Regulation of gene and protein expression : two model systems

Genetic information is expressed by DNA transcription and mRNA translation. With few exceptions, every cell of an organism, or the human body, contains the same genetic information. Yet the structure and function of different tissues are highly variable. The enormous complexity of life that can be produced from just four DNA bases is a result of gene regulation. The cell regulates expression of its genes through complex nucleic acid:protein and protein:protein interactions in the nucleus and the cytoplasm during transcription, translation, and a series of related, closely linked, processes. We explore different methods of regulating gene and protein expression by examining two select model systems.

Viruses are obligate intracellular parasites, and examination of viral models has provided fundamental insights into eukaryotic biology. Human Immunodeficiency Virus type 1 (HIV-1) and Equine Infectious Anemia Virus (EIAV) encode Tat proteins which trans-activate transcriptional elongation by together with host Cyclin T1 (CycT1):Cyclindependent kinase 9 (CDK9) proteins cooperatively binding the trans-activation response (TAR) RNA hairpin elements, resulting in phosphorylation of RNA polymerase II and productive elongation of transcription. Tat is the only known transcription factor to act via an RNA in eukaryotic cells.

Mutational analysis of HIV-1 TAR RNA has revealed critical bulge and loop sequences, and it has been deduced that Tat binds a TAR bulge and that CycT l binds a TAR loop. Using TAR elements chimeric between HIV-1 and EIAV, we demonstrate additional, previously unknown, interactions between HIV-1 Tat and TAR. Using trans-dominant negative Tat and CycT I and kinase inhibitors we show that both HIV-1 and EIAV utilize CycT1 and the same mechanism of activating transcriptional elongation. Animal models for HIV-1 have been difficult to develop because lentiviruses are species specific. We clone CycT1 from dog (cCycT1) and from chimpanzee (panCycT1).

We show that in contrast to human (hCycT1) and mouse CycT 1 (mCycT1), eCycT1 contains critical amino acid residues for function for both HIV-1 (C261) and EIAV (C261 and H79-80R). We show that panCycT1 and hCycT1 are conserved at C261. We show that hCycT1 increases HIV-1 p24 production from primary more than transformed murine cells after infection with an HIV-1 / murine leukemia pseudovirus. These data extend the knowledge about Tat:CycT1:TAR mediated trans-activation of transcriptional elongation, and provide evidence that HIV-1 and EIAV have evolved to utilize a similar mechanism of transcriptional regulation. CycT1 explains part of the species restriction of HIV-1 in mouse, especially in primary cells, but not in chimpanzee.

Like viruses, cancer provides useful models of eukaryotic gene regulation. Carcinoembryonic antigen (CEA) is expressed on human colon cancer and is a potential target for immune therapies. CEA contains signal sequences that targets it to the cell membrane. We clone a DNA vaccine encoding a truncated CEA, devoid of the signal sequences. We achieve altered expression patterns as seen by immunofluorescence, and also protective immune responses. With signal sequences, CEA is targeted to the cell membrane, and without, it is retained in the cytoplasm.

These models illustrate unique and specific aspects of the regulation of eukaryotic gene and protein expression. By addressing them in parallel, one can gain a more comprehensive and generalizable view of the complex steps from gene to protein.

List of scientific papers

I. Lund LH, Wahren B, Garcia-Blanco MA (2003). A functional genetic approach suggests a novel interaction between the human immunodeficiency virus type 1 (HIV-1) Tat protein and HIV-1 TAR RNA in vivo. J Gen Virol. 84(Pt 3): 603-6.
https://pubmed.ncbi.nlm.nih.gov/12604811

II. Lund LH, Garcia-Blanco MA (2005). Functional evidence that HIV-1 and EIAV trans-activate transcription by the same conserved mechanism. [Manuscript]

III. Albrecht TR, Lund LH, Garcia-Blanco MA (2000). Canine cyclin T1 rescues equine infectious anemia virus tat trans-activation in human cells. Virology. 268(1): 7-11.
https://pubmed.ncbi.nlm.nih.gov/10683321

IV. Rollman EN, Lund LH, Sjostrand DE, Leitner T, Wahren BE (2002). A unique amino acid deletion in the chimpanzee Cyclin T1 does not affect Tat trans-activation of HIV. AIDS. 16(17): 2335-7.
https://pubmed.ncbi.nlm.nih.gov/12441807

V. Lund LH, Ljungberg K, Wahren B, Hinkula J (2004). Primary murine cells as a model for HIV-1 infection. AIDS 18(7): 1067-9.
https://pubmed.ncbi.nlm.nih.gov/15096811

VI. Lund LH, Andersson K, Zuber B, Karlsson A, Engstrom G, Hinkula J, Wahren B, Winberg G (2003). Signal sequence deletion and fusion to tetanus toxoid epitope augment antitumor immune responses to a human carcinoembryonic antigen (CEA) plasmid DNA vaccine in a murine test system. Cancer Gene Ther. 10(5): 365-76.
https://pubmed.ncbi.nlm.nih.gov/12719706