NF-kB pathway is activated by inflammation and regulates the expression of different genes involved in different cellular processes such as apoptosis or growth. Upon activation, NF-kB target genes show different kinetics in expression suggesting that different factors might be involved in the regulation. How the expression of those genes is orchestrated is relatively unknown, specially, for the genes that are transcribed later (late genes). We tried to elucidate the mechanisms of gene expression in this pathway using different approaches with special attention to late genes.
NF-kB pathway plays a key role in cancer being one of the main subjects of study for the cure of the disease. In some cancers this pathway is constitutively activated increasing the lethality while in others it might act as a tumor suppressor. So far, the therapies associated to this pathway consist on the inhibition of the full pathway compromising in most cases the immunological system of the patient. The development of a more targeted therapy towards the partial inhibition of NF-kB, particularly to the target of a specific gene or subset of genes, would overcome this problem. In this project, we suggested different methods in order to study the regulation of the expression of particular genes that might be involved in the poor prognosis of the cancer. This knowledge would open possibilities to target with a drug specifically the expression of a gene or a subset of genes without inhibiting completely the NF-kB pathway.
The objectives were:
1. Global profiling of gene expression dynamics after TNF treatment in HeLa and HCT116 cells.
2. Identification of proteins interacting with NF-kB regulated promoters upon TNF treatment.
3. Characterization of the function and recruitment hierarchy of proteins that interact with the NF-kB responsive promoters
4. Investigating gene specificity of the regulatory mechanism
5. Validating discovered mechanism(s) using intestinal organoids.
We have studied the dynamics of the NF-kB gene expression performing RNA-sequencing on two different cell lines. Then, we studied the changes on the accessibility of the chromatin by ATAC-sequencing and we observed also dynamical changes in the accessibility of the chromatin. Both datasets confirm the different kinetics and dynamics that NF-kB target gene shows upon activation at expression and chromatin levels. We could find specific motifs in regions whose accessibility change upon activation of the pathway and identify transcription factors that bind to those regions. Some of those transcription factors might be involved in NF-kB pathway regulation and that is something that needs to be further explored. In summary, now valuable datasets have been generated and analyzed to continue with the NF-kB pathway study.
One of our goals was to purify the proteins bound to particular promoters in order to identify the factors that might be regulating the expression. The purification of the proteins bound to DNA remains so far challenging and it has been performed in repetitive sequences in genome. Although we tried to set up the conditions of this technique for promoters, it seems that further optimizations and/or even new technology would be required to perform single locus purification.
In the same line, as a last conclusion, new technologies to study protein-protein interactions would be required to explore new mechanisms. During this period of time, we deviated from the original proposal and we developed a new technique to study proximal interactions in order to improve the study of the mechanisms regulating NF-kB pathway that can be also applied to the study of other different pathways.
