Variety is the spice of life; and variation is the substrate of evolution. One of the important ways nature creates diversity is through alternative splicing, where different forms of the same gene produce protein variants called isoforms. However, the biological significance of different isoforms is not well understood for many proteins.
For example, a protein known as hepatocyte nuclear factor 4ɑ (HNF4ɑ) is thought to act as a gene regulator when two identical units come together to form a homodimer. But many different isoforms of HNF4ɑ exist, and the impact of each isoform pair is not known.
A recent discovery a team led by Ee Chee Ren from A*STAR’s Singapore Immunology Network (SIgN) has revealed that different HNF4ɑ isoforms can indeed cross-interact, forming heterodimers. These HNF4ɑ heterodimers control different sets of downstream genes, explaining how a single gene regulator can have such a diverse range of targets.
Using reverse transcriptase polymerase chain reaction, the team looked for and found the mRNAs of all 12 isoforms present in 15 different human tissues ranging from the skin to the testes. Together, the entire array of isoforms controlled the expression of 54 different genes.
“Although up to 12 isoforms have been identified in recent years, not all isoforms are known to be transcribed. We took the step to show that the postulated isoforms did exist in human cells, and with that in place, we could see a pattern of potential interaction between the different isoforms,” Ren said.
To prove that different types of isomers can bind to each other and form heterodimers, the researchers used a technique called co-immunoprecipitation, labeling each isomer with a protein ‘tag.’ “When we selectively ‘pull down’ one protein and find that the other tag is also retrieved, we then know that these two proteins are paired together,” Ren explained.
The researchers went on to show that heterodimer combinations such as HNF4ɑ3-8, HNF4ɑ6-12 and HNF4ɑ5-8 activated liver detoxification enzyme gene CYP7A1 and inflammatory genes IL-6 and IL17A more strongly than their corresponding isoform homodimers.
“Diseases like colorectal cancer have altered HNF4ɑ2 and HNF4ɑ8 levels. Knowing that heterodimerization exists allows us to investigate which genes are regulated by both homodimerization and heterodimerization of these isoforms. We also postulate that certain carriers of the hepatitis B virus may have a high viral load resulting from HNF4ɑ heterodimer combinations that favor viral replication,” said Ren.
Ren’s team has also successfully used CRISPR-Cas9 gene editing to create permanent HNF4ɑ knockout cell lines and plans to use them to further characterize the effects of multi-combination HNF4ɑ heterodimers in the absence of basal HNF4ɑ dimer activities.
The A*STAR-affiliated researchers contributing to this research are from the Singapore Immunology Network (SIgN).