PMC

Schematic strategy used to unmask DNA methylation-associated repression of T-UCRs in colon cancer cells. AZA, 5-aza-2′-deoxycytidine.

T-UCR CpG island hypermethylation patterns in human tumorigenesis. (a) Methylation-specific PCR analyses for Uc.160+, Uc.283+A and Uc.346+ methylation in primary human tumors from different types. In vitro methylated DNA (IVD) and normal lymphocytes (NLs) are shown as positive and negative controls for methylated and unmethylated sequences, respectively. (b) Frequency of Uc.160+, Uc.283+A and Uc.346+ hypermethylation in primary human tumors from different types, normal tissues and premalignant lesions (colorectal adenomas). (c) Distribution of Uc.160+, Uc.283+A and Uc.346+ CpG island hypermethylation in primary tumors according to the presence or absence of lymph node metastasis. The presence of hypermethylation of the T-UCRs is significantly associated with the existence of metastasis in human malignancies. (d) Observation of an unmethylated Uc.160+ CpG island in all leukemias that overexpress the miR-155 transcript determined by quantitative RT–PCR in comparison with the presence of Uc.160+ hypermethylation in all leukemias with low levels of miR-155 (P=0.01492, Student's t-test). CRC, colorectal carcinoma.

CpG island methylation and expression of T-UCRs in cancer cell lines and normal tissues. (a) Bisulfite genomic sequencing of the associated CpG islands for the T-UCRs Uc.160+, Uc.283+A and Uc.346+ in the leukemia cell line MOLT16 and normal tissues (colon, NC; lymphocytes, NL; lung, Nlu-1 to Nlu-3). CpG dinucleotides are represented as short vertical lines and the location of the T-UCR probe from the expression microarray is represented as a black arrowhead. The locations of the bisulfite genomic sequencing primers are indicated by black arrows. Eight single clones are represented for each sample. Presence of a methylated or unmethylated cytosine is indicated by a black or white square, respectively. The transcription start sites identified by RACE are represented by vertical black arrows. (b) Expression analyses of T-UCRs by quantitative RT–PCR. Uc.160+ and Uc.346+ show minimal expression in the hypermethylated MOLT-16 cells, while treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine (AZA) induces T-UCR upregulation. The Uc.283+A CpG island is unmethylated in MOLT-16 and the T-UCR is expressed at similar levels in the untreated and demethylating agent-treated cells.

CpG island methylation of T-UCRs in colon cancer cell lines and normal tissues. (a) Bisulfite genomic sequencing of the associated CpG islands for the T-UCRs Uc.160+, Uc.283+A and Uc.346+ in the colorectal cancer cell lines HCT-116 and DKO, normal colon (NC) and normal lymphocytes (NL). CpG dinucleotides are represented as short vertical lines and the location of the T-UCR probe from the expression microarray is represented as a black arrowhead. The locations of the bisulfite genomic sequencing primers are indicated by black arrows. Eight single clones are represented for each sample. Presence of a methylated or unmethylated cytosine is indicated by a black or a white square, respectively. For Uc.283+A and Uc.346+, two regions within the CpG island were analyzed. The transcription start sites identified by RACE are represented by vertical black arrows. (b) Methylation-specific PCR analyses for Uc.160+, Uc.283+A and Uc.346+ methylation in the colorectal cancer cell lines HCT-116 and DKO, normal colon (NC) and normal lymphocytes (NL). Unmethylated (U) or methylated (M) sequences. In vitro methylated DNA (IVD) is shown as a positive control for methylated sequences. The locations of the methylation-specific PCR primers are indicated in panel ‘a' by gray arrows.

T-UCR expression and chromatin environment. (a) Expression analyses of T-UCRs by quantitative RT–PCR. Uc.160+, Uc.283+A and Uc.346+ show minimal expression in the hypermethylated HCT-116 cells, while treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine (AZA) and DKO cells show T-UCR upregulation. (b) Quantitative chromatin immunoprecipitation assay for the histone modification mark trimethylation of lysine 4 of histone H3 (H3K4me3) that is associated with active transcription. The presence of T-UCR CpG island methylation is associated with the lack of H3K4me3 histone, whereas the opposite scenario is observed when DNA demethylation events are present by genetic disruption of the DNMTs (DKO cells) or pharmacological treatment with a DNA-demethylating agent (AZA lane). (c) Chromatin accessibility assay using the MspI restriction enzyme coupled to real-time PCR. The hypermethylated CpG islands of Uc.160+, Uc.283+A and Uc.346+ CpG in HCT-116 are inaccessible to the enzyme, but CpG island demethylation upon 5-aza-2′-deoxycytidine treatment or in DKO cells results in higher accessibility. (d) Quantitative chromatin immunoprecipitation assay for RNA polymerase II (RNA Pol II) shows its absence in T-UCR hypermethylated CpG islands and its enrichment upon DNA hypomethylation events (DKO cells and HCT-116 treated with the DNA-demethylating drug).