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BACKGROUND Chromodomain helicase DNA binding protein 5 (CHD5) is a family member of chromatin remodeling factors. The epigenetic silencing mechanisms of CHD5 in colorectal cancer have not been well studied. METHODS Here we analyzed CHD5 methylation and mRNA expression in vitro and in clinical samples from African American patients. DNA and RNA were isolated from formalin fixed paraffin embedded (FFPE) colon tissues. DNA was tested for methylation using methylation-specific polymerase chain reation (PCR) and bisulfite sequencing. RNA was used for mRNA quantification using qRT-PCR. The RKO cell line was treated with 5-Aza-dC and SAHA. RKO cells were also stably transfected with a CHD5-expressing vector. The transcriptional activity was studied in the 1 kb upstream region of the CHD5 promoter using the dual reporter assay. We performed cell proliferation, migration, and invasion assays using the RKO cell line. RESULTS In most adenoma samples, CHD5 expression was not detected in contrast to normal tissues. In RKO cells, CHD5 silencing was associated with DNA methylation and repressive histone modifications. CHD5 expression was restored after treatment with 5-Aza-dC and SAHA. CHD5 reactivation reduced cell proliferation, migration, and invasion. The reporter assay indicated that the main regulatory region of the CHD5 promoter is encompassed in the −489 to −823 region with important transcriptional regulatory sites (TCF/LEF, SP1, and AP-2). CONCLUSIONS The CHD5 gene is repressed in all types of adenomas, either epigenetically or by chromosomal deletion. CHD5 activity is regulated by DNA methylation and repressive histone modifications. CHD5 likely acts as a tumor-suppressor gene in early colorectal carcinogenesis. Cancer 2014;120:172–180. © 2013 American Cancer Society.

Nicotine in tobacco smoke is thought to stimulate sensory nerve fibers by receptors that are located on airway epithelial cells and on terminal branches of C-fiber afferents, but the exact neurochemical substrate that mediates the sensory effects of nicotine associated with cigarette smoking is not clear. ATP and nitric oxide (NO) have both been implicated in lung responsiveness to airborne chemicals such as nicotine. However, the neuroanatomical and functional relationships between nicotinic acetylcholine receptors (nAChRs), purinergic signaling, and NO are not known, and the main source of NO in the airways is not clear. In the present study, we performed RT-PCR to confirm the presence of mRNA for all three isoforms of nitric oxide synthase (NOS), neuronal (n-NOS), endothelial (e-NOS), and inducible (i-NOS), in the lung. Sequential double labeling was performed to assess the site of expression of the different NOS isoforms with respect to nAChRs and purinergic receptors (P2X3R) of the intrapulmonary airways. RT-PCR confirmed the presence of n-NOS, e-NOS, and i-NOS in the lung, and immunohistochemical studies verified their expression by epithelial cells at all levels of the intrapulmonary airways, including the terminal and respiratory bronchioles. Sequential double labeling demonstrated coexpression of n-NOS and/or i-NOS with nAChR- and P2X3R-expressing cells. These neuroanatomical findings suggest that bronchial epithelial cells may be a primary source of NO in the intrapulmonary airways and that the production and release of NO may be regulated by an autocrine/paracrine signaling system involving nAChRs and P2X3Rs. © 2011 Wiley Periodicals, Inc.

Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo.

The mammalian DNA methyltransferase Dnmt1 is responsible for the maintenance of the pattern of DNA methylation in vivo. It is a large multidomain enzyme comprising 1620 amino acid residues. We have purified and characterized individual domains of Dnmt1 (NLS-containing domain, NlsD, amino acid residues: 1-343; replication foci-directing domain, 350-609; Zn-binding domain (ZnD), 613-748; polybromo domain, 746-1110; and the catalytic domain (CatD), 1124-1620). CatD, ZnD and NlsD bind to DNA, demonstrating the existence of three independent DNA-binding sites in Dnmt1. CatD shows a preference for binding to hemimethylated CpG-sites; ZnD prefers methylated CpGs; and NlsD specifically binds to CpG-sites, but does not discriminate between unmethylated and methylated DNA. These results are not compatible with the suggestion that the target recognition domain of Dnmt1 resides in the N terminus of the enzyme. We show by protein-protein interaction assays that ZnD and CatD interact with each other. The isolated catalytic domain does not methylate DNA, neither alone nor in combination with other domains. Full-length Dnmt1 was purified from baculovirus-infected insect cells. Under the experimental conditions, Dnmt1 has a strong (50-fold) preference for hemimethylated DNA. Dnmt1 is stimulated to methylate unmodified CpG sites by the addition of fully methylated DNA. This effect is dependent on Zn, suggesting that binding of methylated DNA to ZnD triggers the allosteric activation of the catalytic center of Dnmt1. The allosteric activation model can explain kinetic data obtained by others. It suggests that Dnmt1 might be responsible for spreading of methylation, a process that is observed during aging and carcenogenesis but may be important for de novo methylation of DNA.

Methylation of DNA is important in many organisms and essential in mammals. Nucleobases can be methylated at the adenine-N 6, cytosine-N 4, or cytosine-C 5 atoms by specific DNA methyltransferases. We show here that the M.EcoRV, M.EcoRI, and Escherichia coli dam methyltransferases as well as the N- and C-terminal domains of the M.FokI enzyme, which were formerly all classified as adenine-N 6 DNA methyltransferases, also methylate cytosine residues at position N 4. Kinetic analyses demonstrate that the rate of methylation of cytosine residues by M.EcoRV and the M.FokI enzymes is reduced by only 1–2 orders of magnitude in relation to methylation of adenines. This result shows that although these enzymes methylate DNA in a sequence specific manner, they have a low substrate specificity with respect to the target base. This unexpected finding has implications on the mechanism of adenine-N 6 DNA methyltransferases. Sequence comparisons suggest that adenine-N 6 and cytosine-N 4 methyltransferases have changed their reaction specificity at least twice during evolution, a model that becomes much more likely given the partial functional overlap of both enzyme types. In contrast, methylation of adenine residues by the cytosine-N 4 methyltransferase M.BamHI was not detectable. On the basis of our results, we suggest that adenine-N 6 and cytosine-N 4 methyltransferases should be grouped into one enzyme family.

Previous studies have shown that molecules of the taste transduction pathway may serve as biochemical markers for chemoreceptive cells in respiratory and gastrointestinal tracts. In this study, we tested the hypothesis that brainstem neurons contain signaling molecules similar to those in taste buds which may sense the chemical composition of brain extracellular fluids. We used the reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunohistochemical techniques to evaluate presence of different bitter-responsive type 2 taste receptors (T2Rs), their associated G-protein α-gustducin, the downstream signaling molecules phospholipase C isoform β2 (PLC-β2) and transient receptor potential melastatin 5 (TRPM5) in the brainstem of rats. RT-PCR confirmed the mRNA coding for α-gustducin, PLC-β2, TRPM5 and rT2R1 but not that of rT2R16, rT2R26 and rT2R38 in the medulla oblongata. Western blotting confirmed the presence of α-gustducin at the protein level in rat brainstem. Immunohistochemistry identified cells expressing α-gustducin and PLC-β2 at multiple cardiorespiratory and CO 2 /H + chemosensory sites, including rostral ventral medulla, facial, parapyramidal, solitary tract, hypoglossal and raphe nuclei. In the medullary raphe, α-gustducin and PLC-β2 were colocalized with a subpopulation of tryptophan hydroxylase (TPH)-immunoreactive serotonergic neurons, a subset of which has respiratory CO 2 /H + chemosensitivity. Presence of the T2R1 gene and other genes and proteins of the bitter taste transduction pathway in the brainstem implies additional functions for taste receptors and their effector molecules apart from their gustatory function.

Promoters are molecular 'modules', which are con- trolled as individual entities yet are often analyzed by nuclease digestion methodologies which, a priori, destroy this modularity. About 40% of mammalian genes contain CpG islands in their promoters and 15 exonic regions, which are normally unmethylated. We developed a footprinting strategy to map the chromatin structure at unmethylated CpG islands by treatment of isolated nuclei with the CpG- specific DNA methyltransferase SssI (M.SssI), fol- 20 lowed by genomic bisulfite sequencing of individual progeny DNA molecules. This gave single molecule resolution over the promoter region and allowed for the physical linkage between binding sites on individual promoter molecules to be maintained. 25 Comparison of the p16 promoters in two human cell lines, J82 and LD419, expressing the p16 gene at 25-fold different levels showed that the two cell lines contain remarkably different, heterogeneously positioned nucleosomes over the promoter region, 30 which were not distinguishable by standard methods using nucleases. Our high resolution approach gives a 'digitized' visualization of each promoter providing information regarding nucleosome occupancy and may be utilized to define transcription factor binding 35 and chromatin remodeling.

Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo.

The Escherichia coli dam adenine-N6 methyltransferase modifies DNA at GATC sequences. It is involved in post-replicative mismatch repair, control of DNA replication and gene regulation. We show that E. coli dam acts as a functional monomer and methylates only one strand of the DNA in each binding event. The preferred way of ternary complex assembly is that the enzyme first binds to DNA and then to S-adenosylmethionine. The enzyme methylates an oligonucleotide containing two dam sites and a 879 bp PCR product with four sites in a fully processive reaction. On lambda-DNA comprising 48,502 bp and 116 dam sites, E. coli dam scans 3000 dam sites per binding event in a random walk, that on average leads to a processive methylation of 55 sites. Processive methylation of DNA considerably accelerates DNA methylation. The highly processive mechanism of E. coli dam could explain why small amounts of E. coli dam are able to maintain the methylation state of dam sites during DNA replication. Furthermore, our data support the general rule that solitary DNA methyltransferase modify DNA processively whereas methyltransferases belonging to a restriction-modification system show a distributive mechanism, because processive methylation of DNA would interfere with the biological function of restriction-modification systems.

The M. Fok I adenine- N 6 DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine- N 6 DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M. Fok I interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2–3-fold. In addition, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C m ATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein–protein interaction assays show that both domains of M. Fok I are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding of both domains is mutually exclusive in full-length M. Fok I and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification systems are discussed.

Background: CHD5 is a family member of chromatin remodeling factors that functions as a tumor suppressor in neuroblastoma and ovarian cancer. The transcriptional activity of CHD5 is silenced by hypermethylation. It has been found that CHD5 is a biomarker in colon carcinogenesis. In this study, we have investigated the transcriptional regulation and function of the CHD5 in CRC. Methods: The transcriptional activity of CHD5 was tested by reporter luciferase assay. The 1kb large fragment upstream of CHD5 transcription start site (TSS) showed luciferase activity; while the 400 bp subfragment upstream of TSS was inactive in both RKO (CHD5 endogenous non-expressing) and HCT116 (CHD5 endogenous expressing) cell lines. This suggests that the transcriptionally active sites of CHD5 are located within −400 to −1000. There are two consensus TCF binding sites in the 1 kb promoter region. In order to investigate whether Wnt pathway affects the CHD5 transcriptional activity, we treated the RKO and HCT116 cell lines with 6-bromoindirubin-3′oxime (BIO), the GSK-3a inhibitor, which activates the β-Catenin. We tested the Wnt pathway activity in RKO and HCT116 cell lines by using the TCF/LEF responsive luciferase construct. Methylation profiles of CHD5 in 51 CRC and 6 adenomas from African Americans analyzed by MSP. Microdeletion analysis of the CHD5 region (1p36) was performed by aCGH. Results: The activity of TCF/LEF responsive element increased by 17.5 folds in RKO cell line and by 7.3 in HCT116 cell line after BIO treatment. Interestingly, the luciferase activity of 1kb CHD5 promoter increased after treating with BIO, and no changes were observed in activity of the 400 bp fragment. This suggests that the TCF/LEF and β-Catenin might act as coactivators for CHD5 transcriptional regulation. We also analyzed the proliferation of the RKO cell line after stably transfecting with CHD5 expressing plasmid (pcDNA). The results showed that the exogenous expression of CHD5 reduces the proliferation of the RKO cell line by two folds compared to the wild type and the mock controls. CHD5 was hypermethylated in 78% of the tested patients samples and deleted in 36%. Conclusion: The function of CHD5 is not well understood in CRC. Here, we suggest that the wnt/β-catenin pathway may be involved in the transcriptional regulation of CHD5. In addition in vivo CHD5 microdeletion and methylation excrement suggest that CHD5 act as tumor suppressor in CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5018.