The growth and development of eukaryotic organisms is determined by the interactions between their genetic make-up and the environment. At the molecular level this translates into differential gene expression, giving rise to distinctly specialized cells, tissues, and/or organs. Indeed, the complex development of multicellular eukaryotes requires the silencing of many of their genes in different cell types at any given time. Cell and tissue-specific gene inactivation is a characteristic feature of many developmentally regulated genes and is associated with differentiation processes. This gene silencing involves heritable (but reversible) epigenetic mechanisms, in contrast to genetic effects that are based on stable changes at the DNA level. Epigenetic patterns of expression/repression can be altered in response to environmental or physiological signals, repressing genes under conditions where they are not needed and reactivating their expression if internal or external influences change again. Epigenetic mechanisms also play an essential role in defense responses against a variety of intracellular genomic parasites, such as viruses and transposable elements. The long-term goal of our research projects is to understand the molecular basis of these epigenetic phenomena. We are using the unicellular green alga Chlamydomonas reinhardtii and the higher plant Arabidopsis thaliana as model systems to identify and characterize molecular components that determine transcriptional or post-transcriptional gene silencing. We are concentrating on two main areas: RNA-mediated gene silencing and histone modifications associated with euchromatic transcriptional silencing. More recently we have also become interested in the gene regulatory networks that control nonpolar lipid accumulation in microalgae.
RNA-Mediated Gene Silencing
RNA-mediated processes result in suppression of gene expression in eukaryotes, a phenomenon initially termed RNA interference (RNAi) in animals. Double-stranded RNA (dsRNA) appears to be the trigger at the heart of these processes and it can induce, in different eukaryotes, a variety of outcomes such as the degradation of homologous RNAs, translation repression, or heterochromatin formation. The RNAi machinery has also been implicated in the processing and function of microRNAs, a class of endogenous small RNAs that regulate gene expression by translational repression or mRNA cleavage. Despite rapid progress in understanding key steps of these pathways, genetic screens suggest that many factors required for RNAi have not been characterized as yet. We are isolating, by a variety of genetic and functional genomic approaches, mutants defective in RNAi with the purpose of identifying new pathway components as well as characterizing their role(s) in controlling endogenous gene expression. RNA interference is also becoming a valuable tool for loss-of-function high-throughput genomic studies in eukaryotes and for practical applications in animals, plants and algae. Our lab is also involved in the development of effective technical approaches for gene inactivation. Further elucidation of the RNA silencing pathway will likely have an impact not only in basic biology but also in medicine and agriculture.
Histone Modifications Linked to Euchromatic Transcriptional Silencing
In eukaryotes, nuclear DNA is associated with histones and other proteins in a complex structure referred to as chromatin. Histones are subject to multiple post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. These histone PTMs influence chromatin organization and can regulate many DNA-templated processes such as transcription, replication, recombination, and repair. However, relatively little is known about the role of histone methylation or phosphorylation in the regulation of euchromatic genes in photosynthetic eukaryotes, both during development and in response to changing environmental conditions. Our lab is addressing the molecular mechanisms responsible for methylation and phosphorylation of core histones in Chlamydomonas and Arabidopsis. We are particularly interested in the role of histone phosphorylation in mediating rapid changes in gene expression/repression in response to abiotic stresses such as drought.
Gene Regulatory Networks Controlling Neutral Lipid Accumulation
The great potential of microalgae as feedstocks for renewable biofuel production has recently gained recognition. The environmental conditions that trigger lipid accumulation in algae are under careful investigation. However, the complexity of biological systems, including elaborate networks and mutual dependency of cellular processes, makes it difficult to develop the most efficient strategy for biotechnological improvement via traditional approaches. In particular, the gene regulatory systems of microalgae remain virtually unexplored and, yet, represent an interesting target for pathway engineering leading to improved biofuel production. These systems involve not only transcription factors but also a complex array of small RNAs/microRNAs and other post-transcriptional mechanisms. The main goal of this project is to understand the gene regulatory networks that control lipid accumulation in Chlamydomonas reinhardtii. Our specific objectives are to identify transcription factors and miRNAs involved in gene regulation of metabolic components under nitrogen deprivation (with an emphasis on the control of triacylglycerol biosynthesis and competing pathways) and to model gene regulatory networks integrating miRNAs and, to the extent possible, transcription factors.
- Wang Z., Casas-Mollano J.A., Xu J., Riethoven J.-J.M., Zhang C., and Cerutti H. 2015. Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana.Proc. Natl. Acad. Sci. USA, 112:8487-8492.
- Tevatia R., Allen J., Rudrappa D., White D., Clemente T.E., Cerutti H., Demirel Y. and Blum P. 2015. The taurine biosynthetic pathway of microalgae. Algal Res., 9:21-26.
- Knobbe A.R., Horken K.M., Plucinak T.M., Balasssa-Hakim E., Cerutti H. and Weeks D.P. 2015. SUMOylation by a stress-specific SUMO E2 conjugase is essential for survival of Chlamydomonas reinhardtii under stress conditions. Plant Physiol., 167:753-765.
- Voshall A., Kim E.-J., Ma X., Moriyama E.N. and Cerutti H. 2015. Identification of AGO3 associated miRNAs and computational prediction of their targets in the green alga Chlamydomonas reinhardtii. Genetics, 200:105-121.
- Kim E.-J., Ma X. and Cerutti H. 2015. Gene silencing in microalgae: mechanisms and biological roles. Bioresour. Technol., 184:23-32. doi: 10.1016/j.biortech.2014.10.119. Epub 2014 Oct 30.
- Yamasaki T., Voshall A., Kim E.-J., Moriyama E., Cerutti H. and Ohama T. 2013. Complementarity to a miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii. Plant Journal, 76:1045-56.
- Ma X., Kim E.-J., Kook I., Ma F., Voshall A., Moriyama E. and Cerutti H. 2013. siRNA-Mediated Translation Repression Alters Ribosome Sensitivity to Inhibition by Cycloheximide in Chlamydomonas reinhardtii. Plant Cell, 25:985-998.
- Msanne J., Xu D., Konda A. R., Casas-Mollano J. A., Awada T., Cahoon E. B. and Cerutti H. 2012. Metabolic and gene expression changes triggered by nitrogen deprivation in the photoautotrophically grown microalgae Chlamydomonas reinhardtii and Coccomyxa sp. C-169. Phytochemistry, 75:50-59.
- Cerutti H., Ma X., Msanne J. and Repas T. 2011. RNA-Mediated Silencing in Algae: Biological Roles and Tools for the Analysis of Gene Function. Eukaryotic Cell, 10: 1164-1172.
- Cerutti H. and Ibrahim F. 2011. Turnover of mature miRNAs and siRNAs in plants and algae. Adv. Exp. Med. Biol. 700: 124-139.
- Shaver S. S., Casas-Mollano J. A., Cerny R. L. and Cerutti H. 2010. Origin of the Polycomb Repressive Complex 2 and gene silencing by an E(z) homolog in the unicellular alga Chlamydomonas. Epigenetics, 5: 301-312.
- Ibrahim F., Rymarquis L. A., Kim E.-J., Becker J., Balassa E., Green P. J. and Cerutti H. 2010. Uridylation of mature miRNAs and siRNAs by the MUT68 nucleotidyltransferase promotes their degradation in Chlamydomonas. Proc. Natl. Acad. Sci. USA, 107: 3906-3911.
- Kim E.-J. and Cerutti H. 2009. Targeted gene silencing by RNA interference in Chlamydomonas. Methods Cell Biol, 93: 99-110.
- Cerutti H. and Casas-Mollano J. A. 2009. Histone H3 phosphorylation: universal code or lineage specific dialects? Epigenetics 4: 71-75.
- Casas-Mollano J. A., Rohr J., Kim E.-J., Balassa E., van Dijk K. and Cerutti H. 2008. Diversification of the core RNAi machinery in Chlamydomonas reinhardtii and the role of DCL1 in transposon silencing. Genetics 179: 69-81.
- Casas-Mollano J. A., Jeong B.-r., Xu J., Moriyama H. and Cerutti H. 2008. The MUT9p kinase phosphorylates histone H3 threonine 3 and is necessary for heritable epigenetic silencing in Chlamydomonas. Proc. Natl. Acad. Sci. USA 105: 6486-6491.
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- Casas-Mollano J. A., van Dijk K., Eisenhart J. and Cerutti H. 2007. SET3p monomethylates histone H3 on lysine 9 and is required for the silencing of tandemly repeated transgenes in Chlamydomonas. Nucleic Acids Research 35: 939-950.
- Ibrahim F., Rohr J., Jeong W.-J., Hesson J. and Cerutti H. 2006. Untemplated oligoadenylation promotes degradation of RISC-cleaved transcripts. Science 314: 1893.
- Cerutti H. and Casas-Mollano A. 2006. On the Origin and Functions of RNA-Mediated Silencing: From Protists to Man. Curr Genet 50: 81-99.
- van Dijk K.V., Xu H. and Cerutti H. 2006. Epigenetic silencing of transposons in the green alga Chlamydomonas reinhardtii. Nucleic Acids and Molecular Biology 17: 159-178.
- van Dijk K.V., Marley K.E., Jeong B.-r., Xu J., Hesson J., Cerny R.L., Waterborg J.H. and Cerutti H. 2005. Monomethyl histone H3 lysine 4 as an epigenetic mark for silenced euchromatin in Chlamydomonas. Plant Cell 17: 2439-2453.
- Sarkar N., Lemaire S., Wu-Scharf D., Issakidis-Bourguet E., and Cerutti H. 2005. Functional Specialization of Chlamydomonas Cytosolic Thioredoxin h1 in the Response to Alkylation-Induced DNA Damage. Eukaryotic Cell 4: 262-273.
- Rohr J., Sarkar N., Balenger S., Jeong B.-r. and Cerutti H. 2004. Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas. Plant J. 40: 611-621.
- Ebmeier A., Allison L., Cerutti H. and Clemente T. 2004. Evaluation of the Escherichia coli threonine deaminase gene as a selectable marker for plant transformation. Planta 218: 751-758.
- Cerutti H. 2003. RNA interference: traveling in the cell and gaining functions? Trends Genet. 19, 39-46.
- Zhang C., Wu-Scharf D., Jeong B.-r. and Cerutti H. 2002. A WD40-repeat containing protein, similar to a fungal co-repressor, is required for transcriptional gene silencing in Chlamydomonas. Plant J. 31: 25-36.
- Jeong B.-r., Wu-Scharf D., Zhang C. and Cerutti H. 2002. Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements. Proc. Natl. Acad. Sci. USA 99: 1076-1081.
- Wu-Scharf D., Jeong B.-r., Zhang C. and Cerutti H. 2000. Transgene and transposon silencing in Chlamydomonas reinhardtii by a DEAH-box RNA helicase. Science 290: 1159-1162.