In recent years, next-generation sequencing technologies have revealed that the vast majority of the human genome is transcribed into non-coding RNA. Initially, it was thought that these transcripts were 'junk RNA' transcribed from 'junk DNA'. This hypothesis was refuted by thousands of studies reporting that non-coding RNAs regulate a broad spectrum of cellular processes - including transcription and translation. It has also become clear that disregulation of these regulatory elements is associated with human pathologies, such as cancer.
Rui Lopes and his colleagues applied Global Run-on sequencing, a high-throughput sequencing technique that measures nascent RNA transcription, to detect enhancer-associated non-coding RNAs transcribed from active enhancers. Their experiments identified thousands of enhancers that were activated by critical transcription factors (e.g. p53) and might play a role in cancer development.
The team then pioneered the application of CRISPR-Cas9 to test the function of enhancers by mutating them and examining the resulting phenotype in a high-throughput manner (i.e. genetic screening). Their experiments led to the identification of of several enhancers that regulate the expression of critical genes and poorly characterized genes in human cells.
In both cases, they showed that these enhancers are absolutely required for the growth of cancer cells. Their findings provide the basis for better diagnosis and therapies of human cancer.
Rui Lopes defended his PhD thesis on 9 March at Erasmus University Rotterdam.
Title: CRISPRing the Human Genome for Functional Regulatory Elements.
Promotor: prof.dr.Reuven Agami (Netherlands Cancer Institute)