Functional Profiling of Human MeCP2 by Automated Data Comparison Analysis and Computerized Expression Pathway Modeling / 대한의료정보학회지

OBJECTIVES: Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous epigenetic factor that represses gene expression by modifying chromatin. Mutations in the MeCP2 gene cause Rett syndrome, a progressive neurodevelopmental disorder. Recent studies also have shown that MeCP2 plays a role in carcinogenesis. Specifically, functional ablation of MeCP2 suppresses cell growth and leads to the proliferation of cancer cells. However, MeCP2's function in adult tissues remains poorly understood. We utilized a weight matrix-based comparison software to identify transcription factor binding site (TFBS) of MeCP2-regulated genes, which were recognized by cDNA microarray analysis. METHODS: MeCP2 expression was silenced using annealed siRNA in HEK293 cells, and then a cDNA microarray analysis was performed. Functional analysis was carried out, and transcriptional levels in target genes regulated by MeCP2 were investigated. TFBS analysis was done within genes selected by the cDNA microarray analysis, using a weight matrix-based program and the TRANSFAC 6.0 database. RESULTS: Among the differentially expressed genes with a change in expression greater than two-fold, 189 genes were up-regulated and 91 genes were down-regulated. Genes related to apoptosis and cell proliferation (JUN, FOSL2, CYR61, SKIL, ATF3, BMABI, BMPR2, RERE, and FALZ) were highly up-regulated. Genes with anti-apoptotic and anti-proliferative functions (HNRPA0, HIS1, and FOXC1) were down-regulated. Using TFBS analysis within putative promoters of novel candidate target genes of MeCP2, disease-related transcription factors were identified. CONCLUSIONS: The present results provide insights into the new target genes regulated by MeCP2 under epigenetic control. This information will be valuable for further studies aimed at clarifying the pathogenesis of Rett syndrome and neoplastic diseases.

Functional Profiling of Human MeCP2 by Automated Data Comparison Analysis and Computerized Expression Pathway Modeling / 대한의료정보학회지

OBJECTIVES: Methyl-CpG binding protein 2 (MeCP2) is a ubiquitous epigenetic factor that represses gene expression by modifying chromatin. Mutations in the MeCP2 gene cause Rett syndrome, a progressive neurodevelopmental disorder. Recent studies also have shown that MeCP2 plays a role in carcinogenesis. Specifically, functional ablation of MeCP2 suppresses cell growth and leads to the proliferation of cancer cells. However, MeCP2's function in adult tissues remains poorly understood. We utilized a weight matrix-based comparison software to identify transcription factor binding site (TFBS) of MeCP2-regulated genes, which were recognized by cDNA microarray analysis. METHODS: MeCP2 expression was silenced using annealed siRNA in HEK293 cells, and then a cDNA microarray analysis was performed. Functional analysis was carried out, and transcriptional levels in target genes regulated by MeCP2 were investigated. TFBS analysis was done within genes selected by the cDNA microarray analysis, using a weight matrix-based program and the TRANSFAC 6.0 database. RESULTS: Among the differentially expressed genes with a change in expression greater than two-fold, 189 genes were up-regulated and 91 genes were down-regulated. Genes related to apoptosis and cell proliferation (JUN, FOSL2, CYR61, SKIL, ATF3, BMABI, BMPR2, RERE, and FALZ) were highly up-regulated. Genes with anti-apoptotic and anti-proliferative functions (HNRPA0, HIS1, and FOXC1) were down-regulated. Using TFBS analysis within putative promoters of novel candidate target genes of MeCP2, disease-related transcription factors were identified. CONCLUSIONS: The present results provide insights into the new target genes regulated by MeCP2 under epigenetic control. This information will be valuable for further studies aimed at clarifying the pathogenesis of Rett syndrome and neoplastic diseases.

Diagnostic mutational analysis of MECP2 in Korean patients with Rett syndrome

Rett syndrome (RTT) is an X-linked dominant neurodevelopmental disorder affecting 1 per 10,000- 15,000 female births worldwide. The disease-causing gene has been identified as MECP2 (methyl- CpG-binding protein 2). In this study, we performed diagnostic mutational analysis of the MECP2 gene in RTT patients. Four exons and a putative promoter of the MECP2 gene were analyzed from the peripheral blood of 43 Korean patients with Rett syndrome by PCR-RFLP and direct sequencing. Mutations were detected in the MECP2 gene in approximately 60.5% of patients (26 cases/43 cases). The mutations consisted of 14 different types, including 9 missense mutations, 4 nonsense mutations and 1 frameshift mutation. Of these, three mutations (G161E, T311M, p385fsX409) were newly identified and were determined to be disease-causing mutations by PCR- RFLP and direct sequencing analysis. Most of the mutations were located within MBD (42.3%) and TRD (50%). T158M, R270X, and R306C mutations were identified at a high frequency. Additionally, an intronic SNP (IVS3+23C>G) was newly identified in three of the patients. IVS3+23C>G may be a disease-related and Korea-specific SNP for RTT. L100V and A201V are apparently disease-causing mutations in Korean RTT, contrary to previous studies. Disease-causing mutations and polymorphisms are important tools for diagnosing RTT in Koreans. The experimental procedures used in this study should be considered for clinical molecular biologic diagnosis.

Relationships Between the Expressions of CDX1 and CDX2 mRNA and Clinicopathologic Features in Colorectal Cancers

BACKGROUND: CDX1 and CDX2 are members of the caudal-type homeobox gene family and control the proliferation and differentiation of intestinal mucosal cells. Their expressions are commonly reduced in colorectal cancer, but reports about the relationships between their expressions and clinicopathologic features are rare. The aim of this study was to examine the expressions of CDX1 and CDX2 mRNAs in colorectal cancers and to assess the relationships between their expressions and clinicopathologic features. METHODS: CDX1 and CDX2 mRNA expressions were analyzed by real-time polymerase chain reaction in 48 colorectal cancers and in adjacent non-tumorous normal mucosal tissue. RESULTS: CDX1 and CDX2 mRNA expressions were significantly reduced in colorectal cancer tissues versus normal mucosal tissues (p=0.001, p=0.042, respectively). As compared with paired normal mucosal tissues, colorectal tissues showed reduced CDX1 mRNA expression in 64.6% (31/48) and reduced CDX2 mRNA expression in 66.7% (32/48) of cases. A statistically significant positive correlation was found between the expressions of CDX1 mRNA and CDX2 mRNA in colorectal cancer (r=0.543, p< 0.001). However, the expressions of CDX1 and CDX2 mRNAs were not related to age, sex, cancer location, differentiation, lymphatic or vascular invasion, lymph node metastasis, stage or serum carcinoembryonic antigen level. CONCLUSIONS: CDX1 and CDX2 mRNA expressions were found to be significantly reduced in colorectal cancers, but these expressional changes were not found to be related to clinicopathologic features.

Relationship of CDX1 mRNA and CDX2 mRNA expression and clinicopathologic features in colorectal cancers / 대한내과학회지

BACKGROUND: CDX1 and CDX2, members of the caudal-type homeobox gene family, control proliferation and differentiation of intestinal mucosal cells. Their expression is reduced commonly in colorectal cancers, but reports about the relationship between their expression and the clinicopathologic features are rare. The aim of this study was to examine CDX1 mRNA and CDX2 mRNA expression in colorectal cancers and to assess the relationship between their expression and the clinicopathologic features. METHODS: CDX1 mRNA and CDX2 mRNA expression were analyzed by real-time polymerase chain reaction in 48 colorectal cancers and their adjacent non-tumorous normal mucosas. RESULTS: CDX1 mRNA and CDX2 mRNA expression were decreased significantly in colorectal cancers than in normal mucosas (p=0.001, p=0.042, respectively). In comparison with paired normal mucosas, colorectal cancers showed decreased CDX1 mRNA expression in 64.6% (31/48) and decreased CDX2 mRNA expression in 66.7% (32/48). There was a statistically significant correlation between CDX1 mRNA and CDX2 mRNA expression in colorectal cancers (r=0.543, p<0.001). CDX1 mRNA and CDX2 mRNA expression were not related to age, sex, location of cancer, differentiation, lymphatic or vascular invasion, lymph node metastasis, stage and serum carcinoembryonic antigen level in colorectal cancers. CONCLUSION: CDX1 mRNA and CDX2 mRNA expression were decreased significantly in colorectal cancers, but were not related to the clininopathologic features.

Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells

The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.