No abstract

We collected paired samples of tumor and adjacent normal colorectal tissues from 22 patients with colorectal carcinoma to compare the differences in the expression of lysine specific demethylase 1 (LSD1) in these two tissues. The results showed that in 19 paired samples (86.4%), LSD1 is more highly expressed in tumor tissue than in normal tissue. To explore the role of LSD1 in colorectal tumorigenesis, we used somatic cell gene targeting to generate an LSD1 knockout (KO) HCT 116 human colorectal cancer cell line as a research model. The analysis of phenotypic changes showed that LSD1 KO colorectal cancer cells are less tumorigenic, both in vivo and in vitro. The differential expression analysis of the cells by mRNA sequencing (RNA-Seq) yielded 2,663 differentially expressed genes, and 28 of these genes had highly significant differences (Q <0.01). We then selected the 4 colorectal cancer-related genes ADM, DKK1, HAS3 and SMURF2 for quantitative real-time PCR verification. The results showed that the differences in the expression of ADM, DKK1 and HAS3 were consistent with those measured using the RNA-Seq data. As DKK1 was the gene with the most significant differential expression, we analyzed the key proteins of the DKK1-related Wnt/β-catenin signaling pathway and found that, after knocking out LSD1, the amount of free β-catenin translocated to the nucleus was significantly reduced and that the transcription of the signaling pathway target gene c-Myc was down-regulated. Our studies show that LSD1 activates the Wnt/β-catenin signaling pathway by down-regulating the pathway antagonist DKK1, which may be one of the mechanisms leading to colorectal tumorigenesis.

Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non–B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non–B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non–B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non–B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non–B-form DNA in the oat genome and reveals its potential role in centromeres.