- Ph.D., University of Maryland, 1995
- M.S., Clemson University, 1983
My research goal is to clarify interactions between the human LINE1 (L1) retrotransposon and the human genome, which have shaped the architecture of the genome and impacted its functions in mostly negative ways over the course of evolutionary history. Our laboratory uses phylogenetic analyses of ancient and modern L1 sequences resident in the human genome to guide our biochemical studies of one of the two proteins encoded by L1 elements.
L1 (LINE-1) retrotransposons, the major clade of retrotransposable elements in most mammalian genomes, have been replicating and evolving since the onset of the mammalian radiation, and upwards of 40% of genomic DNA in the primate lineage is attributable to L1 activity. L1 encodes two essential proteins, one of which, ORF1p, a nucleic acid chaperone, is a coiled coil-mediated homo-trimer, with 14 amino acid heptad units comprising the coiled coil domain. Coiled coil motifs are highly conserved, structurally, although having repeatedly undergone rapid evolutionary change in the history of ORF1p. Functionally, ORF1p trimers polymerize around the parental m-RNA strand (cis-preference), forming a ribonucleoprotein(RNP) particle. The second of the L1 proteins, ORF2p, joins the complex, and it is this RNP that is thought to bind genomic DNA. Following this nonspecific binding of the RNP, endonuclease and reverse transcriptase activities of ORF2p nick genomic DNA to form a priming site for reverse transcription of the parental mRNA strand, thus inserting a new L1 sequence into genomic DNA. Such copy-paste DNA insertions can damage chromosomal DNA or introduce genetic changes. For the most part, this activity, known as “Target site Primed Reverse Transcription (TPRT) is suppressed in somatic cells, but can be reactivated in some versions of cancer. The preferred environment for L1 activity is during embryogenesis, when retrotransposition enables the L1 element to become part of the genome of the next generation. Numerous defense mechanisms of the host act to inhibit such insertions. Most of the L1 inserts throughout the human genome are inactive due to truncations that occur during TPRT, resulting in L1 fragments rather than full length, active elements. However, these inactive sequences, which account for nearly one half of the human genome, are not excised, so that, over evolutionary history, mammalian genomes have become repositories of L1 fossils. These inactive sequences have accumulated mutations over time, which serve to distinguish older (ancestral) L1 families from the younger (modern) families. The coiled coil domain of ORF1p has been especially amenable to such mutations and the larger number of mutations in this region of ORF1p can be evidence of positive selection. To further investigate these evolutionary changes in the coiled coil motif, we compared two fully active versions of ORF1p, one (named 555) from an ancestral L1pa 5 family and an element (111) from the modern L1pa1 family. Another construct was made by mixing portions of the coiled coil from 555 and from 111. Unlike the ancestral 555 and modern 111, this mosaic (151), had altered polymerization kinetics and was inactive in cell culture retrotransposition assays using HeLa cells. Rescue of retrotransposition activity required restoration of four ancestral amino acids in coiled coil heptads 8-9 of 151to their modern counterparts. Reversion of any one of these four positions in fully modern 111 to ancestral residues inactivated the modern 111 protein. To extend these findings, we started with inactive 151, with its 21 amino acid differences in heptads 1-7 compared to the active ancestral ORF1-555, and used site-directed mutagenesis to reconstruct the retrotranspostion-active version of the coiled coil from this inactive 151 sequence. Reversion of 17 out of 21 residues had no effect, but retrotransposition was restored with the 18th conversion to the ancestral amino acid. This single amino acid change, when introduced singly into the mosaic 151 coiled coil did not restore retrotransposition acitivity, suggesting that the surrounding context of amino acids is important for overall coiled coil influence on L1 activity. In sum, these findings indicate that cross talk between amino acids from heptads 8-9, and in the upstream coiled coil heptads (1-7), is determinative of its effect on ORF1p activity.
Applying our Research
A recent review of the field (Beck et al. (2011), Annu. Rev. Genomics Hum. Genet., 12) estimated that retrotransposition-induced alterations in human genomes are responsible for one of every 1,000 spontaneous, disease-producing mutations in humans. Basic research on the biology and biochemistry of these DNA-altering events informs the ongoing clinical research on these diseases.
Need for Further Study
Areas of retrotransposon biology in need of further study include the insertion mechanism whereby the copied retrotransposon reinserts into the genome, the manner in which mutagenic activity accompanies retrotransposition events, and, from an evolutionary standpoint, how the human “host” has managed to control the ongoing, mostly deleterious activity of LINE1 elements throughout human history.
- Cryptic genetic variation enhances primate L1 retrotransposon survival by enlarging the functional coiled coil sequence space of ORF1p.
- Furano AV, Jones CE, Periwal V, Callahan KE, Walser JC, Cook PR.
- PLoS Genet (2020 Aug) 16:e1008991. Abstract/Full Text
- Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations.
- Shen B, Chapman JH, Custance MF, Tricola GM, Jones CE, Furano AV.
- Elife (2020 Jan 6) 9. Abstract/Full Text
Research in Plain Language
Up until the 1950s, chromosomes were thought to be static molecules, containing genetic material that persisted in an unaltered arrangement from generation to generation. However, thanks to the work of Barbara McClintock, it became clear that significant amounts of DNA are, in fact, capable of relocating (cut & paste) or replicating and re-inserting elsewhere in the genome (copy & paste). Over long periods of time, these mobile elements have adversely affected both the architecture and function of the human genome. Our laboratory is studying the biochemistry and evolutionary history of these elements, the better understanding of which will provide insights that will contribute to efforts to understand the mechanisms behind diseases caused by mutations on DNA.