We have employed 3C to identify long-range regulatory elements that may be involved in controlling the CFTR
gene. We identified four elements that interact with the CFTR
promoter specifically in CFTR
-expressing cells. These looping elements appear to be gene regulatory elements based on the following observations. First, two of the elements had been previously identified as putative regulatory elements and one of these elements (element II) has been found to directly affect CFTR
). Second, these elements contain DHSs and, in the case of elements III and IV patterns of histone modifications in CFTR
expressing cells that have previously been found to be predictive of distal regulatory elements (6
). Third, elements III and IV can synergistically activate the CFTR
promoter in expressing cells indicating that these elements can function as transcriptional enhancers.
Our studies did not identify all previously discovered regulatory elements around the CFTR
locus. There are several reasons for this. First, we did not analyze all restriction fragments throughout the 460 Kb surrounding the CFTR
promoter. For instance, initially we did not analyze interactions between the CFTR
promoter and a known enhancer that is located 10 kb downstream of the promoter in intron 1. The Harris lab has recently shown that this element interacts with and regulates the CFTR
). We have been able to confirm this looping interaction in subsequent 3C experiments using BsrGI (Supplementary Figure S2
). Second, we have focused on only those elements that consistently interact with the CFTR
promoter in CFTR
expressing cell lines (Caco2, HT29, HeLa S3). We did find that in some cell lines (e.g. HT29) there are additional elements that appear to frequently interact with the CFTR
promoter, for instance a region just downstream of the gene. Consistently, the Harris lab has shown that the region contains additional elements that loop to the CFTR
promoter in primary epididymis cells (23
). Thus, we have been able to confirm previously detected regulatory elements (elements I and II, as well as the known enhancer in intron 1), thereby validating our approach, and we have discovered novel CFTR
In this study we focused on elements III and IV. Further 3C experiments allowed us to narrow the regions of interaction of these elements down to ~1 kb. The results of the 3C mapping were fully consistent with reporter assays and both approaches identified the same putative regulatory elements. In addition, these elements coincide precisely with the presence of DHSs present in CFTR
expressing cells and with regions enriched in histone modifications associated with enhancers (6
). Combined, these independent lines of research provide strong evidence that bona fide CFTR
regulatory elements were identified. In a very recent similar study the Harris lab identified the same elements III and IV as CFTR
The long-range looping interactions described here are clearly correlated with expression of the CFTR
gene. However, we note that the frequency of looping is not quantitatively related to the level of CFTR
expression. The frequency of interaction between the CFTR
promoter region and elements III and IV is quite comparable in Caco2 cells and HeLa S3 cells, despite the fact that Caco2 cells express the gene at a much higher level (Supplementary Figure S1
). This indicates that these long-range interactions are not sufficient for high levels of expression. One possible explanation is that in Caco2 cells additional transcription factors and/or co-regulators bind these elements to further activate the gene. We also point out that the long-range looping interactions between the CFTR
promoter and elements I–IV are not required for a low level of expression, as GM06990 cells express very low, but detectable, levels of CFTR
while none of these long-range interactions are detected. It is possible that this low level is simply due to basal promoter activity or is the result of long-range interactions with other distal regulatory element. For instance, we find that in GM06990 cells the promoter is interacting with the enhancer element in intron 1 (Supplementary Figure S2
). Finally, we cannot rule out the possibility that immortalized cell lines such as GM06990 display somewhat altered expression patterns as compared to the primary cells of origin.
The four elements we uncovered in our 3C analysis interact with the CFTR promoter in all three CFTR expressing cells. These elements also all interact with each other, suggesting they form a single cluster of interacting chromatin segments (indicated by the grey circular area in ). We do note that the frequency of interaction between pairs of elements varies (), and is also somewhat different between cell lines. Thus, although the overall conformation of the locus is comparable in Caco2 and HeLa S3 cells, there may be slight differences in the frequencies with which long-range interaction occur.
Figure 5. Different models of the conformation of the CFTR locus. (A) The CFTR locus in a cell line in which the gene is not expressed. The locus assumes a linear conformation without specific long-range interactions. Thick black line indicates the CFTR gene. ( (more ...)
Our reporter assays suggest that elements III and IV activate the CFTR
promoter in a synergistic manner. This is interesting because elements III and IV directly associate with each other, which could provide a mechanism by which widely spaced elements can coordinately control gene expression. It is likely that specific protein complexes associate with these elements and mediate interactions between them and with the CFTR
promoter. Previous work from the Harris Lab has started to identify some proteins that could potentially associate with the DHS that is present in element IV. They found that this element contains two C
T polymorphisms, and these mutations alter the DNA binding of ARP-1 and HNF-4 in vitro
). They also found that C/EBP, CREB/ATF and AP-1 transcription factors can bind this region (22
). In addition, the transcription factor Myc can bind to element III [(10
), Iyer lab (University of Texas, Austin); http://genome.ucsc.edu/ENCODE/
]. Future studies will be aimed at identifying these protein complexes in more detail so that the mechanisms of long-range associations and their effect on transcription can be further dissected.
Identification of extra-genic elements that affect expression of CFTR will not only provide basic insights into spatio-temporal regulation of this important gene, it may also be important for genetic diagnosis of cystic fibrosis. A significant number of patients with cystic fibrosis symptoms do not appear to carry mutations in the CFTR exons or promoter, suggesting that extra-genic or intronic mutations may be present, e.g. in long-range acting gene regulatory elements. Mutations in regulatory elements can also result in disease characteristics that are distinct form the full cystic fibrosis phenotype, such as congenital bilateral absence of the vas deferens. In the absence of information of the positions of distant regulatory elements it is not feasible to screen for mutations in a very large genomic region. Thus, identification of CFTR regulatory elements, as we have described here, provides new targets for mutation screening. Furthermore, gene therapy approaches for cystic fibrosis could benefit from knowledge of gene regulatory elements by including such elements in CFTR gene targeting constructs.
We have shown that 3C technology can be used to discover novel regulatory elements throughout gene loci. A variety of 3C adaptations have recently been developed that allow large-scale detection of chromatin looping interactions (27
). Using these technologies, it may become possible to map the regulatory elements that control genes throughout the genome.