The regulation of gene expression is a complicated affair. A vast array of control mechanisms exist that can adjust the levels of gene expression products to match the needs of the cell. Messenger RNA (mRNA) can be processed to alter its stability, protein translation from mRNA can be controlled, and the stability and/or activity of the protein can be altered via post-translational modifications. The first point of control of gene expression is the initiation of gene transcription.
Setten et al studied an eRNA transcribed from an enhancer near the gene encoding a transcription factor called CEBPA (CCAAT enhancer-binding protein alpha). CEBPA is a transcription factor involved in many processes, including cell cycle inhibition and tumorigenesis, and expressed in specific cell lineage. It also plays a role in maintaining cell identity. To study CEBPA protein levels, the authors carried out a quantitative near-infrared fluorescent Western blot imaged on an Azure Sapphire™ Biomolecular Imager (Figure 7). To quantify changes in protein level between samples, the fluorescent signal from the Western blot was normalized to the total protein loaded, as was visualized on the Sapphire.
The researchers set out to determine whether an eRNA transcribed from an enhancer 9kb downstream from the transcription start site of the human CEBPA gene was involved in regulating CEBPA expression. They called this eRNA CRED9. The authors found that levels of CEBPA mRNA and the CRED9 eRNA were correlated across several different cell lines; when CRED9 was high, CEBPA mRNA was also high. They then knocked down CRED9 in a cell line and found that when CRED9 levels were reduced, CEBPA mRNA and CEBPA protein levels were also reduced.
Finally, knockdown of CRED9 reduced the amount of a histone H3K27ac bound to the enhancer, indicating that the activity of the enhancer region was reduced. These results lead the authors to propose that CRED9 and other eRNAs may have an active role in enhancer function and gene regulation.
Requirements of transcription initiation
Transcription initiation has several requirements. The chromatin structure must open to make the gene accessible to the transcriptional machinery. In eukaryotic cells, the promoter sequence of the gene must be bound by transcription factors that direct RNA polymerase to the gene to begin transcription. Transcription initiation is made more likely by the binding of activator proteins to other DNA regions near the promoter called enhancers, which can be located up- or downstream of the transcription start site.
Genome-wide sequencing experiments have revealed that RNA molecules are transcribed from many enhancer regions, indicating the enhancer regions may not merely be binding sites for activator proteins. These enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) and are not translated into proteins. It is possible that eRNAs may simply be the result of non-specific transcription by RNA polymerase and serve as a sign that chromatin is open and accessible to RNA polymerase in a region or DNA. Alternatively, there is evidence some eRNAs may serve an active role in regulating gene expression by themselves binding to and changing the activity of proteins.
In addition to multichannel and NIR fluorescent imaging, the Sapphire Biomolecular Imager provides chemiluminescence, densitometry, phosphor and white light imaging of blots, gels, tissues, and more. Download a free copy of the Sapphire Applications Booklet and learn about how you can add more applications to your arsenal here.