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Diatom Centromeres Suggest a Mechanism for Nuclear DNA

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Diatom Centromeres Suggest a Mechanism for Nuclear DNA Diatom centromeres suggest a mechanism for nuclear PNAS PLUS DNA acquisition Rachel E. Dinera,b, Chari M. Noddingsc, Nathan C. Lianc, Anthony K. Kangc, Jeffrey B. McQuaida,b, Jelena Jablanovicb, Josh L. Espinozab, Ngocquynh A. Nguyenc, Miguel A. Anzelmatti Jr.b, Jakob Janssonc, Vincent A. Bielinskic, Bogumil J. Karasc,1, Christopher L. Dupontb, Andrew E. Allena,b, and Philip D. Weymanc,2 aIntegrative Oceanography Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92037; bMicrobial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, CA 92037; and cSynthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, CA 92037 Edited by James A. Birchler, Division of Biological Sciences, University of Missouri, Columbia, MO, and approved June 13, 2017 (received for review January 17, 2017) Centromeres are essential for cell division and growth in all eukary- transposons, which can vary substantially in copy number and otes, and knowledge of their sequence and structure guides the organization (16). A common feature of centromeric DNA in many development of artificial chromosomes for functional cellular biol- eukaryotes is low-GC content. Centromeres of Schizosaccharomyces ogy studies. Centromeric proteins are conserved among eukaryotes; pombe and other yeast species feature an unconserved core of AT- however, centromeric DNA sequences are highly variable. We rich DNA sequence often surrounded by inverted repeats (17–20). combined forward and reverse genetic approaches with chromatin The centromeres of the protist Plasmodium have no apparent se- immunoprecipitation to identify centromeres of the model diatom quence similarity besides being 2–4-kb regions of extremely low-GC Phaeodactylum tricornutum . We observed 25 unique centromere content (<3%) (21, 22). Likewise, centromere regions of the red sequences typically occurring once per chromosome, a finding that algal species Cyanidioschyzon merolae contain 2–3 kb of relatively helps to resolve nuclear genome organization and indicates mono- centric regional centromeres. Diatom centromere sequences con- low-GC content but manifest no other apparent pattern (23, 24). tain low-GC content regions but lack repeats or other conserved Centromere identification can also be useful for synthetic bi- ology, enabling further discoveries and biotechnology applica- sequence features. Native and foreign sequences with similar GC PLANT BIOLOGY content to P. tricornutum centromeres can maintain episomes and tions. Artificial chromosomes provide a stable platform for recruit the diatom centromeric histone protein CENH3, suggesting introduction and maintenance of multigene constructs necessary nonnative sequences can also function as diatom centromeres. Thus, for expression of biosynthetic pathways and large complex pro- simple sequence requirements may enable DNA from foreign sources teins (25–28). The experimental identification of eukaryotic to persist in the nucleus as extrachromosomal episomes, revealing a centromeres has been extremely useful for developing molecular potential mechanism for organellar and foreign DNA acquisition. Significance diatom | Phaeodactylum tricornutum | episome | centromere | CENH3 Centromeres are genomic sites facilitating chromosome segre- entromeres play a crucial role in the cellular biology of eu- gation during cell division. We report our discovery of diatom Ckaryotes by acting as a genomic site for kinetochore forma- centromeres and the description of centromere identity in the tion and facilitating effective transmission of replicated nuclear stramenopile protists. We also show that simple requirements DNA to new cells. Centromere-associated proteins are func- for diatom centromeres permit ecologically relevant foreign tionally conserved among eukaryote species (1–3). Nearly all DNA molecules to function as diatom centromeres by “hijacking” eukaryotes studied to date possess a version of a specialized chromosome maintenance features. Because little is known at centromeric histone protein (CENH3, also described as centro- the molecular level about chromosome maintenance in diatoms, mere protein A, CENP-A), which binds to centromeric DNA this paper provides experimental data with broad implications and replaces the histone H3 at the site of kinetochore assembly for cellular biology studies and biotechnology applications. The (4–6). Conversely, the centromeric DNA sequences themselves ability to maintain circular artificial chromosomes using foreign are extremely variable and appear to evolve rapidly, even among DNA sequences is unique among organisms with studied cen- similar organisms (7). tromeres and opens up fascinating evolutionary questions about There are three general types of eukaryotic centromeres: point the mechanisms of nuclear gene acquisition from the multiple centromeres, holocentromeres, and regional centromeres. Point endosymbiotic events characterizing the stramenopile lineage. centromeres are uniquely characterized by specific conserved DNA sequences and are found in limited fungal species including the Author contributions: R.E.D., C.M.N., N.C.L., A.K.K., J.B.M., J. Jablanovic, J.L.E., V.A.B., – B.J.K., C.L.D., A.E.A., and P.D.W. designed research; R.E.D., C.M.N., N.C.L., A.K.K., J.B.M., budding yeast Saccharomyces cerevisiae and close relatives (8 10). J. Jablanovic, J.L.E., N.A.N., M.A.A., J. Jansson, V.A.B., and P.D.W. performed research; In holocentromeric organisms, the kinetochore forms along the R.E.D., C.M.N., N.C.L., A.K.K., J.B.M., J. Jablanovic, J.L.E., N.A.N., M.A.A., V.A.B., B.J.K., entire length of each chromosome; a notable example is the C.L.D., A.E.A., and P.D.W. analyzed data; and R.E.D., J.L.E., C.L.D., A.E.A., and P.D.W. model organism Caenorhabditis elegans (11). Most eukaryotes wrote the paper. have regional centromeres, which are commonly found as a single The authors declare no conflict of interest. large DNA region on each chromosome [reviewed in Sullivan This article is a PNAS Direct Submission. et al., 2001 (12) and Torras-Llortetal.,2009(13)].Regional Freely available online through the PNAS open access option. centromeres are variable in length and sequence even among Data deposition: The sequence reported in this paper has been deposited in the National closely related species; however, there are often predictable ge- Center for Biotechnology Information Sequence Read Archive (accession no. PRJNA357294). 1 netic features. For example, human centromeres contain large Present addresses: Department of Biochemistry, Schulich School of Medicine and Den- tistry, Western University, London, ON N6A 5C1, Canada; and Designer Microbes Inc., stretches of repetitive satellite DNA, ranging in size from hundreds London, ON N6G 4X8, Canada. of kilobases to megabases (12, 14, 15). Centromeres of several 2To whom correspondence should be addressed. Email: [email protected]. plants and the insect model Drosophila melanogaster contain large This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. arrays of satellite repeats interspersed with or adjacent to retro- 1073/pnas.1700764114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1700764114 PNAS Early Edition | 1of10 Downloaded by guest on September 24, 2021 biology tools, particularly in the creation of artificial chromosomes. P. tricornutum chromosome that supported episome mainte- Circular and/or linear artificial chromosomes based on native nance would yield similarly increased colony numbers in our centromeres, origins of replication, and in some cases telomeres standard conjugation assay. Out of the five large fragments span- have been developed for yeast (29), mammalian cells including ning each chromosome, one fragment from each chromosome human cell lines (30), plants (reviewed in ref. 31), and recently produced increased numbers of ex-conjugant diatom colonies: the protist Plasmodium (32). Despite the great potential for plasmid Pt25-100kb-1 (containing the first ∼100-kb fragment of eukaryotic algae in biotechnology, very little is known about algal chromosome 25) (Fig. 1 A and C) and plasmid Pt26-100kb-5 centromeres, and few resources are available to control gene (containing the fifth fragment of chromosome 26) (Fig. 1 B and expression from introduced autonomously replicating genetic D). The plasmid containing Pt25-100kb-1 resulted in 14–32-fold constructs. In 1984, autonomously replicating plasmids using more colonies than plasmids containing other 100-kb fragments chloroplast DNA were described for the green alga Chlamydomonas from chromosome 25 (Fig. 1 A and C), and plasmid containing reinhardii (33). However, these vectors were not maintained stably Pt26-100kb-5 resulted in 26–100-fold higher colony numbers and have not been commonly used. More recently, centromeres than other chromosome 26 fragments (Fig. 1 B and D). have been identified and characterized in the red alga C. merolae Both Pt25-100kb-1 and Pt26-100kb-5 fragments encompass (23, 24), where each of the 20 chromosomes was found to contain regions of low-GC content. We calculated the GC content of the one distinct region recruiting CENH3. However, to our knowl- genome in 100-bp windows overlapping by 50 bp and found that edge, these sequences have not yet been used for the construction windows with the lowest GC content were found on fragments of artificial chromosomes. enabling episome maintenance (Fig. 1 E and F). When calcu- Identifying centromere composition and optimizing artificial lating
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