Transcription of membrane-associated DNA highlights Lieb’s encore
By Jeffrey Stumpf
Shortly after his participation at an NIEHS minisymposium earlier in the week, University of North Carolina at Chapel Hill (UNC-CH) professor Jason Lieb, Ph.D., spoke Jan. 20 at NIEHS about how the organization of DNA in the genome affects gene expression. As part of the Laboratory of Molecular Carcinogenesis (LMC) Seminar Series, hosted by staff scientist Harriet Kinyamu, Ph.D., Lieb demonstrated that the interactions between chromosomes and the nuclear envelope are likely important in orchestrating transcriptional programs.
Transcriptional processes determine the extent to which genes are expressed. The initiation of gene expression is influenced by promoters, which contain DNA sequences that recruit the binding of transcription factors. Understanding how transcription factors bind to the proper DNA sequences is fundamental to future development of cancer therapies, Lieb explained.
“Our studies of gene regulation will lead to better predictions of what happens when transcriptional programs go haywire in cancer,” Lieb said, pointing to Burkitt’s lymphoma as an example of what goes wrong when transcription factors bind to inappropriate targets. “These studies may lead to new therapies, based on maintaining proper chromosome organization and inhibiting transcription factor binding to inappropriate targets.”
Small DNA loops accommodate high levels of transcription
UNC-CH postdoctoral fellow Kohta Ikegami, Ph.D., carried out all of the experiments Lieb discussed, using the simple 959-celled roundworm Caenorhabditis elegans. His work determined that both ends of the autosomal chromosomes are plastered to the nuclear membrane, while the centers looped away. The 1000 kilobase membrane-bound plastered ends contained subdomains of approximately 60 kilobases that were defined by smaller chromatin loops emanating from the membrane. Interestingly, transcription of the loop region increased as the size of the loop decreased.
Lieb asked if the DNA sequence determines the association with the membrane. Results from an elegant chromosome fusion mutant suggested that the formerly end DNA region in the fused chromosome was still membrane-bound despite being in the middle of the chromosome, and that DNA or chromatin composition determined binding to the nuclear envelope.
The increase in transcription at small loops, Lieb hypothesizes, may occur for convenient use of cellular resources. “Small loops may concentrate transcriptional factors, allowing increased residency times and component recycling,” Lieb remarked.
Nuclear pore proteins interact with the transcription machinery
In search of other relationships between transcription and the nuclear membrane, Lieb turned his attention to the nuclear pores, channels that host the active transport of thousands of molecular interactions per second. One hypothesis is that transcription of highly active genes could occur near nuclear pores to facilitate the transport of RNAs to the ribosomes.
Lieb identified 223 locations where nuclear pore proteins bound to DNA. These sites corresponded to binding sites where RNA polymerase III (pol III) initiates transcription of tRNAs and other small RNAs important for normal cell growth.
How important is nuclear pore location to transcription? Reducing the levels of a nuclear pore protein prevented nuclear pore assembly, changing how the chromosomes interact with the nuclear membrane. Also, loss of nuclear pores caused the mistargeting of the alternative RNA polymerase pol II to pol III-specific genes. “To our knowledge, this is the first demonstration of modifying polymerase specificity of a promoter without changing the DNA sequence of the promoter,” Lieb concluded.
Lieb wants to know which of these processes is calling the shots. Does the Pol II transcription machinery peel the chromosome away from the nuclear membrane, or does the membrane actively prevent Pol II from binding to genes? Does the Pol III machinery bring the chromosome to the nuclear pore, or does the nuclear pore function in the initiation of transcription? Lieb’s research will be the foundation for uncovering whether chromosome association with the nuclear envelope is used as a primary mechanism to regulate and coordinate transcription throughout the genome.
(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group.)