The differentiation program of a cell is the result of events affecting the expression levels both of genes involved in the modification of chromatin structure and of genes specific of the cell type. We have utilized human carcinoma colon cells (CaCO-2) because they spontaneously reach the final phenotype of enterocyte-type polarized cells upon confluence. Among genes involved in modification of chromatin structure we have investigated on those controlling the methylation state of DNA. To this aim, we analyzed the levels of gene expression both of methylating enzymes (the maintenance Dnmt1 and the “de novo” methyltransferases Dnmt3a and Dnmt3b) and demethylating enzymes (demethylases MBD2, MBD4 and the T:G mismatch-specific thymine-DNA glycosylase) because DNA methylation state is defined by the equilibrium between methylating and demethylating activities. We have analyzed also the time-courses of apolipoprotein A1, villin (a brush border molecular marker), H-ferritin and angiogenin (typical of late differentiation steps of carcinoma colon cells) as typical of genes specific of the cell type and GAPDH and beta-actin as typical of constitutive genes. During differentiation of CaCO-2 cells to enterocyte-like phenotype, we examined the general methylation pattern of DNA by Southern blot analysis of genomic DNA extracted from cells at different days in cultures, digested with methylation sensitive enzymes and hybridized with Sat II probe. These experiments show that, during the investigated period, no variation of global genome methylation is detectable indicating that no general change of chromatin structure was occurring. Analysis by RT-PCR of gene expression showed a complex pattern consistent with the presence of four different classes of genes with different expression time courses. These are: 1. genes, such as apolipoprotein A-1, villin and H-ferritin, that are expressed already in the seeded cells at 1 day in culture, but their expression increases constantly during differentiation; 2. genes, such as Dnmt1 and MBD4, that switch from a lower to a higher expression level that remains thereafter constant during following days in culture. 3. genes, such as Dnmt3b, MBD2 and T:G mismatch glycosylase, that change in expression levels during differentiation with peculiar patterns in correlation with cell confluence and the appearance of a brush border phenotype typical of enterocyte-type polarized cells, indicating that these genes might be involved in activities controlling the phenotype changes. 4. genes, such as angiogenin, that are specifically expressed only late during differentiation. The invariance of the global methylation pattern indicates that the general chromatin reorganization to produce the final phenotype, has already occurred in the fresh cultured CaCO-2 cells. The variations in the activity of Dnmt3b and MBD2 and T:G mismatch glycosylase suggest that local change might still occur, to modify the expression of specific genes necessary to implement the differentation program. Work is in progress to assess if this is the case.

Distinct time courses of expression correlate with different gene functions in differentiating caco-2 cells

DI GIAIMO, ROSSELLA;IOVINE, BARBARA;DEL GAUDIO, ROSANNA;BEVILACQUA, MARIA ASSUNTA;RUSSO, TOMMASO;GERACI, GIUSEPPE
2004

Abstract

The differentiation program of a cell is the result of events affecting the expression levels both of genes involved in the modification of chromatin structure and of genes specific of the cell type. We have utilized human carcinoma colon cells (CaCO-2) because they spontaneously reach the final phenotype of enterocyte-type polarized cells upon confluence. Among genes involved in modification of chromatin structure we have investigated on those controlling the methylation state of DNA. To this aim, we analyzed the levels of gene expression both of methylating enzymes (the maintenance Dnmt1 and the “de novo” methyltransferases Dnmt3a and Dnmt3b) and demethylating enzymes (demethylases MBD2, MBD4 and the T:G mismatch-specific thymine-DNA glycosylase) because DNA methylation state is defined by the equilibrium between methylating and demethylating activities. We have analyzed also the time-courses of apolipoprotein A1, villin (a brush border molecular marker), H-ferritin and angiogenin (typical of late differentiation steps of carcinoma colon cells) as typical of genes specific of the cell type and GAPDH and beta-actin as typical of constitutive genes. During differentiation of CaCO-2 cells to enterocyte-like phenotype, we examined the general methylation pattern of DNA by Southern blot analysis of genomic DNA extracted from cells at different days in cultures, digested with methylation sensitive enzymes and hybridized with Sat II probe. These experiments show that, during the investigated period, no variation of global genome methylation is detectable indicating that no general change of chromatin structure was occurring. Analysis by RT-PCR of gene expression showed a complex pattern consistent with the presence of four different classes of genes with different expression time courses. These are: 1. genes, such as apolipoprotein A-1, villin and H-ferritin, that are expressed already in the seeded cells at 1 day in culture, but their expression increases constantly during differentiation; 2. genes, such as Dnmt1 and MBD4, that switch from a lower to a higher expression level that remains thereafter constant during following days in culture. 3. genes, such as Dnmt3b, MBD2 and T:G mismatch glycosylase, that change in expression levels during differentiation with peculiar patterns in correlation with cell confluence and the appearance of a brush border phenotype typical of enterocyte-type polarized cells, indicating that these genes might be involved in activities controlling the phenotype changes. 4. genes, such as angiogenin, that are specifically expressed only late during differentiation. The invariance of the global methylation pattern indicates that the general chromatin reorganization to produce the final phenotype, has already occurred in the fresh cultured CaCO-2 cells. The variations in the activity of Dnmt3b and MBD2 and T:G mismatch glycosylase suggest that local change might still occur, to modify the expression of specific genes necessary to implement the differentation program. Work is in progress to assess if this is the case.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/313856
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