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Deciphering transcriptional regulation of NF-kB target genes using integrative omics approaches

Periodic Reporting for period 1 - RegulatioNFkB (Deciphering transcriptional regulation of NF-kB target genes using integrative omics approaches)

Reporting period: 2019-07-01 to 2021-06-30

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.
We have generated two powerful datasets that contain valuable information about the kinetics of the chromatin and gene expression in NF-kB pathway: RNA-sequencing (to study gene expression) and ATAC-sequencing (to study chromatin accessibility). Different timepoints after the activation of the pathway were chosen for the generation of these datasets. Datasets were analyzed accordingly and they will be shared with other scientists by submitting them to the GEO database once the article will be submitted for publication. We have already shown partially our data in conferences by poster presentations. From this analysis we could identify some transcription factors that might be involved in the NF-kB transcription regulation. We chose one transcription factor to follow-up and we generated a GFP tagged cell line on this gene in order to perform some validation experiments, how this transcription factor behaves during the NF-kB activation and the interaction of it with other proteins in the gene regulation.
In parallel, we developed a new method to study the proximal interacting proteins that we called pATurbo method. We fused protein A to the biotinyl transferase, Turbo ID. After permeabilization, cells are incubated with a primary antibody against the protein of interested and protein A Turbo ID, then, the antibody binds to the protein of interest and the protein A binds to the antibody. Upon addition of biotin, the surrounding proteins from the protein of interest are biotinylated by TurboID. Lastly, biotinylated proteins are captured by streptavidin beads and processed to be analyzed by mass spectrometry to unveil the identity of those proteins.
The pAturbo method has been recently accepted in Nature Communications (Off-the shelf proximity labeling for interaction proteomics. Irene Santos-Barriopedro, Guido van Mierlo and Michiel Vermeulen) and it has already being introduced in conferences and poster presentation. Moreover, we have already explained the method to non-scientist public by giving talks to high school students, exposing them to new scientific developments performed in Europe and to the work as a scientist.
The deviation from the initial objectives to the development of pATurbo method has generated the most impact from the project because it is a new technology that can be used for many studies in different fields, not only in the NF-kB context, but also for the study of other cellular mechanisms that are involved in other diseases. The main advantage comparing to other proximity labeling techniques is that no genetic manipulation is required allowing the application of this technique to primary cell lines and even to tissues in the future. We have already presented the method to the scientific community that showed a high interest by asking collaborations to implement the method in their projects and, once our Nature Communications publication will be available online for all of the scientific community, we can predict that the technique will be more extensively used. Moreover, the technique could be further exploited to the development of kits by companies for the identification of interacting proteins.
Furthermore, the data generated for the NF-kB pathway (RNA-sequencing and ATAC-sequencing) gave us information to continue the project for another publication and also can be used for the origin of other projects that might give important information for alternative therapies in cancer. Moreover, once we will submit the paper and the datasets to GEO, the data will be consulted by scientists for future research and increase the knowledge about NF-kB pathway.
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