Lo: Research in Biology
Evolution of Nematode Dosage Compensation Mechanisms
Organisms that utilize chromosome-based mechanisms to determine sex (e.g. XX, females and XY/XO, males) require the chromosome-wide regulatory mechanism of dosage compensation to balance sex-chromosome gene expression between sexes. Strategies to determine sexual fate and to compensate X-chromosome dosage between sexes have evolved independently: mammals, flies, and worms use fundamentally different methods. Therefore, understanding the evolution of dosage compensation requires comparisons over shorter evolutionary time-scales, such as between various nematode species. Understanding the molecular evolution of dosage compensation components and regulatory sequences will provide insights into mechanisms that control sex-specific chromosome-wide gene regulation, and how these mechanisms could facilitate novel functions, X-chromosome evolution, and promote biodiversity.
Comparative studies have illuminated remarkable variability in the conservation of developmental mechanisms. Nematode dosage compensation is a complex process that can be simplified into three distinct parts, the core machinery (the dosage compensation complex), the genetic hierarchy (genes, xol-1 and sdc-2) that regulates dosage compensation, and finally the DNA sequence requirements on the X chromosome. At the start of my post-doc it was of great interest to know how these “three distinct parts” evolved; whether they were all conserved, all diverged, or a combination of the two. This was a relatively unexplored aspect of nematode dosage compensation at the time.
Question 1: What components of C. briggsae dosage compensation complex and genetic hierarchy are conserved?
The C. elegans’ dosage compensation complex is a multi-subunit protein complex. Despite the fact that previous research has revealed at least 10 components in the C. elegans dosage compensation complex, the link between the developmental switch gene, xol-1 that mediates dosage compensation, and the dosage compensation complex is unknown. Furthermore, it has yet to be determined which complex member binds the X chromosome directly, if any. Research in my lab will use an evolutionary approach to identify these unknowns. Using C. briggsae, a sister nematode, we will identify components of the C. briggsae dosage compensation complex. This approach will allow for the identification of both known homologues and novel components. Homlogues of novel components will be tested for a role in C. elegans dosage compensation. Determination of which known components are conserved and which are not, will provide us with a basis to begin to dissect mechanisms involved in dosage compensation complex assembly and targeting to the X chromosome. Projects will include taking a biochemical approach, a classical genetics approach, and a more modern genome editing approach.
Project 1: IP/Mass Spec to identify C. briggsae dosage compensation complex members.
Project 2: Classical genetic screen to identify genetic suppressors of C. briggsae xol-1 XO lethality.
Project 3: Candidate approach to determine C. briggsae complex components using modern genome-editing tools such as TALENs and CRISPR/Cas9.
Once the components of the C. brigssae dosage compensation complex have been identified and verified, we can begin determine the mechanism of dosage compensation complex formation and loading onto the X chromosome. For example, are proteins loaded onto the X chromosme sequentially, or does the complex form and then load onto the X chromosome. These studies will also be extended to additional Caenorhabditis species to further our understanding of not just the dosage compensation complex, but of the mechanism of dosage compensation.
Question 2: Is Dosage Compensation Conserved Between Caenorhabditis and Pristionchus species.
Taken together, evolutionary comparisons among C. elegans, C. briggsae, and P. pacificus will reveal similarities and differences in sex determination and dosage compensation mechanisms across nematodes and determine which traits are the ancestral ones. By understanding the evolutionary trajectory of different aspects of sex determination and dosage compensation such as the core machinery and the X chromosome recruitment elements we will gain functional insights about the co-evolution of regulatory complexes and their cognate binding sites. Furthermore, expanding our studies to include species across greater evolutionary distance will allow us to test whether gene duplications between nematode species have facilitated evolutionary innovation. These studies will also help to further establish P. pacificus as a model organism.
At the moment, little is known about sex determination and dosage compensation in P. pacificus. What is known is that similar to C. elegans and C. briggsae, P. pacificus has two sexes that are determined by different combinations of sex chromosomes. Hermaphrodites (XX) have two X chromosomes, and males (XO) have one. As result, like other heterogametic organisms, P. pacificus most likely also possesses a dosage compensation mechanism to equalize gene expression from the two hermaphrodite X chromosomes to equal the gene expression from the single X in males and this process needs to be carefully regulated.
Project 1: RNA-seq to determine if genes are dosage compensated in P. pacificus similarly to Caenorhabditis species.
Project 2: Use TALENs or CRISPR/Cas9 technology to generate a P. pacificus xol-1 mutant to determine if sex determination and dosage compensation regulation is conserved.
Project 3: Determine if P. pacificus has a conserved dosage compensation complex.
Once the key components and pathways that mediate dosage compensation in P. pacificus have been identified via the experiments described above, we will be well poised to begin to dissect P. pacificus dosage compensation on a mechanistic level.
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