Introduction and Findings
The discovery and validation of human transcriptional silencer elements are scarce. The lack of advance techniques and understanding of their characteristics have resulted in only a handful of silencer discoveries for genes such as human synapsin I, BDNF and CD4. This is in contrast to enhancers and “super-enhancers” of the human genome which have been broadly catagorised as regions with high H3K27ac enrichment, dense long-range chromatin interactions with target genes and transcription factor binding. Previous studies on silencers have involved complex assays and bioinformatic analysis to identify putative silencer regions. A study conducted by the Tucker-Kellog and Fullwood lab at NUS opted for a widely accessible and simpler method of silencer identification. Using a process analogous to finding “super-enhancers”, these researchers define silencers by strong and continuous H3K27me3 signal.
CRISPR excisions of their silencer regions led to an upregulation of tumour suppressor genes in their Leukaemia cancer cell line K562. This was further validated in-vivo by a mouse xenograft assay showing a significant reduction in overall tumour size. Changes in cell identity and differentiation ability were also apparent upon silencer region excision. So far this validates their chosen looping silencers as regions that repress target genes. 4C and histone modification experiments showed that the 3D landscape shifts towards favouring short ranged interactions and this is predicted by their epigenetic states. Another aspect of their study looked into the effects of H3K27me3 depletion on their silencers by inhibiting EZH2. Concordant changes in transcriptional repression and chromatin interactions were found through this assay which indicates the importance of chromatin states in maintaining the identity and characteristics of these silencers.
Opinions
This paper is remarkable in several aspects. First is their simplified approach to classifying human silencers. Functionally validated human distal looping silencers were previously only characterised in Drosophila and mice. Secondly, their functional validation on cancer cells revealed a silenced group of tumour suppressor genes. This highlights the potential of silencers as therapeutic targets for cancers. Lastly, EZH2 perturbation leads to changes in the chromatin landscape and thus silencer identity. This indicates the importance of chromatin states on establishing chromatin architecture.
There were a few things of note that were interesting but rather unexplored in this paper. The authors showed that a diverse range of transcription factors were enriched for different silencer regions but did not go into much detail about what these regulatory elements are doing for the silencers and whether this had any affect on their repressive capabilities or chromatin interactions. Another potential concern is the size of the silencers which are several hundred kilobases long. This raises the question of which regions are essential to silencing and what are the functions of the rest. Therefore, are these canonical silencer regions or “super-silencers”?
Overall, this paper is the first in simplifying the approach to find silencers and in my opinion it would be interesting to use this as a marker for silencers in our future bioinformatic analysis.