1. Programmed DNA Elimination
Genome maintenance and stability are essential, and an organism’s genome rarely changes. Genome instability typically leads to inviability and disease. However, examples are known where genome instability through developmentally regulated DNA loss or rearrangements is integral in the biology of the organism. Chromatin diminution is a form of programmed DNA elimination that leads to the loss of specific sequences from chromosomes. Chromatin diminution occurs during differentiation of the somatic lineage early in embryonic development. In the parasitic nematode, Ascaris, we have shown that 15% of the genome is eliminated in the somatic lineages during the 3rd through 5th cleavage (4 to 16 cell stage), while the germline genome remains intact. Specific repetitive and unique sequences (including ~700 genes) are lost during chromatin diminution to form the somatic genome. The eliminated genes are primarily expressed in the Ascaris germline and early embryo. Overall, these data suggest that chromatin diminution in Ascaris is an extreme and irreversible mechanism for silencing a subset of germline and early embryo expressed genes in somatic tissues and contributes to the distinction between the germline and soma.
Studies are now underway to determine
- What is the mechanism of chromosome breakage? What proteins are involved?
- How are the breakpoints defined?
- Given that nematodes have holocentric chromosomes (multiple centromeric/kinetochore complexes), what determines which chromosomal regions are retained and which are lost?
- Are small RNAs involved in chromatin diminution?
- Are there discrete epigenetic changes associated with chromosome diminution?
- Given that chromosome breaks are healed by telomere addition, what recruits telomerase and what factors prevent other repair mechanisms, apoptosis, or cell cycle arrest from initiating?
- What are the somatic consequences of DNA elimination?
2. Spliced Leader RNA trans-splicing in metazoa
Spliced leader (SL) RNA trans-splicing generates the mature 5’ ends of mRNAs by addition of a spliced leader sequence to the 5’ end of a pre-mRNA. Addition of the SL sequence also brings a new and an atypical cap to the RNA, a trimethylguanosine cap (m2,2,7GpppN) compared to the typical m7GpppN eukaryotic cap. Current studies involve analysis of
- Functional significance of trans-splicing
- Protein and mRNA metabolism adaptation to spliced leader trans-splicing
- Post-transcriptional role of trans-splicing in mRNA translation and stability
- Structure/function of mRNA cap-interacting proteins in trans-splicing and mRNA metabolism (eIF4E, eIF4G, nuclear cap binding complex, decapping, etc.)
3. Regulation of Ascaris RNA metabolism during gametogenesis, zygote maturation and early embryo development
Estimates suggest that Ascaris produces ~ 1 million eggs/day. Zygote maturation prior to pronuclear fusion and early development in Ascaris are very slow and synchronous compared to the model nematode C. elegans. These two attributes enable the staging of large amounts of material for developmental studies and other studies (see Information on Ascaris). We have shown that significant and diverse RNA polymerase II transcription occurs prior to pronuclear fusion during maturation of the zygote in the uterus and prior to the 4-cell stage during the first two divisions. This transcription occurs earlier and is more robust and diverse than any transcription previously observed in early animal development. Ascaris early development and cell lineage appears identical to that in C. elegans. However, the timing of zygotic gene activation and the maternal to zygotic transition occur much earlier and immediately following fertilization in Ascaris. Furthermore, although early gene expression is post-transcriptionally regulated and derived from maternal contributions in C. elegans, gene expression have been re-wired in Ascaris and early development is driven transcriptionally. Studies are underway to examine this transcriptional re-wiring and epigenetic changes. Depositied fertilized eggs have not undergone pronclear fusion are quiescent until incubated under appropriate conditions. Studies are underway to examine possible mechanisms of mRNA masking and translational control in these zygotes. The unqiue properties of Ascaris enables us to carry out analysis of early gene transcription and post-transcriptional gene regulation.
A variety of small RNAs including endogenous siRNA (22G-RNAs and 26G-RNAs) and miRNAs play key roles in silencing in the Ascaris germline, gametogenesis, and early development. We are interested in investigating the role and function of these small RNAs in chromatin diminution, development, gametogenesis, and the germ line.