Final Report Summary - CHR_LNCRNA (Identification and functional characterization of lncRNA-chromatin protein complexes associated with specific chromatin modifications in breast cancer)
Great advances in sequencing technology in early-mid 2000 have led to the discovery of thousands of non-coding RNAs (ncRNAs) in mouse and human cells. Unlike ‘conventional’ messenger RNAs, ncRNAs do not encode proteins.
They, however, seem to regulate the processes involved in messenger RNA synthesis (transcription) or the fate of messenger RNAs (e.g. stability, translation into proteins) through multiple mechanisms. Importantly, a number of ncRNAs have been implicated in normal development and pathogenesis including cancers, immunological defects and neurological disorders. Therefore, it is important to understand how ncRNAs function in cells.
Despite intense efforts over the past years, biological functions of many ncRNAs, especially a class of ncRNAs known as long non-coding RNAs (lncRNAs), still remain unclear. Recent studies, however, suggest that some of development- or disease-associated lncRNAs are bound to chromatin factors and modulate transcriptional activity by regulating recruitment/activity of chromatin-binding/ modifying proteins at target genes. Consistent with this, it has been shown that several chromatin factors are capable of binding to RNAs in vitro and/or in vivo.
Thus, in this project I focused on the relationship between lncRNAs and chromatin factors. I used a breast cancer cell line, MCF7 cells and aimed:
- to develop a methodology for systematic identification of RNA-binding chromatin factors that specifically associate with a chromatin mark (H3K9me3)
- to identify lncRNA species associated with several identified RNA-binding chromatin factors
- to investigate biological roles of identified lncRNA-chromatin factor interactions in breast cancer formation/progression.
I first attempted to develop a new, versatile methodology for identification of potential RNA-binding chromatin factors. This methodology is hereon referred as photoactivatable RNA crosslinking and pulldown (PAR-CLPD). PAR-CLPD is based on several pre-existing state-of-the-art experimental techniques: UV-mediated crosslinking of RNA and proteins, non-radioactive in situ labeling of proteins, peptide pulldown and quantitative SILAC-based mass spectrometry. In general, experimental parts of technology development have progressed steadily after overcoming several technical difficulties. Analysis part of this task happened to be more challenging since this was entirely new and we dealt with very complex samples. After multiple optimization procedures, we identified more than 100 RNA-binding proteins using the methodology. The large majority of identified factors were known RNA-binding proteins (including splicing factors, ribosomal proteins, RNA helicases etc.) and were mostly 'background' proteins which are non-specifically associated with histone H3 tails. We were also able to obtain more than 10 novel RNA-binding chromatin protein candidates that specifically bind to methylated lysine 9 residue of histone H3 tail (H3K9me3). Interestingly, deregulation of a subset of these novel RNA-binding candidates have also been implicated in cancers.
We are now performing a few control and verification experiments as well as trying to improve the reproducibility of the results. I am confident that these tasks will be completed and the outcome of this research will be put together into a publication in near future.
Although the methodology development has taken much longer than initially planned, I believe that perfecting this task is the most crucial part of this project as the success of any follow-up experiments will highly depend on the reliability or quality of this part. Since nature and functions of many lncRNAs are still enigmas, lncRNA research is likely to remain as one of the 'hottest' research areas for next decade. I expect that the outcome of the methodology development and publication of the results/datasets will greatly accelerate lncRNA research in both basic and clinical sciences in EU countries. A collective outcome of these researches can be translated into novel therapy/drug development for cancers and other diseases in future.
They, however, seem to regulate the processes involved in messenger RNA synthesis (transcription) or the fate of messenger RNAs (e.g. stability, translation into proteins) through multiple mechanisms. Importantly, a number of ncRNAs have been implicated in normal development and pathogenesis including cancers, immunological defects and neurological disorders. Therefore, it is important to understand how ncRNAs function in cells.
Despite intense efforts over the past years, biological functions of many ncRNAs, especially a class of ncRNAs known as long non-coding RNAs (lncRNAs), still remain unclear. Recent studies, however, suggest that some of development- or disease-associated lncRNAs are bound to chromatin factors and modulate transcriptional activity by regulating recruitment/activity of chromatin-binding/ modifying proteins at target genes. Consistent with this, it has been shown that several chromatin factors are capable of binding to RNAs in vitro and/or in vivo.
Thus, in this project I focused on the relationship between lncRNAs and chromatin factors. I used a breast cancer cell line, MCF7 cells and aimed:
- to develop a methodology for systematic identification of RNA-binding chromatin factors that specifically associate with a chromatin mark (H3K9me3)
- to identify lncRNA species associated with several identified RNA-binding chromatin factors
- to investigate biological roles of identified lncRNA-chromatin factor interactions in breast cancer formation/progression.
I first attempted to develop a new, versatile methodology for identification of potential RNA-binding chromatin factors. This methodology is hereon referred as photoactivatable RNA crosslinking and pulldown (PAR-CLPD). PAR-CLPD is based on several pre-existing state-of-the-art experimental techniques: UV-mediated crosslinking of RNA and proteins, non-radioactive in situ labeling of proteins, peptide pulldown and quantitative SILAC-based mass spectrometry. In general, experimental parts of technology development have progressed steadily after overcoming several technical difficulties. Analysis part of this task happened to be more challenging since this was entirely new and we dealt with very complex samples. After multiple optimization procedures, we identified more than 100 RNA-binding proteins using the methodology. The large majority of identified factors were known RNA-binding proteins (including splicing factors, ribosomal proteins, RNA helicases etc.) and were mostly 'background' proteins which are non-specifically associated with histone H3 tails. We were also able to obtain more than 10 novel RNA-binding chromatin protein candidates that specifically bind to methylated lysine 9 residue of histone H3 tail (H3K9me3). Interestingly, deregulation of a subset of these novel RNA-binding candidates have also been implicated in cancers.
We are now performing a few control and verification experiments as well as trying to improve the reproducibility of the results. I am confident that these tasks will be completed and the outcome of this research will be put together into a publication in near future.
Although the methodology development has taken much longer than initially planned, I believe that perfecting this task is the most crucial part of this project as the success of any follow-up experiments will highly depend on the reliability or quality of this part. Since nature and functions of many lncRNAs are still enigmas, lncRNA research is likely to remain as one of the 'hottest' research areas for next decade. I expect that the outcome of the methodology development and publication of the results/datasets will greatly accelerate lncRNA research in both basic and clinical sciences in EU countries. A collective outcome of these researches can be translated into novel therapy/drug development for cancers and other diseases in future.