Hints that we can change non-genetic aspects of the cell code come from phenomena like paramutation and RNA interference, where changes in gene regulatory information can persist across generations. Both phenomena rely on RNA for sequence specificity and the details of how they affect gene expression are still being worked out. But, this ability to pass non-genetic information across generational boundaries and recent advances in single cell analyses, genome editing, and epigenome engineering enable us to begin deciphering the cell code that perpetuates life.
Our research using the simple worm C. elegans in the following four areas provides tools and a framework that guides our approach to this central problem.
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Overview: We found that double-stranded RNA (dsRNA) expressed in neurons can enter the germline and silence a gene of matching sequence and that the silencing can last for more than 25 generations (Devanapally et al., 2015). The dsRNA exported from neurons in an animal, injected into an animal, or ingested by an animal are all expected to reach the body cavity that surrounds most tissues in C. elegans. Analysis of genetic requirements for silencing by ingested dsRNA revealed that each somatic cell needs long dsRNA for silencing (Raman et al., 2017). Yet, we found that any extracellular material including RNA can directly reach progeny after entry into oocytes (Marré et al., 2016), suggesting that dsRNA made in neurons could similarly enter oocytes, reach progeny, and then initiate transgenerational gene silencing. The multi-generational maintenance of gene silencing requires a nuclear Argonaute protein (Devanapally et al., 2015) that binds antisense small RNAs (~22 nucleotides), which are likely made in every generation. Quantifying small RNAs associated with silencing is possible through next-generation RNA sequencing (RNA-seq) but the analysis of the resultant data can be challenging. We developed bioinformatic approaches for the clear analysis of RNA-seq data and discovered a new class of small RNAs that are shorter than 18 nucleotides (Blumenfeld & Jose, 2016). Experimental and theoretical considerations suggest that there are additional such tiny RNAs and we have improved northern blotting to enable their independent analysis (Choi et al., 2017). Even if the information to express or silence a gene is passed from one generation to the next, in every generation, animals face the challenge of keeping the expression of a gene similar in cells within a tissue despite cell divisions. We found that a variable set of cells become susceptible to silencing of repetitive DNA in the absence of factors that inhibit RNA silencing and that the dsRNAs that trigger such silencing can move between cells in the embryo (Le et al., 2016). Thus, we have begun to learn how to use RNA in one generation to affect the expression of a matching gene in subsequent generations and have developed tools to effectively analyze small RNAs associated with gene silencing in every generation. Building on these past studies, we plan to change the expression of a specific gene without affecting its sequence and discover the factors that restrict or allow the persistence of such changes for multiple generations. As a complementary approach, we plan to use CRISPR-based genome editing to alter DNA to discover the sequence characteristics that restrict or allow transgenerational reprogramming. If you are curious about the evolution of research programs or simply want more, check out our previous research statement.