1. Chromatin Diminution
Genome maintenance and stability are essential, and an organism’s genome rarely changes. But a few organisms undergo chromatin diminution, a programmed process that eliminates specific DNA sequences from the genome. In the parasitic nematode, Ascaris, 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. Both repetitive and unique sequences (genes) are lost during chromatin diminution. The elimination results in chromosome breakage and the loss of chromosome termini as well as the generation of new chromosomes. We recently have sequenced and compared the Ascaris germline and somatic genomes and defined the chromosome breaks and sequences lost.
Studies are underway to determine
What is the mechanism of chromosome breakage? What proteins are involved?
How are the breakpoints defined?
Are there discrete epigenetic changes associated with chromosome diminution?
Are small RNAs involved in chromatin 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?
Given that nematodes have holocentric chromosomes, what determines which chromosomal regions are retained and which are lost?
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
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. A goal is to use novel cell-free systems to study the mechanisms of small RNA silencing and biogenesis.
Zygote maturation prior to pronuclear fusion and early development in Ascaris are very slow compared to C. elegans. This enables the staging of material for developmental studies and large amounts of synchronously developing stages can be obtained (see Information on Ascaris). We have shown that significant and diverse Pol 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. Early development and cell lineage appear identical between C. elegans and Ascaris although the timing of zygotic gene activation and the maternal to zygotic transition occur much earlier and immediately following fertilization. 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.