Cheung lab research

Genetics of human gene expression

It is well known that individuals differ at the DNA sequence level; however, the effect of DNA sequence variants on phenotypes remains largely unknown. Since the expression level of genes has important effects on cellular phenotypes, we examined the extent of individual variation in gene expression. We found extensive variability and a heritable component to this variation. This allows us to treat expression levels of genes as quantitative traits and to screen the genome for variants that influence these gene expression phenotypes.

Using genome-wide linkage and association analyses in large families as well as molecular studies, we have identified polymorphic regulators that influence expression levels of a few thousand human genes. These include cis- and trans-acting regulators, as well as regulators that influence the expression of many genes. More than 60 percent of the regulators were not known to play a role in gene expression regulation. Gene knockdown, metabolic perturbation assays and studies of RNA-protein and protein-protein interactions have allowed us to uncover how these regulators influence gene expression.  We are continuing these genetic studies and extending our approach to measure gene expression more precisely and characterize the regulatory landscape of gene expression in human cells at baseline and following cellular stress.

 

RNA-DNA Differences

It is generally believed that RNA sequences are identical to their corresponding DNA sequences.  We uncovered RNA-DNA sequence Differences (RDDs) in human cells beyond the known ADAR and APOBEC-mediated RNA  editing.  We found all 12 types of RDDs, including transversions.  Based on preliminary data, we estimate RDD frequency as 1 per 10,000.  We are leveraging additional samples and advances in DNA and RNA-sequencing to  improve the precision of this estimate.  Our results show individual differences in the frequencies and levels of RDDs, thus, demonstrating a layer of genetic variation beyond those in the DNA sequences.  

In collaboration with Professor John Lis, we studied the sequences of nascent RNAs and found that RDD formation occurs soon after transcription, about 55 nucleotides from the RNA polymerase II active site.  RDDs are formed after  RNA synthesis and they are not likely a direct consequence of modified base incorporation.  However, they are formed before ADAR-mediated RNA editing.  We are now examining other co-transcriptional events such as R-loop  formation to determine if they co-occur with RDD formation. 

We found RDDs lead to peptides that differ from their corresponding DNA.  Using mass spectrometry, we identified peptides that correspond to both the RNA and DNA sequences at RDD sites.  In addition, preliminary results show  that like splicing, the frequency of some RDDs is affected by cellular perturbations.  Our on-going study focuses on identifying the mechanisms that lead to RDD formation.

 

Genetics of Cellular Response to Stress

Appropriate response to stress is critical for maintaining cellular homeostasis.  Our work focuses on two types of stress: endoplasmic reticulum (ER) stress as a result of excessive unfolded protein, and radiation-induced DNA and other cellular damages.  Secretory cells such as human B-cells must be able to handle large protein loads.  Failure to respond to ER stress can lead to diseases such as immune-deficiency and neurodegenerative diseases.  Similarly, human cells are increasingly exposed to radiation through the environment and in medical settings.  Radiation-based tools have significant medical benefits; however, cellular damage can result from exposure to radiation.  We and others have identified genes that change in expression in response to ER stress and radiation exposures.  We have shown that these gene expression responses differ across individuals.  Treating these changes in gene expression as quantitative traits, we have identified polymorphic regulators that influence gene expression response and determined gene interactions by network analyses.  To advance these findings into mechanistic understanding of how stress response affects cell fate, we are identifying the transcriptional steps that regulate these responses.  We are studying how RNA polymerase activity and RNA stability are regulated to fine tune cellular responses to stress.  The results should advance our basic understanding of transcription and identify risk factors of diseases characterized by inadequate stress responses.