Epigenetic modifications and conserved, non -coding DNA play: A role in regulation of type IV collagen gene expression
Type IV collagens are components of basement membranes throughout the body and are involved in maintenance of the structural integrity of tissues as well as cellular differentiation, growth, and adhesion. Members of this collagen family are uniquely arranged in pairs in a head-to-head orientation and share a proximal promoter region. The COL4A5-COL4A6 gene pair is involved in numerous human diseases and cancer metastasis. For these reasons, defining the mechanisms that regulate collagen gene expression is of specific interest. To study type IV collagens, an in vitro model system was characterized. Comparative genomics was utilized to identify conserved, non-coding DNA in COL4A5 and COL4A6. These sequences were transfected into cell lines differing in type IV collagen expression and tested for the ability to regulate transcription of a reporter gene. Each cell line was also treated with the epigenetic modifying agents, 5-Aza and TSA. The effects on type IV collagen expression were determined. The COL4A5-COL4A6 promoter region was extensively characterized using ChIP analysis; antibodies against RNAPII, acetylated histone H3, and H3K9me2 were used. Additionally, bisulfite sequencing was carried out on each cell line to determine the methylation status of CpG dinucleotides in the promoter.
Cell lines differing in expression of COL4A5 and COL4A6 were identified: (1) SCC-25 keratinocytes and HEK-293 cells transcribed both COL4A5 and COL4A6, (2) HT-1080 cells selectively activated COL4A5, and (3) SK-N-SH neuroblastoma cells did not express either gene. In SK-N-SH cells, histone modifications were shown to facilitate formation of condensed chromatin to prevent transcription initiation; repression was independent of DNA methylation. Activation of COL4A5 and COL4A6 in SCC-25 and HEK-293 cells involved acetylation of histones, although differences between the two cell types were seen. In addition, conserved, non-coding sequences were shown to affect transcription of a reporter gene; these sequences may be interacting with the transcription machinery to modulate collagen expression. Finally, repression of COL4A6 in HT-1080 cells appeared to be mediated through DNA methylation of the promoter; selective activation of COL4A5 may involve conserved, non-coding DNA. In summary, epigenetic modifications as well as conserved sequences are intimately involved in regulation of type IV collagen gene expression.
0379: Cellular biology