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Eukaryotic gene expression is developmentally regulated, in part by chromatin remodelling, and its dysregulation has been linked to cancer. CHD5 (chromodomain helicase DNA-binding protein 5) is a tumour suppressor gene (TSG) that maps to a region of consistent deletion on 1p36.31 in neuroblastomas (NBs) and other tumour types. CHD5 encodes a protein with chromatin remodelling, helicase and DNA-binding motifs that is preferentially expressed in neural and testicular tissues. CHD5 is highly homologous to CHD3 and CHD4, which are the core subunits of nucleosome remodelling and deacetylation (NuRD) complexes. To determine if CHD5 forms a similar complex, we performed studies on nuclear extracts from NBLS, SY5Y (both with endogenous CHD5 expression), NLF (CHD5 null) and NLF cells stably transfected with CHD5 cDNA (wild-type and V5–histidine-tagged). Immunoprecipitation (IP) was performed with either CHD5 antibody or antibody to V5/histidine-tagged protein. We identified NuRD components both by GST–FOG1 (Friend Of GATA1) pull-down and by IP. We also performed MS/MS analysis to confirm the presence of CHD5 or other protein components of the NuRD complex, as well as to identify other novel proteins. CHD5 was clearly associated with all canonical NuRD components, including metastasis-associated protein (MTA)1/2, GATA zinc finger domain containing 2A (GATAD2A), histone deacetylase (HDAC)1/2, retinoblastoma-binding protein (RBBP)4/7 and methyl DNA-binding domain protein (MBD)2/3, as determined by Western blotting and MS/MS. Our data suggest CHD5 forms a NuRD complex similar to CHD4. However, CHD5–NuRD may also have unique protein associations that confer functional specificity and may contribute to normal development and to tumour suppression in NB and other cancers.

CHD5 was first identified because of its location on 1p36 in a region of frequent deletion in neuroblastomas. CHD5 (chromodomain–helicase–DNA–binding-5) is the fifth member of a family of chromatin remodeling proteins, and it probably functions by forming a nucleosome remodeling and deacetylation (NuRD) complex that regulates transcription of particular genes. CHD5 is preferentially expressed in the nervous system and testis. On the basis of its position, pattern of expression, and function in neuroblastoma cells and xenografts, CHD5 was identified as a tumor suppressor gene (TSG). Evidence soon emerged that CHD5 also functioned as a TSG in gliomas and a variety of other tumor types, including breast, colon, lung, ovary, and prostate cancers. Although one copy of CHD5 is deleted frequently, inactivating mutations of the remaining allele are rare. However, DNA methylation of the CHD5 promoter is found frequently, and this epigenetic mechanism leads to biallelic inactivation. Furthermore, low CHD5 expression is strongly associated with unfavorable clinical and biologic features as well as outcome in neuroblastomas and many other tumor types. Thus, based on its likely involvement as a TSG in neuroblastomas, gliomas, and many common adult tumors, CHD5 may play an important developmental role in many other tissues besides the nervous system and testis. Cancer Res; 74(3); 652–8. ©2014 AACR.

Purpose: Neuroblastomas (NBs) have genomic, biological, and clinical heterogeneity. High-risk NBs are characterized by several genomic changes, including MYCN amplification and 1p36 deletion. We identified the chromatin-remodeling gene CHD5 as a tumor suppressor gene that maps to 1p36.31. Low or absent CHD5 expression is associated with a 1p36 deletion and an unfavorable outcome, but the mechanisms of CHD5 inactivation in NBs are unknown. Experimental Design: We examined (i) the CHD5 sequence in 188 high-risk NBs investigated through the TARGET initiative, (ii) the methylation status of the CHD5 promoter in 108 NBs with or without 1p36 deletion and/or MYCN amplification, and (iii) mRNA expression of CHD5 and MYCN in 814 representative NBs using TaqMan low-density array microfluidic cards. Results: We found no examples of somatically acquired CHD5 mutations, even in cases with 1p36 deletion, indicating that homozygous genomic inactivation is rare. Methylation of the CHD5 promoter was common in the high-risk tumors, and it was generally associated with both 1p deletion and MYCN amplification. High CHD5 expression was a powerful predictor of favorable outcome, and it showed prognostic value even in multivariable analysis after adjusting for MYCN amplification, 1p36 deletion, and/or 11q deletion. Conclusions: We conclude that (i) somatically acquired CHD5 mutations are rare in primary NBs, so inactivation probably occurs by deletion and epigenetic silencing; (ii) CHD5 expression and promoter methylation are associated with MYCN amplification, suggesting a possible interaction between these 2 genes; and (iii) high CHD5 expression is strongly correlated with favorable clinical/biological features and outcome. Clin Cancer Res; 18(6); 1588–97. ©2012 AACR.

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.

In this study, a recombinant pET28c-gBTB/POZ was constructed by cloning the sequence of the BTB/POZ domain of the zebrafish gcl (germ cell-less) into the expression vector pET28c, and pET28c-gBTB/POZ was transformed into BL21(DE3) pLysS strain to express the fusion protein for the preparation of antibody. Polyclonal-antibody against the GCL-BTB/POZ domain was prepared by immunizing rabbit with the fusion protein, and the Western Blot and immuno-histochemical analysis were performed to detect the quantity of the polyclonal-antibody. The result indicates that the polyclonal-antibodies were of good quantity and specification. Further studies will be performed to demonstrate the function and expression pattern of the GCL protein during the development process of zebrafish with the polyclonal-antibody.

Background Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines. Methods NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1–10 μM 13-cis-retinoic acid (13cRA) for 3–12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies. Results Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor. Conclusions Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0425-y) contains supplementary material, which is available to authorized users.

Effect of 13cRA on cell proliferation by SRB assay. SY5Y and NB69 cells were plated on 96 well plates and treated with or without 10Â ÎźM retinoic acid. Plates were harvested after 2, 5, and 7Â days of 13cRA treatment. Cell viability was analyzed by an SRB assay measuring optical density (OD). Cell lines treated with 13cRA showed lower OD, indicating reduced cell number compared to untreated control cells. (DOC 46 kb)

(A) Neurite inhibition by CEP-701 in SY5Y-TrkA cells. Representative phase contrast images of SY5Y-TrkA cells depicting extension of neurites in response to 13cRA treatment after 4 days, and subsequent neurite inhibition following 100 nM CEP-701 treatment (μm-micrometer) (B) Statistical analysis of neurite outgrowth following 13cRA as well as CEP-701 treatment of SY5Y-TrkA cells. Length of neurites (μm-micrometer) from three to five representative fields of 13cRA and CEP-701 treated cells were measured in μm and compared with neurites from untreated cells. P-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 148 kb)

(A) Detection of neurites in NB69 cells in response to retinoic acid. Representative phase contrast images of NB69 cells showing long neurites upon 13cRA treatment for 6 and 10 days. (B) Statistical analysis of various neurites upon 13cRA treatment in NB69 cells. Length of neurites from various selected fields were measured in μm and compared with neurites from untreated cells. Neurites were measured (μm-micrometer) from at least three independent experiments and p-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 296 kb)

(A) Neurite formation in SY5Y cells in response to 13cRA. Representative phase contrast images depicting extension of neurites in response to 13cRA treatment after 6 days and 10 days. Long neurites were observed when cells were treated with 13cRA. (B) Statistical analysis of neurite outgrowth following 13cRA treatment in SY5Y cells. Length of neurites from three to five representative fields of 13cRA treated cells were measured in μm and compared with neurites from untreated cells. Neurites were measured (μm-micrometer) from at least three independent experiments, and p-values were calculated by one-way ANOVA using Prism followed by Tukey’s post-test. (DOC 208 kb)

Spermatogenesis is a highly specialized process of cellular differentiation to produce spermatozoa. This differentiation process accompanies morphological changes that are controlled by a number of genes expressed in a stage-specific manner during spermatogenesis. Here we show that in mice, the absence of a testis-specific, cytoplasmic polyadenylate [poly(A)] polymerase, TPAP, results in the arrest of spermiogenesis. TPAP-deficient mice display impaired expression of haploid-specific genes that are required for the morphogenesis of germ cells. The TPAP deficiency also causes incomplete elongation of poly(A) tails of particular transcription factor messenger RNAs. Although the overall cellular level of the transcription factor TAF10 is unaffected, TAF10 is insufficiently transported into the nucleus of germ cells. We propose that TPAP governs germ cell morphogenesis by modulating specific transcription factors at posttranscriptional and posttranslational levels.

Neuroblastoma (NB) is the most common and deadly solid tumor in children. Despite recent improvements, the long-term outlook for high-risk NB is still50%. Further, there is considerable short- and long-term toxicity. More effective, less toxic therapy is needed, and the development of targeted therapies offers great promise.Relevant literature was reviewed to identify current and future therapeutic targets that are critical to malignant transformation and progression of NB. The potential or actual NB therapeutic targets are classified into four categories: i) genes activated by amplification, mutation, translocation or autocrine overexpression; ii) genes inactivated by deletion, mutation or epigenetic silencing; iii) membrane-associated genes expressed on most NBs but few other tissues; or iv) common target genes relevant to NB as well as other tumors.Therapeutic approaches have been developed to some of these targets, but many remain untargeted at the present time. It is unlikely that single targeted agents will be sufficient for long-term cure, at least for high-risk NBs. The challenge will be how to integrate targeted agents with each other and with conventional therapy to enhance their efficacy, while simultaneously reducing systemic toxicity.

Review on MYCN (v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)), with data on DNA, on the protein encoded, and where the gene is implicated.

Review on CHD5 (chromodomain helicase DNA binding protein 5), with data on DNA, on the protein encoded, and where the gene is implicated.

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme and forms antioxidant bile pigments as well as the signaling molecule carbon monoxide. HO-1 is inducible in response to a variety of chemical and physical stress conditions to function as a cytoprotective molecule. Therefore, it is important to maintain the basal level of HO-1 expression even when substrate availability is limited. We hypothesized that the HO-1 protein itself could regulate its own expression in a positive feedback manner, and that this positive feedback was important in the HO-1 gene induction in response to oxidative stress. In cultured NIH 3T3 cells, transfection of HO-1 cDNA or intracellular delivery of pure HO-1 protein resulted in activation of a 15-kb HO-1 promoter upstream of luciferase as visualized by bioluminescent technology and increased HO-1 mRNA and protein levels. These effects were independent of HO activity because an enzymatically inactive mutant form of HO-1 similarly activated the HO-1 promoter and incubation with HO inhibitor metalloporphyrin SnPP did not affect the promoter activation. In addition, HO-1-specific siRNA significantly reduced hemin and cadmium chloride-mediated HO-1 induction. Furthermore, deletion analyses demonstrated that the E1 and E2 distal enhancers of the HO-1 promoter are required for this HO-1 autoregulation. These experiments document feed-forward autoregulation of HO-1 in oxidative stress and suggest that HO-1 protein has a role in the induction process. We speculate that this mechanism may be useful for maintaining HO-1 expression when substrate is limited and may also serve to up-regulate other genes to promote cytoprotection and to modulate cell proliferation.

Background: Neuroblastoma (NB) is a tumor of the sympathetic nervous system that is the most common extracranial solid tumor of childhood. NBs show remarkable clinical heterogeneity, and we, along with others, have identified different patterns of genomic change that underlie these diverse clinical behaviors. We first identified 1p deletion as a characteristic change in advanced stage NBs. CHD5, a tumor suppressor gene, was first identified because of its location on 1p36. However, mutation of the remaining allele of CHD5 is rare in these tumors. Therefore, it is likely that epigenetic mechanisms play important roles in CHD5 downregulation. MicroRNAs (miRNAs) are small RNAs that bind to the 3′ UTR to mediate downregulation of target gene expression by translational repression or cleavage of the target gene message. Dysregulation of CHD5 via miR-211 has been reported in colon cancer. In order to understand the role of miRNA regulation of CHD5 in NBs, we wanted to assess the function of all miRNAs that are predicted to target CHD5. Method/approach: We used TargetScan, miRanda, MirTarget2 and other prediction algorithms to identify miRNAs that were predicted to bind to the CHD5-3′UTR. We identified 18 miRNAs that were predicted by 2 or more programs: miR-204-5p, -211, -216b, -17, -19ab, -20ab, -93, -106ab, -130ab, -301ab, -454, -519d, -3666. We used a renilla-luciferase reporter plasmid that contains 103 bp of the 3′UTR of CHD5 targeted by all miRNAs except miR-204, -211, - 219b, and -3666. We also created control plasmids with no CHD5 3′UTR insert and a mutated CHD5 3′UTR with mutated miRNA binding sites. Allstar siRNA served as an additional negative control. We performed transient transfections in two NB cell lines, NLF (a MYCN amplified cell line) and SY5Y (a MYCN nonamplified cell line), with the reporter plasmid and miRNA mimic. We measured luciferase (internal control) and renilla (reporter) activity. Results: Our results from the luciferase reporter assay indicate that four miRNAs are strong candidates for CHD5 downregulation in NB: miR-17, -93, -20b, and miR-106b. Each exhibited a significant decrease of fluorescence activity in cells transfected with CHD5 3′UTR in comparison to cells transfected with either of the control plasmids. Interestingly, MYCN upregulates three of the four miRNA candidates: miR-17, -93, & -20b. We further confirmed consistent downregulation of the CHD5 by all four miRNAs. Conclusion: CHD5 expression is downregulated by miR-17, -93, -2b, and 106b. MYCN amplification and overexpression leads to upregulation of miR-17, -93, and -20b, and this may lead to down regulation of CHD5. Citation Format: Koumudi Naraparaju, Venkatadri Kolla, Tiangang Zhuang, Mayumi Higashi, Gerd A. Blobel, Garrett M. Brodeur. The role of microRNAs in the epigenetic silencing of CHD5, a tumor suppressor in neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 191. doi:10.1158/1538-7445.AM2015-191

We have previously identified a testis-specific poly(A) polymerase, TPAP (PAPbeta), involved in poly(A) tail extension of specific mRNAs in the cytoplasm of round spermatids. Targeted disruption of the mouse TPAP gene resulted in the arrest of spermiogenesis due to reduced expression of haploid-specific genes required for morphogenesis of germ cells. To further elucidate the role(s) of TPAP in spermatogenesis, transgenic mice expressing an exogenous TPAP transgene on wild-type and TPAP-deficient backgrounds were generated and characterized. The transgenic mice overexpressing TPAP exhibited normal spermatogenesis and fertility. The sizes of some transcription factor mRNAs as the substrates of TPAP were also unaffected. Transgenic expression of the TPAP gene in the TPAP-deficient mice complemented both the incomplete elongation of the poly(A) tails of specific transcription factor mRNAs, and reduced expression of haploid-specific genes, resulting in the resumption of normal spermiogenesis. These data conclusively show that spermatogenesis requires the cytoplasmic elongation of the mRNA poly(A) tails catalyzed by TPAP, and imply the presence of a regulatory mechanism(s) defining the extent of the cytoplasmic mRNA polyadenylation.

We have identified cDNA clones encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contained 642 residues, and shared a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system was capable of incorporating the AMP moiety from ATP into an oligo(A)12 RNA primer in the presence of MnCl2. Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognized a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs.

Nucleotide sequences of primers used in this study (DOCX 15 kb)

Nucleotide sequences of primers used in this study (DOCX 15 kb)

NB69 morphology in response to 13cRA. (A) (A) Representative phase contrast images showing NB69 morphology change with or without 13cRA treatment (10 μM, 12 days). Two very different morphologic populations of cells were observed: a neuronal morphology with neurite outgrowth (N-type) (Red arrows), and flat and substrate adherent (S-type) (Blue arrows). (B) Immunofluorescence of NB69 cells with or without 13cRA treatment. Representative immunofluorescence images obtained when cells were treated with 13cRA and stained with neuronal markers TH, SYN, and βIII-Tubulin. (DOC 395 kb)

NB69 morphology in response to 13cRA. (A) (A) Representative phase contrast images showing NB69 morphology change with or without 13cRA treatment (10 μM, 12 days). Two very different morphologic populations of cells were observed: a neuronal morphology with neurite outgrowth (N-type) (Red arrows), and flat and substrate adherent (S-type) (Blue arrows). (B) Immunofluorescence of NB69 cells with or without 13cRA treatment. Representative immunofluorescence images obtained when cells were treated with 13cRA and stained with neuronal markers TH, SYN, and βIII-Tubulin. (DOC 395 kb)