The collection of genes expressed, when they are expressed and how much they are expressed are what give a cell its personality. Consequently, regulating gene expression is very important and there are many molecular mechanisms through which it is achieved. Understanding these mechanisms provides a clearer picture to deciphering what goes wrong in cells when this regulation is lost, for example in cancer cells. A recent paper published in Cell by Cho et al. 1 has provided evidence for which expression of a commonly mutated gene in cancer cells, MYC, is regulated, involving the promoter of the gene PVT1.
So, how are genes expressed?
A key initiating event of gene expression is the assembly of the transcriptional apparatus at and near the promoters of target genes. A ‘promoter’ refers to a DNA sequence that is recognised by proteins such as the general transcription factors that can bring in the RNA polymerase (RNAP) which catalyses the production of mRNA from the DNA gene sequence. To aid recruitment of RNAP, there is another nearby DNA sequence called the enhancer. Enhancer sequences can be bound by other proteins known as transcription factors that by interacting with the GTFs and other proteins bring in RNAP to initiate transcription (Figure 1). Enhancers do more than just enhance RNAP recruitment though. Interactions made between the enhancer-recruited factors and the gene also aid the transition to transcriptional elongation (the bulk of transcription).
In normal cells the process of transcription is tightly regulated to ensure that the right genes are expressed at the right time and at the right amount. However, this process becomes aberrant and ‘out of control’ in cancer cells – expression of genes may increase or decrease, or just occur continuously.
Tumour suppressors and oncogenes
Certain genes are commonly referred to as tumour suppressors or oncogenes. The terms are used to describe how a gene influences the potential for tumour development. Unsurprisingly, tumour suppressors are genes whose expression reduces the potential for tumours to develop. Conversely, oncogenes (or technically proto-oncogenes that become oncogenes when mutated) are genes whose aberrant expression can increase the chance of tumour development. For example, MYC is an oncogene, whose overexpression has been implicated in >50% of human cancers.
Expanding the role for non-coding RNA
PVT1 is a non-coding RNA (ncRNA), meaning the gene product doesn’t code for a protein but functions in its RNA form after transcription. There are many examples of ncRNAs that are expressed and many functions for them have been characterised. For example, they can act as a scaffold for proteins or they can recognise complementary RNA sequences. PVT1 has previously been shown to act as an oncogene, since the RNA can stabilise MYC protein2. However, the interesting finding by Cho’s team was that it wasn’t the PVT1 ncRNA that was acting as a tumour suppressor to prevent MYC expression, it was the PVT1 promoter.
To distinguish between the effects of PVT1-ncRNA expression and the PVT1 promoter in control of MYC expression, the team exploited the CRISPR technologies. CRISPR interference (CRISPRi)*, to reduce PVT1 expression, enhanced cell proliferation in multiple cancer cell lines. But, what causes this increased cellular proliferation?
To tackle this question the team used RNA-sequencing (RNA-seq) to identify genes whose RNA levels increased in the CRISPRi-PVT1 cells. After confirming that PVT1 levels were reduced, the gene whose RNA levels increased the most was the protooncogene MYC. Increased levels of MYC mRNA and cell proliferation were not seen however when truncated transcripts of PVT1 were transcribed (i.e non-functional PVT1-ncRNA). This supports the role of the PVT1 promoter as causal instead of PVT1 itself.
An intragenic enhancer
Enhancer elements reside within the PVT1 DNA locus that regulate expression of the gene. These enhancers promote expression of PVT1 through DNA looping (Figure 1). MYC is a neighbour of PVT1 and so putting these facts together started to suggest that DNA looping and enhancer-promoter contacts may be key to the regulation of PVT1 and MYC.
To test this the team used a combined technique of Chromatin Immunoprecipitation (ChIP) (targeting the enhancer mark H3K27ac) and Chromatin conformation capture (Hi-C) called HiChIP to map the enhancer interactome. Compared to a control, the CRISPRi-PVT1 cells showed a clear decrease in contacts made between the PVT1 intragenic enhancers and PVT1 promoter with an increase in contacts made instead with the MYC promoter (Figure 2). In this way, the promoters of MYC and PVT1 are competing to interact with the enhancers, with PVT1 ‘winning’ in normal cells. DNA elements that act in this manner to prevent gene expression are referred to as boundary elements (or silencers). Thus, here the PVT1 promoter is acting as a boundary element. Since it also controls aberrant expression of the proto-oncogene MYC, the PVT1 promoter is acting as a tumour suppressor boundary element… cool!
A TAD more specific
The role of the PVT1 promoter was found in some cancer cells lines… emphasis on the some. Other cell lines such as HeLa cervical carcinoma cells did not show MYC upregulation when CRISPRi-PVT1 was used. So, what is causing these cell-specific effects?
Put simply, the looping is different.
Instead of using the PVT1 intragenic enhancer elements, in other cell types, the MYC promoter forms alternative DNA loops, forming interactions with a different enhancer – the CCAT1 enhancer.
So, what can we take from this study?
The structure of the human genome is becoming increasingly more linked and implicated as causal in numerous human diseases. This study on the PVT1 promoter and MYC expression further adds to our growing knowledge into the complexities of chromatin conformation.
PVT1 is often mutated in cancer. It is a common breakpoint for DNA translocations in Burkitt’s lymphoma. These translocations and mutations can disrupt promoter-enhancer contacts, consequently disrupting the regulation of gene expression. Examining this link more carefully through cancer genome sequencing, in conjunction with the results of this study, will aid a fuller understanding of causes and potential therapies for cancer patients.
- (Read the paper to find out how the regulation lies at promoter proximal pause release!!) Cho, S. W. et al. Promoter of lncRNA Gene PVT1 Is a Tumor-Suppressor DNA Boundary Element. Cell 0, 1398–1400.e22 (2018).
- Tseng, Y.-Y. et al. PVT1 dependence in cancer with MYC copy-number increase. Nature 512, 82–86 (2014).
*More on CRISPR and CRISPRi: https://asheekeyscienceblog.com/2017/09/07/crispr-just-got-snappy/