CTCF, a Novel Regulator of Alternative Splicing
By binding to the promoter regions of some alternative exons, CTCF causes RNA polymerase II (pol II) to pause giving components of the splicing machinery time to incorporate the alternative exon. When the CTCF binding site is methylated, however, CTCF cannot bind, pol II does not pause, and the alternative exon is not incorporated into the transcript.
Alternative splicing, or the inclusion of different patterns of exons from the same gene, plays an important role in expanding the coding possibilities of a limited genome. The immune system is an ideal system to study this since alternative splicing is used to generate an almost unlimited number of antibodies against any pathogen we might encounter.
In a study published in the November 3, 2011, issue of Nature, Shalini Oberdoerffer, Ph.D., Head of RNA Processing in the Cellular Development Section of CCR’s Mouse Cancer Genetics Program, and Sanjeev Shukla, Ph.D., a Postdoctoral Fellow in the lab, investigated how exons with weak splicing signals are included using the CD45 gene as a model system. At different stages of lymphocyte development, exons 4, 5, and 6 are specifically incorporated or excluded from the CD45 mRNA. These researchers previously found a protein, hnRNPLL, regulates exons 4 and 6, but the mechanism for regulating exon 5 was unclear.
The researchers first analyzed previously published data describing protein associations with various regions of DNA. They found that DNA-binding protein CTCF, which is thought to shield inactive regions, had a strong interaction with CD45 exon 5, even in cells expressing high levels of CD45 protein. This interaction was also observed in mouse immune cells. Contrary to previous studies, this data suggested that CTCF binding may play an important role in exon 5’s inclusion in the CD45 mRNA transcript.
Next, by examining Burkitt lymphoma cell lines expressing high or low levels of CD45 that incorporates exon 5 (CD45-5), the researchers found that exon 5 was more likely to be included when CTCF was bound and that loss of CTCF protein in these cells reduced CD45-5 expression. One way CTCF binding might help incorporate exon 5 is by affecting the activity of RNA polymerase II (pol II), the enzyme that produces mRNA transcripts. More active pol II was associated with exon 5 in cells expressing high levels of CD45-5, which suggests that pol II spends more time, or pauses, at exon 5 in these cells. Depleting CTCF protein reduced pol II binding at exon 5. By slowing down pol II, CTCF could provide time for the splicing machinery to recognize the exon 5 splice site and incorporate exon 5 into the CD45 mRNA transcript.
To determine whether other factors are involved in CTCF-mediated incorporation of exon 5, the investigators used a cell-free assay system with purified DNA that included a CTCF binding site, RNA building blocks (oligonucleotides), and CTCF and pol II proteins. CTCF was able to bind the DNA directly, and bound CTCF caused pol II to pause in front of the CTCF binding site. Extending the incubation time or adding additional transcription factors allowed pol II to overcome the CTCF-induced pause, indicating that CTCF alone is capable of slowing, but not completely inhibiting, pol II transcription.
The researchers then investigated how CTCF binding to exon 5 is regulated since CTCF is always expressed but exon 5 is only included in the CD45 transcript at certain stages of lymphocyte development. The addition of a methyl-group to DNA nucleotides is known to interfere with CTCF binding, and in cell lines with methylated DNA at exon 5, CTCF failed to bind. To see whether this was also the case in normal lymphocytes, the investigators studied T cells that expressed higher or lower levels of CD45-5. In agreement with their cell line results, the researchers observed increased methylation and reduced CTCF binding in the T cells expressing less CD45-5. Inhibiting DNA methylation in the cells expressing lower CD45-5 increased CTCF binding and pol II pausing at exon 5. Importantly, these results are the first to link the processes of DNA methylation and alternative mRNA splicing.
Since CTCF binding sites are located in the exons of genes other than CD45, the researchers wondered whether CTCF binding to these other locations could also affect their splicing. To address this question, they reduced CTCF levels in cell lines and then looked for RNA sequences that differed with the loss of CTCF. The researchers determined that exons with a CTCF binding site downstream were preferentially excluded when CTCF was depleted. Similar to CD45 exon 5, CTCF binding downstream of these newly identified exons induced pol II pausing.
These studies have revealed the importance of CTCF in the inclusion of alternative exons. Changes in CTCF function may play a critical role in diseases such as cancer where altered splicing and DNA methylation patterns have been observed.Summary Posted: Tue, 11/01/2011
Shukla S, Kavak E, Gregory M, Imashimizu, M, Shutinoski B, Kashlev M, Oberdoerffer P, Sandberg R, Oberdoerffer S. CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature. November, 3, 2011. PubMed Link