Abstract
The human CYP19 gene encodes aromatase, which converts androgens to estrogens. CYP19 mRNA variants are transcribed mainly from three promoters. Quantitative RT-PCR was used to measure the relative amounts of each of the three transcripts and determine the on/off state of the promoters. While some of the promoters were silent, CYP19 mRNA production differed among the other promoters, whose estimated transcription levels were 0.001% to 0.1% of that of the TUBB control gene. To investigate the structural aspects of chromatin that were responsible for this wide range of activity of the CYP19 promoters, we used a fractionation protocol, designated SEVENS, which sequentially separates densely packed nucleosomes from dispersed nucleosomes. The fractional distribution of each inactive promoter showed a similar pattern to that of the repressed reference loci; the inactive regions were distributed toward lower fractions, in which closed chromatin comprising packed nucleosomes was enriched. In contrast, active CYP19 promoters were raised toward upper fractions, including dispersed nucleosomes in open chromatin. Importantly, these active promoters were moderately enriched in the upper fractions as compared to active reference loci, such as the TUBB promoter; the proportion of open chromatin appeared to be positively correlated to the promoter strength. These results, together with ectopic transcription accompanied by an increase in the proportion of open chromatin in cells treated with an H3K27me inhibitor, indicate that CYP19 mRNA could be transcribed from a promoter in which chromatin is shifted toward an open state in the equilibrium between closed and open chromatin.
Highlights
Transcription is often defined as the first regulatable step in gene expression, and in this step a specific gene is targeted within the genome
Another assay for chromatin structure, called FAIRE, can concentrate a protein-free and open region of chromatin into an aqueous phase following extraction with phenol [36]
Because sheared chromatin obtained from more than a million cells is used as starting materials in the assay, data represent the proportion of graded structures in a mixture of multiple chromatin fragments
Summary
Transcription is often defined as the first regulatable step in gene expression, and in this step a specific gene (or set of genes) is targeted within the genome. When lysine residues of histones become acetylated, nucleosomes comprising these acetylated histones lose affinity for DNA; the chromatin structure loosens, and a respective promoter becomes more accessible [15] This type of structural alteration occurs at activated promoters following TF-mediated recruitment of histone acetyltransferase (HAT). A recent statistical analysis indicates that the level of expression of a gene is related to the binding of TFs [20] This finding suggests that chromatin structure at physically distinct promoters that are combinatorially affected by multiple TFs could be responsible for the transcription level, namely the strength of a respective promoter. To investigate a relationship between the transcriptional state and the chromatin structure of promoters with different activity, we compared the activity of CYP19 promoters in three human cell lines in which the CYP19 gene is transcribed or not transcribed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
