Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2β1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3–6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth. We found that CHAD plays roles in the cartilage development and maturation of the growth plate at young age and in the molecular composition of articular cartilage in adults as well as in bone homeostasis and function.Bone and cartilage are both made up of relatively few cells embedded in an abundant extracellular matrix (ECM). In cartilage, collagen fibrils and the negatively charged proteoglycan aggrecan, forming large aggregates with hyaluronic acid, constitute the major structural assemblies of the matrix. These two components provide tissue with tensile strength and resistance against compressive forces, respectively. The members of the small leucine rich repeat proteins (SLRPs) regulate assembly and function of the ECM, particularly the collagen networks, and include decorin, biglycan, asporin, fibromodulin, lumican, keratocan, PRELP (proline arginine-rich end leucine-rich repeat protein), osteoadherin (OSAD) and chondroadherin (CHAD) CHAD null mice showed normal embryological development and appeared healthy after birth. Macroscopically no phenotypic abnormalities were visible and the mice appeared healthy up to more than one year of age. Both female and male mice were fertile and the CHAD−/− breeding pairs did not differ in litter sizes compared to WT.In initial experiments a procedure for identification of CHAD−/− and WT mice was established by the use of PCR of tail samples prepared by routine procedures  with primers selected to give different products when CHAD was present or not. These products were distinguished by Agarose gel electrophoresis . WT miceHeart, lung including bronchial cartilage, kidney, liver and spleen showed no histopathological changes by systematic investigation at the light microscopic level of paraffin sections. Since chondroadherin is expressed in the eye, a more detailed study by semi thin epon sections was undertaken but showed no differences (data not shown).BMD, lean and fat content were measured in mice 6 weeks, and 3, 5 and 8 months of age. Results showed only very small differences in 6 week-old males lacking CHAD compared to controls. In this group the BMD/mg body weight was slightly lower , so was the fat content measured in the whole mouse . Apart from these differences, the DXA data did not reveal any abnormalities in CHAD-null mice (data not shown).Overall the CHAD−/− mice presented a 35% increase in mean height of the femoral epiphyseal growth plate at 3 weeks of age  mRNA was primarily detected in the proliferative chondrocytes of the epiphyseal growth plate, with no significant difference between the groups. Osteopontin (OPN) and BSP were both detected in hypertrophic chondrocytes, while there was no detectable difference. Immunostaining for COMP was almost exclusively localized to the epiphyseal growth plate, in addition to some cartilaginous remnants in trabecular areas. The staining was most intense in the territorial matrix along the columns of proliferative chondrocytes, although pericellular staining was observed to various degrees in all zones, and weaker staining was observed in the interterritorial matrix  . HistoloInitially there were 226 proteins identified by MASCOT 2.1 from 5457 peptide matches above homology or identity threshold. After filtering the data removing obvious false positives (13 proteins) and protein hits with only one peptide hit, it was possible to measure and calculate the relative ratios of 178 proteins detected in all extracts . The proScreening of mice aged 5 days, 3 week and 4 months showed that the length of the femora  increased with time but was not significantly different between CHAD−/− and WT mice .The 4 months old mice were analysed further. A number of parameters expectedly showed significantly higher values for the male wild type mice . ParticuQualitative electron microscopic analysis of 6 weeks old CHAD−/− mice showed no abnormalities in the structure, tissue organization and thickness of collagen fibrils in calvarial bone compared to WT mice (data not shown).Tartrate resistant acid phosphatase (TRAP) mRNA was primarily detected in metaphyseal osteoclasts; in addition, some resting and hypertrophic chondrocytes in the growth plate also expressed TRAP. Cathepsin K (CTK) mRNA was solely present in multinucleated cells in the metaphyseal region. OPN mRNA was detected primarily in osteoblasts lining the metaphyseal trabecular surfaces. As for OPN, BSP mRNA was highly expressed in osteoblasts lining trabeculae.The relative number of BSP mRNA expressing cells was significantly lower in the metaphysis of CHAD−/− mice compared with WT (p = 0.01) . Thus, tThe most intense accumulation of gold particles for both BSP  and OPN All mechanical properties increase significantly from 6 weeks to 4 months of age . FemoralCHAD deficient mice did not show gross anatomical defects, grew to normal size, were fertile, and had a normal life span up to 2 years of age, which is in line with other studies with SLRPs-null mice vis-à-vis collagen The CHAD −/− mice presented a widened epiphyseal growth plate. This was most pronounced in the proliferative zone at 3 and 6 weeks of age which fits well with data showing that CHAD is synthesized mainly by late proliferative chondrocytes Significant differences in trabecular/cortical parameters were apparent at the age of 4 months, where the null mice presented significantly higher BMD, lower cortical thickness, increased trabecular thickness, and increased structure model index (SMI) by micro-CT. SMI is a measure of the ratio of “plate-like” to “rod-like” trabecula within a trabecular bone specimen and higher density cancellous bone generally shows more “plate-like” trabecula. Interestingly, osteoporotic trabecular bone transits from plate-like to rod-like, increasing the SMI in vitro. BSP for the most part promotes the process Non collagenous proteins of the SIBLING  family , which includes OPN and BSP, are believed to play key biological roles in the development, turnover and mineralization of bone .A mouse genomic cosmid library was screened using a 887-bp CHAD rat cDNA fragment as described Approximately 20×106 semi-confluent R1 embryonic stem (ES) cells (kindly provided by professor Reinhard Fässler)  were electroporated with 80 µg linearized targeting vector. The ES cells were cultured on feeder cells in DMEM supplemented with fetal calf serum and leukemia inhibitor factor  mouse and a 13 kbp fragment in the targeted mouse. GGT CTT TCT TGC CA was used together with a 3′primer 5′ATG TCG TTG TGG GAC AGG TA. This detects a 320 bp fragment in the WT mouse. An additional primer corresponding to the sequence 5′CGC CTT CTT GAC GAG TTC TT in the neo-cassette was used to detect a fragment of 650 bp corresponding to homologous recombination in the knock-out.PCR was used to detect homologous recombination of the mouse CHAD gene. A 5′primer 5′CAG TCTBone mineral density (BMD), fat and lean content were examined with dual-energy X-ray absorption (DXA) using the Lunar PIXImus Densitometer . Measurements were performed on null and WT mice at the age of 6 weeks, and 3, 5, and 8 months of age . At each time-point at least 6 CHAD−/− and 6 WT animals were measured. The measurements were performed on anaesthetized living animals.Micro computed tomography (micro-CT) was performed as two experiments: first, femora of mice sacrificed at 5 days, 3 weeks and 4 months of age, respectively, were included as a general screening. In the second experiment, in depth analyses were performed in both sexes of 4 months old mice . All specimens were scanned by the use of high-resolution micro-CT . Dissected whole femora were affixed to the scanning stage and projection images were obtained at a resolution of 8.03 µm and reconstructed by use of manufacturer-provided software . After calibration of the standard unit of X-ray CT density , the conversion from HU to volumetric bone mineral density (vBMD) was done. Reconstructed images were analyzed by use of manufacturer-supplied software. Three sections as shown in At sacrifice the mice were subjected to macroscopic work up aiming at detection of malformation. Moreover, samples from heart muscle, kidney, spleen, liver and lung were fixed in formalin, paraffin-embedded, sectioned and stained with haematoxylin & eosin (H&E) according to a routine protocol. Intact eyes were fixed in 2% glutar aldehyde and embedded in an epoxy resin  and equatorial semi thin sections were stained with toluidine blue according to a routine protocol. Three to five coded sections per organ and animal were subjected to conventional light microscopy by an experienced surgical pathologist and evaluated for structural tissue changes. Six animals (CHAD−/− and WT) were investigated at each age, i.e. 3 and 6 weeks as well as 3 and 8 months.Femora from 3 and 6 weeks old animals were fixed in glutar aldehyde, decalcified in 7% EDTA for 15 days and embedded in an epoxy resin as above. Semi thin longitudinal sections of distal femoral metaphyses were cut and stained with toluidine blue, and histomorphometric analysis was performed on digital images (resolution 2576×1932 pixels) using image analysis software . Mean height of the epiphyseal growth plate was calculated for each section using the mean of 10 randomly placed lines for measurement. The relative zonal distribution of the resting, proliferative and hypertrophic zone was estimated by point counting.Femora for measurement of osteoid were fixed in 4% buffered formalin, embedded in a methyl methacrylate resin  without prior decalcification, sectioned and stained with Masson-Goldner's trichrome. Relative osteoid surface (% of trabecular surface) was estimated by point counting. For each animal, a minimum of 3 non-overlapping visual fields of vision were analyzed.in situ hybridization with riboprobes for OPN, CTK, BSP, TRAP and COMP. Gene sequences for TRAP, CTK, COMP, OPN and BSP were amplified by conventional PCR using cDNA from mouse osteoblasts  or IMAGE clones using the oligonucleotide primers listed in Five to six 6 weeks old animals from each group were subjected to Ten 6 week old animals from each group were included in the analysis. Immunohistochemistry of COMP was performed using the peroxide technique with diaminobenzidine (DAB) as the chromogen according to a routine protocol . Longitudinal sections of formalin-fixed femoral bone sections embedded in paraffin were used. Following permeabilisation by digestion with chondroitinase ABC  in tris/acetate buffer, the sections were incubated with rabbit polyclonal antiserum raised to rat COMP Tibias from 6 weeks old mice were immediately dissected free and fixed by immersion in a solution of 2% paraform aldehyde and 0.5% glutar aldehyde (GA). Subsequently, the tissue was embedded at low temperature in a freeze substitution device according to our established protocol Coded ultrathin sections from GA-fixed, epon-embedded samples of calvarial bone of 6 weeks old CHAD −/− and WT mice were subjected to electron microscopy of collagen fibrils. The fibrils were evaluated semi-quantitatively for thickness and spatial orientation. Sections from two blocks of each of 6 animals (3 CHAD−/− and 3 WT) were investigated and categorized as normal or pathological.Immunogold labelling with antibodies against BSP and OPN was performed as previously described Samples were obtained from G3 mice 4 months old. Full thickness femoral head cartilage was dissected from 8 female CHAD −/−, 7 female wild type, 7 male CHAD −/−, and 7 male wild type. The tissue from each group was pooled separately and homogeneous powder was made in liquid nitrogen. The samples were extracted with guanidine hydrochloride with added proteinase inhibitors  and extracts were collected after centrifugation (IEC Micromax) at 13200 rpm for 30 min.TM was then used to enable simultaneous analysis of a mix of all the four samples. Trypsin digests of the four pools of cartilage were separately labelled using standard protocols according to the manufacturer. The trypsin digested labelled extracts were combined and chromatographed on a SCX cation exchange column. The 29 fractions collected were separately applied to and analyzed using a reversed phase C18 nano-LC column online with a QTOF mass spectrometer as described .Procedures for quantitative proteomics were the same as those described for the analyses of a set of human cartilage tissues. Essentially, proteins in extracts were reduced and alkylated, followed by trypsin digestion . An isobaric 4-plex ITRAQThe mass spectrometric raw data was processed using Protein Lynx 2.1 with internal calibration. The processed files were searched with taxonomy mus musculus using MASCOT 2.1. The ratios of individual peptides between female CHAD −/− vs. wild type, male CHAD −/− vs. wild type, female CHAD−/− vs. male CHAD −/−, and female wild type vs. male wild type mice were calculated by MASCOT.iTRAQ quantification parameters: significant threshold p<0.05; weighted average ratios; minimum number of peptides of 2, minimum precursor charges of 2; at least homology of 0.05. The searched list was manually inspected for errors and a limited number of obviously incorrectly identified proteins were removed e.g. non-collagenous proteins identified with hydroxylation on proline residues.Cartilage from the femoral head, trachea and nose were dissected clean, cut into small pieces and extracted with guanidine-HCl (GuHCl) containing a proteinase inhibitor cocktail according to standard procedures in vivo. Using an Instron® 8511.20 biaxial load frame with an MTS® TestStar II controller, displacement was applied to the femoral head, using a flat ended indenter, at 0.1 mm s−1 until fracture occurred. Care was taken to ensure that the load was applied to the top of the femoral head, such that the loading indenter was not touching the greater trochanter. Fracture lead to a drop in the applied load. After removal from the mechanical test machine the specimen was checked visually to ensure that the fracture had occurred through the femoral neck, the data was rejected ff the fracture had occurred outside the femoral neck. The load at fracture of the femoral neck was registered. Tibias were used for cortical bone testing after dissection and removal of the fibula just proximal to its insertion into the tibia. The tibia was then placed on two supports of an 8 mm span three point loading rig. The bone was positioned so the supports were under the curve in the proximal tibia and at the distal tibia so that when the load was applied at the mid point between the supports it was through the point of insertion of the fibula. Loading was applied at 0.1 mm s−1. In preliminary tests it was found that this position was stable, reproducible and that the tibia did not rotate during the test. The stiffness was measured over the linear portion of the loading curve and the load at failure was recorded.Mice were sacrificed at 6 weeks, 4 or 8 months of age and frozen at −20°C. At the time of testing the mice were thawed, the femora and tibiae were dissected out and kept wet being wrapped in saline soaked tissue at 4°C overnight prior to testing. Two different mechanical tests were performed aimed at measuring femoral neck for the properties of cancellous bone and tibia for cortical bone. The strength of the cancellous bone of the femoral neck was measured using a miniaturised version of the mechanical test previously developed to test total hip replacements . The bone was gripped in a cylindrical holder, which was then held at 9° to the vertical, orientated so that the bone was vertical in the sagittal plane and in valgus in the frontal plane, thus similar to the position of the femur p-value of <0.05 was considered significant for all analyses.Morphological and micro-CT results are given as mean ± standard deviation (SD) and differences between CHAD−/− and WT animals were tested using a two-tailed independent Student's t-test. A multivariate analysis of variance (MANOVA) was used to compare immunogold data. For the latter, interest was focused on whether the overall distribution pattern for each of the two proteins differed between the groups. Thus, for a protein, only differences in overall comparison between the groups using MANOVA, and not difference in tests between subjects, were considered. A Figure S1Immunostaining for COMP in the epiphyseal growth plate at 6weeks of age. The epiphyseal growth plate (EGF) showed intense staining for COMP in both interterritorial and territorial matrix, although the staining did not differ by histologic scoring between CHAD−/− (3a) and wild type mice (3b). Non-immune control was negative (3c) (×10). Counterstained with H&E and phloxine B.(TIF)Click here for additional data file.Figure S2Ultrastructural protein distribution of BSP in bone at 6weeks of age. Sections incubated with anti-BSP showed distinct accumulation of gold particles over cement lines/mineralization fronts (arrows) in areas of mineralized bone but quantitative comparison revealed no differences in distribution pattern between the CHAD−/− (4a), wild type mice (4b) and non-immune control (4c) .(TIF)Click here for additional data file.Figure S3Positions of the 3 sections analyzed in the femur of 4months old mice by micro-CT.(TIF)Click here for additional data file.Figure S4Threshold levels in comparison to the original grey scale scan for the 3 different age groups in the screening micro-CT experiment.(TIF)Click here for additional data file.Table S1The proteins identified by MASCOT 2.1 from 5457 peptide matches above homology or identity threshold, and ratios in CHAD−/− (KO) versus wild type (WT) mice. Proteins identified by only one peptide were excluded. Proteins that changed ≥50% are underlined.(DOCX)Click here for additional data file.Table S2Micro-CT cortical/trabecular bone parameters at different ages. Bone length (femur), cortical/trabecular thickness (Ct.Th/Tb.Th), cortical volumetric BMD (Ct. vBMD), cortical/trabecular bone volume (Ct.BV/Tb.BV) cortical/trabecular porosity (Ct.P/Tb.P), trabecular separation (Tb.Sp), degree of anisotropy (DA) and structure model index (SMI) in proximal (P), middle (M) or distal (D) femur. * p<0.05, **p<0.01 between CHAD−/− and wild type (WT) mice in the age-group.(DOCX)Click here for additional data file.Table S3Oligonucleotide primer sequence for DIG-labeled cRNA probes.(DOCX)Click here for additional data file.


Carcinogenesis is thought to occur through a combination of mutational and epimutational events that disrupt key pathways regulating cellular growth and division. The DNA methylomes of cancer cells can exhibit two striking differences from normal cells; a global reduction of DNA methylation levels and the aberrant hypermethylation of some sequences, particularly CpG islands (CGIs). This aberrant hypermethylation is often invoked as a mechanism causing the transcriptional inactivation of tumour suppressor genes that directly drives the carcinogenic process. Here, we review our current understanding of this phenomenon, focusing on how global analysis of cancer methylomes indicates that most affected CGI genes are already silenced prior to aberrant hypermethylation during cancer development. We also discuss how genome-scale analyses of both normal and cancer cells have refined our understanding of the elusive mechanism(s) that may underpin aberrant CGI hypermethylation. It is widely accepted that carcinogenesis necessitates multiple genetic alterations that either drive cellular division or remove checkpoints regulating this process in normal cells. These same disruptions could potentially also be caused by epimutations. Epigenetic events have been strictly defined as heritable changes in gene function that are not explained by changes in DNA sequence but also more recently as ‘the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states’ . Here, wStrong support for the possibility that epimutations play a significant role in cancer comes from the recent discovery that epigenetic regulators are recurrently mutated in cancer genomes and observations that the levels and distributions of epigenetic marks are altered in cancer . ParticuTP53 (p53) mutations [de novo methyltransferases DNMT3A and DNMT3B [In normal mammalian somatic genomes, DNA methylation mainly occurs at cytosines in a CpG dinucleotide context . Around utations . Methylautations . During d DNMT3B . The maid DNMT3B , 11. Regd DNMT3B , 13. At d DNMT3B  and signd DNMT3B .Figure In cancer cells, genomic levels of DNA methylation are frequently reduced compared with their normal counterparts . The undHere, we review our current understanding of aberrant CGI hypermethylation as a paradigm of a potential epimutation in cancer. In particular, we focus on how recent data from the study of cancer methylomes demonstrates that the majority of genes that are aberrantly hypermethylated in cancer are in fact already repressed in preneoplastic cells. This parallels the view that most CGI hypermethylation in normal development occurs subsequent to silencing by other means. Based on these findings, we present alternative hypotheses as to the impact of aberrant CGI hypermethylation on the growth and development of cancers. Finally, we describe how the genome-scale analysis of cancer has advanced our understanding of the potential molecular mechanisms behind this epigenetic reprogramming.RB1 [MLH1 [BRCA1 [Evidence that aberrant CGI hypermethylation might act as an epimutation directly driving carcinogenesis is based primarily on studies of individual candidate genes. The aberrant hypermethylation of genes such as RB1 , MLH1 [3B1 [MLH1 , 32 and 1 [BRCA1 , whose m1 [BRCA1 , can be CDNK2A (p16/ARF) where the presence of a mutation in the first exon in a colon cancer cell line facilitated the direct demonstration that hypermethylation occurs only on the wild-type allele [BRCA1 have failed to observe instances of LOH through hypermethylation, suggesting that it might be a very rare event [First, hypermethylation of TSGs has been observed alongside inherited germline mutations . This sue allele . In the re event , 39.MLH1 mutations predispose to colorectal cancer and MLH1 hypermethylation is largely limited to colorectal tumours [BRCA1 mutations predispose specifically to breast and ovarian cancer and hypermethylation is limited to cancer of these tissues [RB1 mutated and hypermethylated retinoblastomas phenocopy each other [MLH1 have microsatellite instability (MSI) [VHL hypermethylation occurs in renal cancers of the clear cell histological subtype as do VHL mutations [MLH1 and MSH2 also confer a predisposition to developing MSI colorectal cancer as do mutations in these genes, although these apparent epimutations are tightly associated with genetic variants [BRCA1 mutations develop particular types of breast cancer, specifically estrogen receptor negative (ER−ve) tumours that are often classified as belonging to one of a few special histological types [BRCA1 hypermethylation was reported to be more frequent in medullary carcinomas which are ER−ve and often occur in BRCA1 carriers, but is also observed in mucinous carcinomas which are ER+ve [BRCA1 hypermethylation is not specific to ER−ve breast cancers [BRCA1 mutated and methylated cancers differ [BRCA1 hypermethylated serous ovarian adenocarcinomas also displayed a clinical course that was more similar to BRCA1 wild type than BRCA1 mutant tumours [CDNK2A predispose to melanoma and pancreatic tumours [CDNK2A mutations occur in a variety of other cancer types including non-small cell lung cancer [Second, the tissue specificity of TSG hypermethylation in sporadic cancer overlaps with the tissue-specific predispositions caused by inherited mutations in these same genes. Inherited  tumours . Similar tissues . Within ch other , colorecty (MSI)  and VHL utations . Rare cavariants , 43. Theal types . BRCA1 hre ER+ve , 45, 46. cancers  and thats differ . BRCA1 h tumours . It shou tumours  but somag cancer .VHL [MLH1 [BRCA1 [CDNK2A in a colorectal cancer cell line [The final strong piece of evidence underpinning the causative role of aberrant DNA hypermethylation in silencing tumour suppressor genes in cancer is that they can be reactivated when methylation is removed from their promoters. This is most often achieved by treatment with the drug 5′-aza-2′-deoxycytidine (5-Aza) which causes the degradation of DNMT1, the maintenance methyltransfrase . TreatmeVHL , MLH1 [3HL [MLH1 , 52 and 1 [BRCA1 . Also, gell line , 55. Theell line , but silell line , 58 and ell line , 60. TheCALCA , the first gene reported to become hypermethylated in cancer [The evidence for aberrant CGI hypermethylation as a direct silencer of TSGs is mostly correlative leading many to question its direct role in carcinogenesis . Althougn cancer . Early un cancer . The mosn cancer  and moren cancer . The fren cancer , but thin cancer , 73.RUNX3, which is frequently hypermethylated in gastric cancers, conclusively demonstrated that it is in fact never expressed in the cells that give rise to these tumours and supports the hypothesis that hypermethylation prone genes are fully repressed rather than expressed to a low level [In order for aberrant hypermethylation to directly drive cancer by silencing genes, the affected genes must be expressed prior to hypermethylation. Transcriptionally repressed genes have been known to undergo hypermethylation in tissue culture for many years . Recent ow level .de novo methyltransferases initiate X-inactivation normally [Dnmt1 does not preclude X-inactivation but instead results in sporadic reactivation of the Xi later in development [Meg3 (Gtl2) promoter in mouse development occurs prior to its hypermethylation [Oct-3/4 is also silenced before becoming hypermethylated during differentiation and functional studies implicate hypermethylation in stabilising its silencing [in vitro differentiation of mouse ES cells [These recent findings from the study of cancer methylomes draw parallels with our understanding of CGI hypermethylation during normal development which, most evidence suggests, occurs at genes already repressed through other mechanisms , 84. As normally  and geneelopment . Represshylation . The CGIilencing , 87. It ES cells  and sileES cells . A stabiES cells  and thatES cells . Taken tAPC in gastric cancer [Although the analysis of cancer methylomes demonstrates that the vast majority of genes whose CGI promoters become hypermethylated in cancer are repressed prior to hypermethylation, the possibility remains that occasionally active genes become hypermethylated and repressed . Such gec cancer . Clarific cancer , and carMLH1 is frequently hypermethylated in colorectal tumours with a CIMP phenotype [MLH1 becomes hypermethylated through the same mechanism affecting passenger genes. BRCA1 is repressed in sporadic breast cancer in the absence of hypermethylation [BRCA1 in cell lines can be initiated by the transcription factors SNAI1 and SNAI2  in partnership with the histone lysine demethylase LSD1 [CDKN2A which is frequently hypermethylated in a variety of tumour types [CDKN2A is normally only expressed in cells following replicative or oncogenic stress and it remains silent in most normal cells [CDKN2A is normally repressed by polycomb repressive complexes (PRCs) [DNMT1 and DNMT3B in a colon cancer cell line, CDKN2A is silenced before DNA hypermethylation is re-established [CDNK2A silencing also occurs prior to hypermethylation in colonies of human mammary epithelial cells (HMECs) that escape replicative arrest in culture [The strong phenotype of cancer predisposition genes which are also hypermethylated , 33 demohenotype  supportihylation  suggestiase LSD1 . An inteur types . CDKN2A al cells . This sus (PRCs) , 102 whiablished  and CDNK culture .MLH1 and MSH2 which are tightly associated with sequence variants [CDNK2A suggests that prior expression in normal tissue is not a perquisite for the hypermethylation of a gene playing a role in promoting carcinogenesis. Also, even in cases of driver genes where hypermethylation was not the initiating silencing event, its role in maintaining silencing might be important for the continued growth of the cancer. Potential epigenetic driver genes have been identified by screening for genes whose promoters remain aberrantly hypermethylated after genetic ablation of DNA methyltransferase activity in a hypomorphic colorectal cancer cell line [Taken together, these observations suggest that although some hypermethylated genes might be expressed in normal tissues, their hypermethylation could follow aberrant down-regulation by other means. Another possibility is that separate mechanisms result in the hypermethylation of active and repressed genes. A recent study demonstrated that the hypermethylation of initially active genes on the Xi requires additional factors compared with initially silent genes supporting the idea that alternative mechanisms might underpin the hypermethylation of different gene types . Non-codvariants , 43. Theell line . The ideWhile the potential that the hypermethylation of some genes is subject to positive selection because it facilitates carcinogenesis fits current data from methylome studies, a number of questions remain. One prediction of the driver and passenger model is that tumours with methylator phenotypes would demonstrate more aggressive clinical behaviour because they would be statistically more likely to have hypermethylated more TSGs or drivers. Methylator phenotypes in colorectal tumours, breast tumours and glioblastomas, however, all coincide with better clinical prognoses , 71, 107Rather than playing an evolutionary neutral passenger role and representing a surrogate of general epigenetic dysregulation, the widespread hypermethylation of normally repressed genes in cancer could have other impacts on carcinogenesis and progression. As discussed above, hypermethylation of CGIs in normal development results in stable gene silencing and prevents ectopic activation . It is cIDH1 R132H mutations correlate with a hypermethylator phenotype in acute myeloid leukaemia (AML) [Many groups have noted that genes repressed by PRCs in embryonic stem (ES) cells are frequently hypermethylated in cancer . Other sia (AML)  and knocia (AML) . These mia (AML) , 118 andia (AML) , 71, 107Instead of maintaining a stem-cell-like state, a restriction of epigenetic plasticity induced by widespread hypermethylation could act as a check on cancer progression B. The diCancers are, however, very heterogenous with respect to their genetic and epigenetic profile and the environment in which they grow; the two hypotheses outlined above may, therefore, reflect differing roles of widespread aberrant hypermethylation in different cancer types or subtypes. They both may even play a part in the same cancer at different stages of progression. For example, a hypermethylation-mediated block on differentiation may promote the initial growth of a tumour but later this restricted epigenetic landscape might prevent its metastasis. Furthermore, neither of these hypotheses is incompatible with the occasional hypermethylation of driver genes outlined above. Differentiating these possibilities requires an understanding of the mechanism responsible for aberrant CGI hypermethylation.de novo methylation.Although the mechanism(s) responsible for aberrant promoter hypermethylation in cancer remains elusive, potential hypotheses have emerged from genome-scale analyses of both normal and cancerous cells. Two main types of mechanisms have been proposed; active processes mediated by targeting of specific factors to CGIs or passive mechanisms resulting from a loss of protection against min/+Apc mouse model of colorectal cancer demonstrate that higher DNMT levels promote carcinogenesis [Dnmt3b over-expression in this model is also associated with promoter hypermethylation of the murine homologues of genes hypermethylated in human colorectal tumours [DNMT3A mutations occur in AML and other haematological cancer genomes [DNMT3B mutations in cancer have not currently been found but aberrant splicing of DNMT3B frequently gives rise to cancer-specific isoforms of the enzyme [Mbd2 and Kaiso appear have contributary roles in intenstinal tumorogenesis as their deletion in mice result in reduced tumour numbers in min/+Apc mice [One hypothesis is that aberrant CGI hypermethylation in cancer results from the over-expression or increased activity of DNMTs. Such increases were initially reported , 125 butogenesis . Dnmt3b  tumours . On the  genomes , 134 and genomes . They ha genomes , 136. DNe enzyme . These ie enzyme , 139. Aln/+ mice , 141. AsAs noted above, genes marked by PRCs in ES cells are frequently hypermethylated in cancer. DNMT3A and 3B biochemically interact with EZH2, a member of the PRC2 complex , leadingCfp1 knockout in mouse ES cells does not result in CGI hypermethylation [Histones and their associated marks may play other roles in protecting CGIs from hypermethylation. In normal cells, the histone mark H3K4me3 is anti-correlated with DNA methylation . H3K4me3hylation . The varhylation , fish [1hylation  and humahylation . Mutatiohylation  but the IDH1 or 2 mutations and one of its effects is the inhibition of TET enzyme activity [IDH mutations in AML correlate with a similar hypermethylator phenotype to TET2 mutations [IDH1 mutations [Tet1 knockdown in mouse ES cells led to CGI hypermethylation [The analysis of cancer methylomes has also linked dysfunction in DNA demethylation pathways to aberrant CGI hypermethylation. DNA demethylation is proposed to be initiated by the conversion of mC to hmC by the ten-eleven translocation (TET) family of enzymes , 159. Teactivity . IDH mututations  and CIMPutations . Althoughylation , the samhylation . As TET1vice versa [de novo methylation by transcription during the developmental window in which the genome is remethylated [RIL gene confers resistance to hypermethylation [MLH1 promoter that reduce promoter activity are associated with soma-wide mosaic hypermethylation [Epigenomic analyses of normal cells point towards DNA sequence determining genome methylation patterns through sequence-specific transcription factors (TFs) rather than ce versa . DNMT3A ce versa  and abnoce versa . Althougce versa , 169. Asce versa , 170 whice versa . In addithylated . An analthylated . Variatihylation  and sequhylation . It is uhylation , 174.One of the main results to emerge from the systematic analysis of cancer methylomes is the finding that cancer-associated hypermethylation does not occur at random but affects a distinct set of genes. As noted above, several groups have documented the overlap between PRC-marked genes in ES cells and hypermethylation in cancer and this has been reproduced extensively in genome profiling studies , 175. RaTaken together, these studies suggest that many factors could be involved in the reprogramming of repressed CGIs to a hypermethylated state in cancer. It is clear that a number of different factors found at active CGI promoters are capable of maintaining them in a hypomethylated state including H3K4me3, H2A.Z, TFs, TET enzymes and active RNA transcription A. RepresThe recent characterisation of cancer methylomes has demonstrated, contrary to the prevailing view, that the hypermethylation of CGI promoters in cancer parallels CGI hypermethylation during normal development and is secondary to silencing by other means. For most aberrantly hypermethylated promoters, this silencing occurs as a result of normal development and subsequent methylation represents an epigenetic reprogramming event. Potential driver genes that are expressed in normal cells could conceivably be directly silenced by aberrant hypermethylation. The overwhelming tendency for hypermethylation to occur as a secondary event, however, strongly suggests that such drivers are subject to primary aberrant silencing in cancer through other means. Hypermethylation of isolated individual repressed genes may also contribute a growth advantage to cancer by preventing their activation at later stages of carcinogenesis or progression. We should also consider whether the widespread hypermethylation of CGIs in cancer has other impacts on the growth of cancers, in particular by blocking differentiation or restricting epigenetic plasticity and adaptive potential.The analysis of cancer genomes and methylomes have helped refine our definition of the type of gene affected by aberrant promoter hypermethylation and generated new hypotheses as to the molecular defect underpinning this epigenetic reprogramming but many questions remain to be answered. Dissection of this mechanism is also likely to lead to new insights regarding the biology of CGIs, the most abundant promoter type in our genome . The reiCXCR4, which has a CGI promoter, was shown to facilitate metastasis in a renal cancer cell line [As this review was going to press, de-repression of ell line . This fuCpG islands (CGIs) frequently become aberrantly hypermethylated in cancers.Analysis of cancer methylomes has shown that aberrant CGI hypermethylation occurs primarily at genes that are already silent in the host tissue and is therefore not generally linked to transcriptional silencing of tumour suppressor genes.The predominant view is that CGI hypermethylation in normal development is also secondary to prior silencing through other mechanisms.Several hypotheses now exist as to the impact of CGI hypermethylation on carcinogenesis and progression. The occasional hypermethylation of rare driver genes might directly promote carcinogenesis. Widespread CGI hypermethylation could also result in more aggressive cancers by blocking cellular differentiation or act as a protective mechanism hindering progression by preventing epigenetic adaptation to changing conditions.The mechanism underpinning aberrant CpG island hypermethylation remains elusive but genome-scale studies have refined our hypotheses and demonstrated that a distinct gene set is affected.Medical Research Council, the Biotechnology and Biological Sciences Research Council and by the Innovative Medicine Initiative Joint Undertaking (IMI JU) under grant agreement number 115 001 (MARCAR project). D.S. is supported by the Medical Research Council, Breakthrough Breast Cancer and Breast Cancer Campaign.Work in R.M.’s lab is supported by the


PTEN pseudogene, PTENP1. Our analysis of the ENCODE project data also found many transcriptionally active pseudogenes in the GM12878 and K562 cell lines; moreover, it showed that many human pseudogenes produced small RNAs (sRNAs) and some pseudogene-derived sRNAs, especially those from antisense strands, exhibited evidence of interfering with gene expression. Further integrated analysis of transcriptomics and epigenomics data, however, demonstrated that trimethylation of histone 3 at lysine 9 (H3K9me3), a posttranslational modification typically associated with gene repression and heterochromatin, was enriched at many transcribed pseudogenes in a transcription-level dependent manner in the two cell lines. The H3K9me3 enrichment was more prominent in pseudogenes that produced sRNAs at pseudogene loci and their adjacent regions, an observation further supported by the co-enrichment of SETDB1 (a H3K9 methyltransferase), suggesting that pseudogene sRNAs may have a role in regional chromatin repression. Taken together, our comprehensive and systematic characterization of pseudogene transcription uncovers a complex picture of how pseudogene ncRNAs could influence gene and pseudogene expression, at both epigenetic and post-transcriptional levels.Thousands of pseudogenes exist in the human genome and many are transcribed, but their functional potential remains elusive and understudied. To explore these issues systematically, we first developed a computational pipeline to identify transcribed pseudogenes from RNA-Seq data. Applying the pipeline to datasets from 16 distinct normal human tissues identified ∼3,000 pseudogenes that could produce non-coding RNAs in a manner of low abundance but high tissue specificity under normal physiological conditions. Cross-tissue comparison revealed that the transcriptional profiles of pseudogenes and their parent genes showed mostly positive correlations, suggesting that pseudogene transcription could have a positive effect on the expression of their parent genes, perhaps by functioning as competing endogenous RNAs (ceRNAs), as previously suggested and demonstrated with the  Pseudogenes are genomic sequences with high sequence similarity to functional genes but have been presumed to be “non-functional” via their non-coding RNA (ncRNA) products, may play regulatory roles in modulating the expression of their parental genes, as well as non-parental genes cis-effect) has not yet been investigated, although trans-effects have been suggested. For example, the Oct4 pseudogene ncRNA was shown to direct epigenetic remodeling complexes to the Oct4 parent gene Thousands of pseudogenes are found in the human genome; some of them have been suggested to have critical regulatory functions PTENP1, a pseudogene derived from the tumor suppressor gene PTEN, was first shown to act as a competitive decoy for several miRNAs that target PTEN mRNA, thus stabilize expression of its parental PTEN gene PTENP1 and their role in regulating PTENPseudogene transcripts functioning by other mechanisms have also been reported In this study, we have surveyed the landscape of pseudogene transcription across a large number of human tissues and cell lines and begun to explore potential functional and cellular activities of pseudogene ncRNAs from several perspectives. We found that a few thousand human pseudogenes were transcribed and their transcription was overall correlated with increased expression levels and expression diversity of their parental genes. Some pseudogenes, on the other hand, displayed evidence of siRNA production and function, potentially by either interfering with parental gene expression or mediating local epigenetic silencing. Taken together, our results suggest that pseudogene transcription is likely an important process that has provided novel ncRNA elements for modulating the transcriptional fluctuation of protein-coding genes.A major challenge in detecting transcribed pseudogenes is how to map RNA-Seq reads back to their genuine origins when both pseudogenes and their parents are candidates because of their high sequence similarity. The lack of introns may even make a processed pseudogene the preferred candidate for reads originating from exon-exon junctions of the parent. To address these issues directly, we have designed a new computational method to filter out RNA-Seq signals that are likely to have originated from coding genes but can be mapped to pseudogenes due to ambiguity , bottom,To determine pseudogene transcription systematically, we first applied our method to analyze RNA-Seq data from 16 normal human tissues in the Illumina Human Body Map 2 Project, and then to the data from GM12878 and K562 cell lines (see below) in the ENCODE project p<2.2e-16, Wilcoxon test) . A hierarchical clustering analysis showed that a subset of pseudogenes was nearly exclusively transcribed in testis . White bon test) . LincRNAon test) . The tison test) .parents that had a JS score >0.1 for all studies described below unless mentioned otherwise, which effectively excluded nearly all (n = 745) pseudogenes derived from ribosomal protein genes.To determine to what extent the high JS scores for pseudogenes could be explained by their low transcription, we computed JS scores for randomly selected coding genes with maximal FPKMs matched to those of pseudogenes. We found that the JS scores of processed pseudogenes remained lower than their expression-matching coding genes , but for duplicated pseudogenes the difference was more significant at high expression level , suggestIn summary, the above results for the extent of pseudogene transcription and their tissue expression pattern are consistent with previous reports p<0.02 and <2.2e-16 for pseudogenes and genes, respectively; data not shown). The difference remained statistically significant  when the JS score cutoff was set to 0.25 or 0.35. In addition, changing the promoter definition slightly from ±2 kb to ±5 kb or ±10 kb produced similar statistics . Since the data capture a mixture of TF events in 91 cell types, this result suggests that pseudogenes transcribed more broadly contain more potential regulatory sites, but the functional importance of this observation needs further investigation.To explore transcriptional regulation potentially contributing to tissue specificity, we examined the number of transcription factor (TF) binding events in the promoters (−2 kb to transcription start sites (TSS)) of pseudogenes and protein coding genes, using the integrated ChIP-Seq data from the ENCODE project pg:g). The resulting correlation coefficients for both processed and duplicated pseudogenes showed a distribution that was deviated from the theoretical normal distributions  test) and biased towards positive numbers . Since some pseudogenes are close to their parents on chromosomes  and adjacent genes tend to be co-regulated pg:g values for the t2 group remained skewed to positive for both processed and duplicated pseudogenes . In summary, our results indicate that pseudogene transcription is positively correlated with the expression of their parents.The evidence of pervasive pseudogene transcription is compelling, but more important questions are what kinds of biological functions pseudogene ncRNAs can have. Note that the term “biological function” in this report is used in a loose sense, whereas “biochemical activity” may arguably be more appropriate, in accordance with the source of our experimental data and the computational nature of our work. The first obvious question is how pseudogene and parent gene transcription are related, as this information may shed light on how pseudogenes could regulate their most conceivable targets. To this end, we computed the Spearman rank correlation of the 16 tissue transcription levels for each of the 1,270 pseudogene-parent pairs .Although our observation is based on correlation, it is consistent with the ceRNA hypothesis pg:g values than those producing antisense ncRNAs , while duplicated pseudogenes followed the same trends  to each of the miRNAs that it can putatively bind. Likewise, we calculated ρmiRNA:pg. This produced three-way pairwise correlations. To plot the data, we binned genes to groups based on ρmiRNA:g numbers  and ρmiRNA:g  . This pattern implies that the miRNA sponge effect of a pseudogene ncRNA is potentially more significant, manifested as a large and positive ρpg:g, if the shared miRNA shows a larger inhibition to the parent, indicated by a small and negative ρmiRNA:g, further supporting the idea that a miRNA sponge effect could partially contribute to the observed transcriptional correlation between pseudogenes and their parents, especially in the cases when miRNA regulatory effects were large  from a previous study  numbers , x-axis in vivo miRNA target sites in HEK293 cell line as determined by AGO and TNRC6 occupancy using the PAR-CLIP technology PTENP1, and other candidates in To seek additional experimental support, we examined a total of 19,184 high confident pg:g, both the means and variances of parental gene expression were greater in the tissues where pseudogene ncRNA levels were higher , but pseudogene transcripts can also be used to produce small RNAs, which can potentially execute a variety of functions via dsRNA intermediates formed either between pseudogene ncRNAs and parental mRNAs, or in the hairpin loops of pseudogene ncRNAs, whereas “group II” pseudogenes probably can generate sRNAs independent of their parents in a manner similar to repeats and transposons located at heterochromatin regions (p<2.2e-16). We hypothesized that the parental genes of group I pseudogenes would be expressed at lower levels than the parents of both group II pseudogenes and the pseudogenes that did not produce sRNAs . This trend persisted if the threshold was changed to >10 sRNA per kb . Two subsets of such pseudogenes were selected for comparison, based on whether sRNAs were also detected in the flanking regions , with th regions . InteresA per kb , althougA per kb . In TablFor the small RNAs generated from pseudogenes independently of their parents . Furthermore, the extent of H3K9me3 within 15 kb of the transcription start sites showed a clear enrichment that was dependent on pseudogene transcription levels . We hypothesize that these results suggest pseudogene-derived sRNAs can play an active role in the establishment of broad but local silencing chromatin environment for repressing pseudogene transcription, a phenomenon that has been documented in yeast and plants In order to address whether H3K9me3 enrichment was related to pseudogene-derived sRNAs, we compared the H3K9me3 levels between the two groups of sRNA-producing pseudogenes . As showOur findings suggest that many human pseudogenes are transcribed and the transcripts exhibit evidence for various biological activities. It is possible that the human transcriptome and its regulation are sufficiently robust to tolerate the small perturbation introduced by pseudogene transcription. If so, transcribed pseudogenes would not show significantly different evolutionary constraints compared with non-transcribed ones. Therefore, we analyzed nucleotide diversity (based on two population datasets from the HapMap project) and cross-species conservation (based on 46 way phastcon scores) within pseudogenes. The data show that transcribed pseudogenes exhibit significantly higher evolutionary constraints than non-transcribed ones, as suggested by the lower degree of polymorphism and greater phastcon scores  of human pseudogenes found to be transcriptionally active in the current study is consistent with previous estimates  parents . Neverthbona fide pseudogene-originating reads could be removed. Although further detailed assessment is needed when new computational algorithms or experimental technologies for better quantifying pseudogene expression become available, we believe these factors have not introduced significant bias to our results. For instance, we examined the expression levels of 161 pairs of pseudogenes, in which each pair were derived from the same parental gene but they had distinct identities to the parent. These pseudogene pairs exhibited no expression difference in all the 16 tissues except two , with larger FPKMs for the pseudogenes that were more similar to their parents. We also found that pseudogenes with higher sequence similarity to their parents had lower JS scores and greater ρpg:g values, indicating that young pseudogenes are more widely transcribed than the old ones, perhaps due to less decay of their promoters.We should point out that pseudogenes with sequences identical to their parents would not have any mapped RNA-Seq reads and thus would be treated as non-transcribed by our current approach, even though they may well be transcribed in the tissue samples. Another caveat of our approach is potential underestimation of expression levels for certain pseudogenes, because some Xist modulating X chromosome inactivation Our study has systematically explored the potential functional activities of transcribed pseudogenes from several perspectives. Our finding of transcription-dependent H3K9me3 enrichment in some pseudogenes suggests that pseudogene-derived sRNAs may play a role in modulating epigenetic repression of pseudogene transcription, probably by the same molecular mechanism(s) underlying sRNA-mediated heterochromatin formation vs 86% .We should point out that high levels of H3K9me3 have also been observed in transcriptionally active genes, particularly at the 3′ exons of zinc finger genes ψPPM1KIn addition to silencing pseudogenes, pseudogene-derived ncRNAs could be a good source of endogenous siRNAs that interfere with the expression of protein-coding genes. We uncovered evidence for this , but thiWe were unable to address whether human pseudogene-derived sRNAs could function as siRNAs to interfere with parental gene expressions or to mediate epigenetic silencing under normal physiological conditions, since the necessary sRNA-Seq and histone modification ChIP-Seq data had not been available for all the 16 tissues in our current study. It will be interesting to revisit this critical issue when the relevant data become available for all or a subset of these tissues, so that data from the same tissues can be studied in order to reduce biological variations.PTENP1, whose transcripts have been shown by an extensive array of genetic and biochemical experiments to compete with its parental gene PTEN for several miRNAs Our study indicates that the predominant effect of pseudogene transcription, however, appears to be related to the increase in the expression levels and diversity of the parental coding genes. Approximately 64% of the ∼4,000 transcribed pseudogenes (FPKM >1) exhibited a transcriptional profile that was positively correlated with that of their parental genes. Furthermore, pseudogenes with higher correlation were found to have more predicted miRNA-targeting sites. More importantly, the expression of parental genes was significantly higher and more variable in tissue where pseudogene ncRNAs are more abundant. All of these observations are consistent with previous reports suggesting that cellular RNAs could serve as miRNA sponges (or “target mimicry”) and regulate the stability of other transcripts In summary, consistent with previous work, our study demonstrates that pseudogene transcription is genuine and prevalent; its impact on other cellular RNAs appears complicated and diverse and in some cases one pseudogene may play multiple molecular roles. Whether these result passively from transcriptional leakage, incomplete chromatin silencing, or represent an active process of nurturing novel ncRNAs certainly will require a more systematic investigation and experimental verification. Our results, on the other hand, suggest that pseudogenes with major regulatory roles are unlikely ubiquitous, since under normal physiological conditions most of the pseudogene ncRNAs are present at much (∼20x) lower levels than their coding counterparts and the majority of them did not display evidence of strong interaction with their parents. Nevertheless, perturbation of pseudogene transcription can affect the homeostasis of gene expression and lead to human diseases and cancers as previously reported.http://www.ebi.ac.uk/arrayexpress), and transcriptomic and epigenomic data from the ENCODE project (http://genome.ucsc.edu/ENCODE/) Our primary datasets are RNA-Seq data from the Illumina Body Map 2.0 Project  and then collected reads mapped to pseudogene loci. These reads were then compared to cDNA sequences of human protein coding genes, extracted from the Ensembl database , using the program Bowtie (version 0.12.7) vs sequence identities using a 200-bp window sliding across the alignment. Noted that all pseudogenes with an annotated parent  were included in this analysis. According to pseudogene identification strategies, the aligned regions are essentially the “exons” of pseudogenes. A moderate correlation between read numbers and identities  was detected before the application of our filtering process, but none existed after filtering   The read filtering step for computing FPKMs from RNA-seq data was not applied to either the ChIP-seq or sRNA-seq alignment data since the alignments of those reads were not be affected by exon-exon junction and a single and best-matched location was kept for each of the ChIP-seq reads.JS (Jensen-Shannon) divergence has been found to be a good metric for quantifying the tissue specificity of a transcript pg:g). As a control, we computed ρ by pairing up each pseudogene with a randomly chosen coding gene. Exclusion from this analysis were the pseudogenes without parent information from GENCODE or whose parents had 0 FPKMs in all the 16 tissues. The same method was applied to compute the correlation between miRNAs and their putative targets.We computed Spearman correlation coefficient (ρ) of the 16 tissue FPKMs to determine the relationship between the transcription profiles of a pseudogene and its parent (ρStrand-specific RNA-Seq data were obtained from a previous study http://www.targetscan.org) We downloaded data of miRNA sequence families and target prediction tools (TargetScan V5.0) from the TargetScan web site  were considered as candidates that could produce small RNAs. A subset of these candidates were defined as group I or II sRNA-generating pseudogenes if the sRNA read densities at their flanking 1 kb regions were  = 0 or >5, respectively ; the resvs non-transcribed pseudogenes, we evaluate the difference by considering the number of mapped ChIP-Seq reads at ±2.5kb of pseudogene TSS (In the comparison of transcribed gene TSS . The sighttp://www.hapmap.org) i-th and j-th individual sequences from YRI population, i-th and j-th individual sequences in each pseudogene locus Sequence conservation was determined by the Phastcon scores downloaded from the UCSC genome browser; the scores were derived from sequence comparison of 46 species including primates, mammals and vertebrates Figure S1Most pseudogenes share <90% sequence similarity with their parents and our method for filtering RNA-Seq reads does not affect the quantification of parental gene expression. Left, histogram of human pseudogene distribution shows the number of pseudogenes (y-axis) at different levels of sequence identity to the parental genes (x-axis). Right, the FPKM values for the parental genes is not affected by our method of filtering and remapping of RNA-Seq reads. Data shown is for brain sample, but results from other tissues yielded the same pattern.(PDF)Click here for additional data file.Figure S2Tissue specificity of pseudogene transcription. A) Three examples of tissue-restrictively transcribed pseudogenes. B) Distribution of the JS scores computed with all RNA-Seq reads Click here for additional data file.Figure S3(A–B) QQ-plot analysis of transcriptional correlation coefficients. The ρpg:g values (y-axis) for transcribed duplicated pseudogenes (A) and processed pseudogenes (B) were significantly deviated from the ρpg:g values (x-axis) calculated for pairs of each transcribed pseudogenes with a randomly chosen coding genes. C) Distinct effect on parental gene expression between sense and antisense pseudogene ncRNAs. The parents of the antisense transcribed pseudogenes  exhibited significantly lower expression than those of sense transcribed pseudogenes  in all the 16 tissues .(PDF)Click here for additional data file.Figure S4Increased levels and variations of parental gene expression in relation to pseudogene transcription. QQ-plot analysis shows that both differences in mean (A) and variance (B) of the parental gene expression between tissues of high  and low  pseudogene transcription were significantly deviated from the normal distribution; the Kolmogorov-Smirnov (KS) statistics are shown at top. The means (C) and variances (D) of the expression of the parent genes (x-axis) across all 16 human tissues also increased as the transcription levels of pseudogenes increased. Color lines plot the distributions of parental genes with pseudogenes transcribed at different levels, defined by the maximal FPKMs among the 16 tissues.(PDF)Click here for additional data file.Figure S5Size distribution of small RNAs from the group I and II pseudogenes. The data were derived from the sRNA reads that were perfectly matched to pseudogene sequences without any gap. Data from GM12878 and K562 are plotted in  and , respectively.(PDF)Click here for additional data file.Figure S6Enrichment of active histone modifications and depletion of repressive histone modifications at transcribed pseudogene loci. Comparison of eight histone modifications between transcribed (‘y’) and non-transcribed (‘n’) pseudogenes was shown by boxplot analysis. The y-axis shows numbers of ChIP-Seq reads mapped to +/− 2.5 kb to TSS in GM12878 (A) and K562 (B) cell lines. C). The average densities of three types of repeats at pseudogenes transcribed in GM12878 (FPKM>1) in 500-bp bin windows, with no enrichment observed at pseudogene loci when compared to adjacent genomic regions.(PDF)Click here for additional data file.Figure S7Correlation of the nucleotide diversities computed from two distinct human populations. For every pseudogene, we determined its nucleotide diversities in the YRI or CEU populations and the results show a high correlation between the data derived from these two populations, indicating the reduction in diversity is not due to a few genes that recently become pseudogenes in human. A), duplicated pseudogenes; B), processed pseudogenes.(PDF)Click here for additional data file.Table S1List of transcribed pseudogenes and their associated features and groups.(XLSX)Click here for additional data file.


APP and/or PRNP homologs in the zebrafish  produces a dose-dependent phenotype characterized by systemic morphological defects, reduced cell adhesion and CNS cell death. This genetic interaction is surprisingly exclusive in that prp1 genetically interacts with zebrafish appa, but not with appb, and the zebrafish paralog prp2 fails to interact with appa. Intriguingly, appa & appb are largely redundant in early zebrafish development yet their abilities to rescue CNS cell death are differentially contingent on prp1 abundance. Delivery of human APP or mouse Prnp mRNAs rescue the phenotypes observed in app-prp-depleted zebrafish, highlighting the conserved nature of this interaction. Immunoprecipitation revealed that human APP and PrPC proteins can have a physical interaction. Our study reports a unique in vivo interdependence between APP and PRP loss-of-function, detailing a biochemical interaction that considerably expands the hypothesized roles of PRP in Alzheimer Disease.Genetic and biochemical mechanisms linking onset or progression of Alzheimer Disease and prion diseases have been lacking and/or controversial, and their etiologies are often considered independent. Here we document a novel, conserved and specific genetic interaction between the proteins that underlie these diseases, amyloid-β precursor protein and prion protein, APP and PRP, respectively. Knockdown of  C) is a conserved GPI-anchored membrane protein that, when misfolded into an aberrant conformation (PrPSc), is able to recruit and template the misfolding of normal PrPC. This initiates the pathological events in Creutzfeldt-Jakob disease, scrapie, and bovine spongiform encephalopathy Amyloid-β precursor protein (APP) is a highly conserved type Ι transmembrane protein that liberates Aβ peptides into the extracellular space when it is sequentially cleaved by β- and γ-secretases C and oligomerized Aβ C suggested by interactome studies APP and PRNP may be in a common genetic pathway. We examined the functional significance and conservation of this hypothetical interaction via concerted knockdown of these proteins in zebrafish.Intense interest has recently focused on a high-affinity interaction between PrPDanio rerio, has two paralogs of APP and two paralogs of PRNP. Zebrafish paralogs both show ∼70% predicted identity to human APP, increasing to ∼90–100% identity in the regions that encode the amyloidogenic, transmembrane and intracellular domains appa and appb are homologs of mammalian APP, and are less similar to mammalian APP-like proteins . The two zebrafish paralogs of PRNP are not similar to mammalian homologs at the level of amino acid identity, but share a substantial conservation of protein domain architecture APP or PRNP to replace zebrafish homologues argues strongly for an impressive conservation of function  designed to bind the exon-intron boundary between exon 2 and intron 2–3. For appb the site chosen was the exon-intron boundary between exon 3 and intron 3–4. In both cases these targeted sites are upstream of highly conserved regions of the genes that are present in all splice isoforms. Delivery of appa or appb MO at high (effective) doses resulted in fish displaying a neurodevelopmental phenotype comprised of overt physiological malformations; cranial edema, reduced body size, improper CNS development and structure, and the presence of CNS cell death evident as dark necrotic-like regions disrupting transmission of light through the otherwise transparent embryo (t embryo .efficacy was confirmed by multiple methods: (i) RT-PCR demonstrated altered mRNA splicing in the presence of MO, producing PCR products of a size that indicated retention of the expected MO-adjacent intron (ii) Sequencing of these RT-PCR products demonstrated that multiple termination codons are present in the altered transcript, predicting a truncated protein (iii) There was a dose-response relationship between the amount of injected MO and appearance of the phenotypes (iv) by quantifying protein knockdown from three biological replicates, representing delivery of all MO reagents on three separate days (Morpholino  protein . (iii) Tenotypes , S2, andate days , S3. Proate days .specificity was confirmed by (i) multiple trials quantifying the significant rescue of MO-induced phenotypes with MO-insensitive mRNA from the cognate gene  Failure to rescue the MO-induced phenotypes when these same mRNAs had termination codons engineered into them (iii) Failure to rescue phenotypes when the mRNA from related genes were delivered; (iv) Delivery of a second MO reagent designed to block appa translation by binding to a disparate portion of the gene, the 5′UTR, produced the same phenotypes (appa translation blocking (TB) MO in reducing protein abundance). The quantity of MO and/or mRNA reagent delivered was kept consistent between trials via the calibration of injection volumes using an ocular micrometer. Overall these tests of reagent efficacy and specificity represent the quantification of phenotypes in several thousand individual fish involved in more than 100 separate trials  began to show signs of apoptotic cell death   . When thhenotype , Table 1henotype . The quace versa . We concC proteins may physically interact APP and PRNP genes. When sub-effective doses of the appa (0.5 ng) and prp1 (0.5 ng) MOs were injected alone many fish displayed slight morphological malformations  and prior reports in the literature indicating that murine APP and PrP p<0.01, . This ovnslation –K.appa and appb demonstrated above during normal development  were combined there was no significant change in the percentage of fish displaying CNS malformations  an idiosyncrasy of our MO reagents affecting different exons, or (ii) a result of different spatiotemporal expression domains between appa and appb. Concerted injection of appa mRNA was able to rescue the Appa plus Prp1 knockdown phenotype  having a direct effect on the system by itself , nor an appa mRNA with a single nucleotide insertion producing a frameshift (appa14_15 insT), were able to rescue the concerted Appa plus Prp1 co-knockdown phenotype  and this independent MO showed the same phenotypes and knockdown of APP immunoreactivity  increase in the average number of activated-caspase 3-positive cells  as did fish injected with the appb or prp1 MOs  when compared to control injected fish   . Apoptotprp1 MOs .appa and prp1 MOs were injected and the aggregating ability of cells was examined. Cells from embryos that had been injected with MO solutions and dextran dyes were first dissociated such that less than 10% remained aggregated (appa or prp1 had no significant effect on the aggregating ability of cells (a 9% increase or 11% decrease respectively in aggregation compared to control MO), but when sub-effective doses were combined there was a 33% decrease in the number of cells in aggregates . Combining these same doses of appa and prp1 mRNAs had a tendency to increase cell aggregation, but did not reach significance .To test the hypothesis that APP and PrP interactions play a role in cell adhesion, gregates ; P<0.05.prp1’s paralog-specific interactions with appa versus appb, we similarly tested genetic interactions in regards to PrP paralogs. Concerted mRNA injections, in a background of Appa plus Prp1 knockdown, demonstrated that 100 pg of prp1 mRNA could rescue the phenotype (P<0.05)  . In conthenotype . Thus apparalogs , these dprp1 in its interaction with appa. We found that 100 pg of mouse Prnp mRNA was able to rescue the phenotypes observed during concerted delivery of appa plus prp1 MOs, and while this was not to the same extent as zebrafish prp1 mRNA, it was significantly more efficacious than zebrafish prp2 in this role  rescue the phenotypes caused by concerted delivery of appa plus prp1 MOs   were employed  transgenic fish harbored no intrinsic susceptibility to MO injection as these fish developed normally , and this could be through a direct interaction of proteins or their common occurrence within a scaffolded protein complex. Alternatively, they may independently affect a downstream pathway; these hypotheses are not mutually exclusive. Past work describing the APP interactome identified PrPC as one of the hundreds of proteins hypothesized to interact with APP in mice, and similar work suggests APP as a hypothetical member of PrPC’s interactome e.g. they can replace each other during development, see C was observed only in N2a cells that were transfected with both human APP and with human PrPC . Considering the immunoprecipitation of mammalian homologs , we interpret this to mean that APP and PrP interactions are highly conserved through at least 450 million years of evolutionary time, and thus important and worthy of further detailed study. The interaction we report, underpinning cell adhesion and CNS apoptosis, appears to be entirely relevant to mammalian orthologues in-so-much that human APP and murine PrP can replace their zebrafish orthologues during the APP-PrP interaction in our neurodevelopmental assays. The conservation is further underlined by our findings that (i) appa and appb can replace each other during normal zebrafish development, i.e. they are redundant in our assays; (ii) appb cannot replace appa during CNS development if PrP protein levels are reduced; (iii) in a surprising contrast to the latter, human APP mRNA is able to replace zebrafish appa during development of CNS with reduced PrP levels; (iv) similar to the latter point, in a context of reduced Appa levels, mouse Prnp is better able to replace zebrafish prp1 compared to related zebrafish prp2 or zebrafish shadoo.We sought to validate and expand upon putative APP-PrP interactions through an independent method. Our concerted appa with prp1 is a specialized niche event during neurodevelopment, disruption of which has substantive consequences on cell adhesion and neuron survival. Third, the ability of human APP (but not zebrafish Appb) to replace zebrafish appa in the context of reduced PrP protein argues strongly that human APP interacts with PrP in a conserved neuroprotective role. We validated just such an interaction of human APP with human PrP using co-immunoprecipitations. Finally, this data sets the stage for comparisons amongst APP proteins to define which residues and domains are critical for the interactions with PrP and the effects on cell adhesion and neuron survival. We are actively pursuing the latter by investigating residues that are shared by human APP and zebrafish Appa but that are not shared by zebrafish Appb. Appa and human APP are more similar to each other than to Appb in only one contiguous location greater than three amino acids - the amino-terminal end of Aβ, i.e. the site of β secretase (BACE) cleavage  cleavage of APP and thus affect levels of Aβ42 associated with increased Alzheimer Disease risk P102L is also observed CSecondly, human genetics has frequently, though inconsistently, described a controversial association of the human in vivo, though it cannot yet be excluded that intermediary binding partners are required. Protein interactomes of APP and PrPC each independently annotate high-quality data that make APP and PrPC likely interactors in rodent brains and cell culture paradigms C, representing Type I transmembrane and GPI-anchored proteins respectively, both being localized to the external leaf of cell membranes, at synapses and within lipid rafts C have in common, including APLP1, neural cell adhesion molecule 1, integrins, and contactins, supporting the validity of the biochemical interaction and a common role for APP and PrPC proteins in modulating cell adhesion in vivo paradigm for their investigation.Finally, systems biology approaches have prompted the hypothesis that APP and PrP interact biochemically In sum, our comparison between APP and PrP paralogs has identified a conserved and specific niche role for APP-PrP interactions that are required for vertebrate CNS development, and the effects of disrupting this interaction are not the result of generalized decrements in neurodevelopmental integrity but are required for cell adhesion events. Considering the well-established role for APP and PrP  in synaptic plasticity we speculate upon a role for APP-PrP interactions in modulating learning and/or excitotoxicity.C’s high-affinity binding of an APP catabolite, i.e. oligomerized Aβ  were cloned from wild type zebrafish into a PCS2+ or pCR2.1TOPO vector (primers in appa pre-processed mRNA (APPa_SB 5′ TAG TGT TGC TTC ACC TCC TGG CAG T 3′). A translation blocking MO to the 5′UTR of appa designated APPa_TB (5′ GCT TCT GCT CCT CTT TAT TTC GCC T 3′). A splice-blocking morpholino designed to specifically bind the exon-intron boundary of exon 3-intron 3 of the zebrafish appb mRNA (5′ CAC ACA CAT ACA TAC CCA GGC AAC G 3′), and a previously published prp1 mRNA (5′ TGA GCA GAG AGT GCT GCG GGA GAG A 3′). A standard negative control morpholino was obtained from Gene Tools (5′ CCT CTT ACC TCA GTT ACA ATT TAT A 3′). All morpholino injection solutions also contained a standardized dose (3 ng) of tp53 morpholino  or sub-effective  concentrations, mRNAs as appropriate, and nuclease-free water to 10 µL. One cell stage embryos were staged on agarose plates, and injection volume calibrated to 1 nL using an ocular micrometer immediately prior to injection. Zebrafish injected with MOs and control MOs were staged at 24 hours post-fertilization (hpf) based on body morphology and screened for the presence of CNS cell death. Observer was blinded to treatment groups during screening of all phenotypes.Six antisense morpholino oligonucleotides (MO) purchased from Gene Tools, LLC  were used during these experiments. These MOs were all designed to not bind the cognate mRNA constructs we produced, thus enabling rescue experiments. A splice-blocking morpholino was designed to specifically bind the exon-intron boundary of exon 2-intron 2 of the zebrafish Tg(gfap:GFP)mi2001Zebrafish were maintained at 28.5°C in standard conditions Figure S1appa morpholino (MO) injection leads to a dose-dependent disruption in appa mRNA processing. A. Zebrafish embryos were injected with increasing doses of appa MO and scored for malformations and CNS cell death. B. Same experiments as panel A revealed doses that were toxic to the developing fish. C.appa splice block MO is efficacious, as it leads to disruption of appa mRNA. RNA was isolated from fish injected with appa MO, an equivalent dose of control MO, and subjected to RT-PCR. Fish injected with appa MO show a band at ∼300 bp corresponding to mRNA with intron 2 retained. This band is absent in when fish are injected with the control MO, or when standard Taq is used in place of reverse-transcriptase. D. Sequencing of the aforementioned ∼300 bp band confirms the retention of intron 2–3 in mature mRNA, and confirms our MO produces a truncated protein. Our sequence (top) was an exact match to zebrafish genomic clone NW_003336735 (bottom). Translation of the sequence, immediately 5′ of appa exon 3 (annotated in yellow at bottom right), predicts two termination codons . E–J.A second MO reagent against appa was used to test specificity of the phenotypes observed. Designed against a disparate portion of the gene, the 5′UTR and thus is a translation blocking (TB) MO. The efficacy of this MO is demonstrated in appa MO we primarily use in this work. The low dose of appa TB MO also showed a genetic interaction with the low dose of prp1 MO, equivalent to results in K. Quantification of appa-TB MO demonstrates a dose-dependant effect by itself and an additive effect with prp1 MO during concerted delivery at sub-effective doses. Colour coding in the histogram is as per (TIF)Click here for additional data file.Figure S2appb morpholino (MO) injection leads to a dose-dependent disruption in appb mRNA processing. A. Zebrafish embryos were injected with increasing doses of appb MO and screened based on presence of morphological malformations and CNS cell death. B. The same experiments as in panel A revealed doses that were toxic to the developing fish. C.appb splice block MO is efficacious, as it leads to disruption of appb mRNA. RNA was isolated from fish injected with 2.5 ng appb MO, 1.0 ng appb MO, or an equivalent dose of control MO, and subjected to RT-PCR. Fish injected with 2.5 ng of the appb MO show a band at ∼300 bp corresponding to retention of intron 3–4 in mRNA. This band is reduced when the dose of the MO is reduced, and absent when fish are injected with the control MO. Sequencing of the band confirmed the retention of intron 3–4 in mature mRNA, and predicted STOP codons in the modified mRNA. D. Embryos injected with the appb MO alone or with 200 pg of cognate appb mRNA. The instance of fish displaying a severe phenotype was significantly reduced and the number of normal fish was significantly increased upon inclusion of appb mRNA. **p<0.01. Colour coding in the histogram D is as per (TIF)Click here for additional data file.Figure S3Efficacy of appa and appb MO’s assessed by western blot. MO knockdown reagents were assessed by Western blot to quantify protein abundance. Parts of this data appear in (TIF)Click here for additional data file.Figure S4Conservation of App between zebrafish paralogs and human APP. Amyloid β Precursor Protein (APP) is processed to Aβ (red), the major protein constituent of plaques in AD, by sequential enzyme cleavage. Zebrafish have two gene paralogues, appa & appb, wherein most residues of C99 at least one of them is a perfect match to human. Human APP is able to replace Appa in this interaction, Appa is more similar to human . Residues responsible for familial AD mutations (pink) are conserved in all three proteins.(TIF)Click here for additional data file.Figure S5appa and appb mRNAs that rescue observed phenotypes must be translated to have their effect. The phenotypes produced by knockdown of appa or appb can be rescued by co-injection of wild type appa or appb mRNA, respectively (A & B here), but not by mutant mRNAs. A, B. Assessment of mutant mRNAs. Mutant mRNAs possessing point mutations in the start of the coding region fail to rescue phenotypes that are rescued by wildtype (WT) mRNA. C. The same result is found regarding the ability (or lack thereof) of these mRNAs to rescue the joint knockdown of prp1 and appa  that ablate capacity to encode a functional full-length protein. In appa, we changed two basepairs (highlighted) at the start of the CDS that are predicted to create large changes in the protein by creating stop codons  (asterisks in protein represent stop codons) inducing a truncation. Alternatively, we modified the appa CDS by a single basepair insertion (14_15 insT) inducing a frameshift of 72 residues (grey shading) before a premature stop codon, and with only one of 72 residues having sequence identity with the parental wt mRNA. Similarly in appb, changing four base pairs created stop codons  and a truncated protein. We altered the appb mRNA in a second way, inducing a single basepair deletion (del8A) to create stop codons and a truncated protein. Stop codons are represented by asterisks, and residues N-terminal to stop codons are emphasized with strikethrough. Colour coding of histograms as per (TIF)Click here for additional data file.Figure S6mRNAs envisaged following injection of splice blocking morpholinos do not have a dominant effect. Injection of the splice blocking MOs leads to inappropriate retention of the adjacent intron (with STOP codons); this is predicted to encode a truncated protein along with a portion of the retained intron. This altered mRNA may be degraded, but the kinetics are unknown so potential dominant effects of the predicted protein were tested, by delivering an mRNA encoding the truncated mRNA with retained intron. A. Delivery of mRNA encoding the first two exons of appa plus intron 2 . B. Delivery of mRNA encoding the first three exons of appb plus intron 3 . Data obtained from injecting mRNAs encoding the full length appa and appb proteins are presented for ease of comparison, replicated from appa-i2 or appb-i3 mRNA compared to injecting the cognate full length mRNA, or compared to injecting control MOs, or compared to uninjected fish. Thus MO injection likely leads to the phenotypes observed through reduction of protein abundance, and is not due to dominant effects of products from mis-spliced mRNAs. Colour coding of histograms as per (TIF)Click here for additional data file.Figure S7High doses of MOs affect apoptotic cell death, and Human APP rescues apoptotic cell death. High doses of prp1, appa or appb MO are used here to show their individual effects on apoptotic cell death. Some combinations of these MOs co-injected at low doses show that these MOs can synergize to produce this effect . Brightfield images of the area above the yolk sac extension of fish injected with effective doses of control, appb, appa and prp1  MOs. Compared with control fish, prominent anti-activated caspase 3 staining is apparent in appb, appa, or prp1 MO injected fish . E. Number of caspase 3 positive cells were counted above the yolk sac extension in fish treated as per those in A–D. N = 5. Panels F–K show examples of human APP rescuing the concerted appa plus prp1 knockdown. appb is not able to rescue the phenotype in wildtype fish, nor in transgenic fish labelling the CNS with GFP  as we noted in APP is able to rescue these phenotypes . Panels L–N show the yolk-sac extension of fish in F, H & J. L′–N′ show examples of activated caspase labelling during rescue with human APP. The latter treatments were quantified in O. N = 5. * = P<0.05. ** = P<0.01.s effect . Apoptos(TIF)Click here for additional data file.Protocol S1Supplemental description of Methods.(DOCX)Click here for additional data file.