies at room temperature for 1 hour, while the localization of these factors is largely limited to the cortex when the oocytes are fixed and incubated in primary antibody at 4 °C overnight. We then probed sections of fixed/embedded ovaries and isolated two-cell embryos with specific antibodies and found that, under these conditions, PADI6 and MATER were again primarily cytoplasmically localized, although the staining for these factors is slightly more cortical at the two-cell stage. Taken together, our results suggest that the localization of CPL-associated proteins by confocal IF is particularly affected by processing conditions. Further, based on our current observations, it appears that PADI6 and MATER are primarily distributed throughout the cytoplasm as opposed to the oocyte subcortex.

Reovirus infection or ectopic expression of outer capsid protein micro1 induces apoptosis independently of the cellular proapoptotic proteins Bax and Bak.

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein µ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed µ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of µ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for µ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of µ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing µ1, indicating that caspases were not required. Additionally, µ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.

An ADAM9 mutation in canine cone-rod dystrophy 3 establishes homology with human cone-rod dystrophy 9.

PURPOSE: To identify the causative mutation in a canine cone-rod dystrophy (crd3) that segregates as an adult onset disorder in the Glen of Imaal Terrier breed of dog. METHODS: Glen of Imaal Terriers were ascertained for crd3 phenotype by clinical ophthalmoscopic examination, and in selected cases by electroretinography. Blood samples from affected cases and non-affected controls were collected and used, after DNA extraction, to undertake a genome-wide association study using Affymetrix Version 2 Canine single nucleotide polymorphism chips and 250K Sty Assay protocol. Positional candidate gene analysis was undertaken for genes identified within the peak-association signal region. Retinal morphology of selected crd3-affected dogs was evaluated by light and electron microscopy. RESULTS: A peak association signal exceeding genome-wide significance was identified on canine chromosome 16. Evaluation of genes in this region suggested A Disintegrin And Metalloprotease domain, family member 9 (ADAM9), identified concurrently elsewhere as the cause of human cone-rod dystrophy 9 (CORD9), as a strong positional candidate for canine crd3. Sequence analysis identified a large genomic deletion (over 20 kb) that removed exons 15 and 16 from the ADAM9 transcript, introduced a premature stop, and would remove critical domains from the encoded protein. Light and electron microscopy established that, as in ADAM9 knockout mice, the primary lesion in crd3 appears to be a failure of the apical microvilli of the retinal pigment epithelium to appropriately invest photoreceptor outer segments. By electroretinography, retinal function appears normal in very young crd3-affected dogs, but by 15 months of age, cone dysfunction is present. Subsequently, both rod and cone function degenerate. CONCLUSIONS: Identification of this ADAM9 deletion in crd3-affected dogs establishes this canine disease as orthologous to CORD9 in humans, and offers opportunities for further characterization of the disease process, and potential for genetic therapeutic intervention.

Potential role for MATER in cytoplasmic lattice formation in murine oocytes.

BACKGROUND: Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to, and is required for the formation of, abundant fibrillar Triton X-100 (Triton) insoluble structures termed the oocyte cytoplasmic lattices (CPLs). Given their similar expression profiles and mutant mouse phenotypes, we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function. METHODOLOGY/FINDINGS: Herein, we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction, suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally, the solubility of PADI6 was dramatically increased in Mater(tm/tm) oocytes following Triton extraction, suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater(+/+) and Mater(tm/tm) germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater(tm/tm) oocytes compared to Mater(+/+) oocytes. CONCLUSIONS: Taken together, these results suggest that, similar to PADI6, MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility, these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.