bioRxiv preprint doi: https://doi.org/10.1101/2021.09.01.458626; this version posted September 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. The chromatin organization of a chlorarachniophyte nucleomorph genome Georgi K. Marinov1,#, Xinyi Chen2, Tong Wu3, Chuan He3,4,5, Arthur R. Grossman6, Anshul Kundaje1,7, and William J. Greenleaf1,8,9,10,# 1Department of Genetics, Stanford University, Stanford, California 94305, USA 2Department of Bioengineering, Stanford University, Stanford, California 94305, USA 3Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, 60637, USA 4Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, 60637, USA 5Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, 60637, USA 6Carnegie Institution for Science, Department of Plant Biology, Stanford, California 94305, USA 7Department of Computer Science, Stanford University, Stanford, California 94305, USA 8Center for Personal Dynamic Regulomes, Stanford University, Stanford, California 94305, USA 9Department of Applied Physics, Stanford University, Stanford, California 94305, USA 10Chan Zuckerberg Biohub, San Francisco, California, USA #Corresponding author Abstract Nucleomoprhs are remnants of secondary endosymbiotic events between two eukaryote cells wherein the endosymbiont has retained its eukaryotic nucleus. Nucleomorphs have evolved at least twice independently, in chlorarachniophytes and cryptophytes, yet they have converged on a remarkably similar genomic architecture, characterized by the most extreme compression and miniaturization among all known eukaryotic genomes. Previous computational studies have sug- gested that nucleomorph chromatin likely exhibits a number of divergent features. In this work, we provide the first maps of open chromatin, active transcription, and three-dimensional organi- zation for the nucleomorph genome of the chlorarachniophyte Bigelowiella natans. We find that the B. natans nucleomorph genome exists in a highly accessible state, akin to that of ribosomal DNA in some other eukaryotes, and that it is highly transcribed over its entire length, with few signs of polymerase pausing at transcription start sites (TSSs). At the same time, most nucleo- morph TSSs show very strong nucleosome positioning. Chromosome conformation (Hi-C) maps reveal that nucleomorph chromosomes interact with one other at their telomeric regions, and show the relative contact frequencies between the multiple genomic compartments of distinct ori- gin that B. natans cells contain. Introduction the evolution of eukaryotes 3, usually resulting in retention of the plastid of the photosynthetic eukaryotic endosym- Endosymbiosis, especially between a eukaryotic host and biont (as a secondary plastid) while the nucleus of the en- a prokaryote, is a common event in the evolution of eu- dosymbiont is lost entirely. However, several notable excep- karyotes, and subsequent changes in the host and endosym- tions to this general rule do exist. One is the dinotoms, the biont genomes often follow similar general trends. One such result of an endosymbiosis between a dinoflagellate host and trend is the reduction of the endosymbiont's genome due to a diatom, in which the diatom has not been substantially gene loss and endosymbiotic gene transfer 1,2 (EGT) into reduced 4,5. More striking are the nucleomorphs, which are the host's nucleus, the classic example of which are the ex- best known from the chlorarachniophytes and the crypto- tremely reduced genomes of plastids and mitochondria that phytes (but may in fact have arisen in other groups too, such evolved as the bacterial progenitors of these organelles un- as some dinoflagellates 6,7). Nucleomorphs retain a vestigial derwent organellogenesis. This trend is also strongly man- nucleus with a highly reduced but still functional remnant ifested in the fate of secondary endosymbionts (eukaryotes of the endosymbiont's genome 8,9. that become endosymbionts of other eukaryotes). Such en- dosymbiotic events have occurred on multiple occasions in A remarkable feature of chlorarachniophyte and cryp- 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.09.01.458626; this version posted September 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. tophyte nucleomorphs is that they have evolved indepen- may display atypical signatures of nucleosome depletion dently, from a green and a red alga, respectively, yet their and positioning, histone modifications, etc., and relation genomes exhibit surprisingly convergent properties 10,11. In of these marks to transcriptional activity, or they may ex- both cases, the genomes of their nucleomorphs are the hibit unique 3D genomic organization. However, none of smallest known among all eukaryotes, usually just a few these features associated with nucleomorph chromatin or hundred kilobases in size (∼380 kbp for the chlorarach- gene expression regulation has been directly studied. niophyte B. natans). All sequenced nucleomorph genomes In this work we map chromatin accessibility, active tran- are organized into three highly AT-rich chromosomes, in scription, and three-dimensional (3D) genome organization which arrays of ribosomal RNA genes form the subtelom- in the chlorarachniophyte Bigelowiella natans to address eric regions. These genomes are also extremely compressed, these gaps in our knowledge of nucleomorph biology. We exhibiting very little intergenic space between genes, with find that nucleomorph chromosomes exist in a highly acces- genes even overlapping on occasions. The genes themselves sible state, reminiscent of what is observed for ribosomal are also often shortened 12{18. DNA (rDNA) in other eukaryotes, such as budding yeast, A number of important questions about the biology as- where rDNA is thought to be fully nucleosome-free when ac- sociated with the extremely reduced nucleomorph genome tively transcribed 26{28. However, nucleomorph promoters remain unanswered, including the extent of conservation are associated with strongly positioned nucleosomes, and and divergence of chromatin organization and transcrip- they exhibit a distinct nucleosome-free region upstream of tional mechanisms of these extremely reduced nuclei rel- the transcription start site (TSS). Active transcription is ative to that of a convention eukaryotic genome. Previous nearly uniformly distributed across nucleomorph genomes, computational analysis of nucleomorph genome sequences 19 with the exception of elevated transcription and chromatin has suggested that a considerable degree of deviation from accessibility at the subtelomeric rDNA genes. We find few the conventional eukaryotic state is likely to have devel- signs of RNA polymerase pausing over promoters. Nu- oped in nucleomorphs. For example, histone proteins are cleomorph chromosomes form a network of telomere-to- ancestral to all eukaryotes, and the key posttranscriptional telomere interactions in 3D space, and also fold on them- modifications (PTMs) that they carry also date back to selves, but centromeres do not preferentially interact with the last eukaryotic common ancestor (LECA) and are ex- each other. Curiously, the genome of the B. natans mi- tremely conserved in nearly all branches of the eukaryotic tochondrion, which derives from the host, exhibits an ele- tree 20, with the notable exception of dinoflagellates 21. This vated Hi-C trans contact frequency with the genomes of the is likely because these PTMs are deposited in a highly reg- endosymbiont compartments (the plastid and the nucleo- ulated manner on specific residues of histones, and are then morph) than it does with the host genome. These results read out by various effector proteins, thus playing crucial provide novel insights into chlorarachniophyte nucleomorph roles in practically all aspects of chromatin biology, such as chromatin structure and a framework for future mechanistic the regulation of gene expression, the transcriptional cycle, studies of transcriptional and regulatory biology in nucleo- the formation of repressive heterochromatin, mitotic con- morphs. densation of chromosomes, DNA repair, and many others 22 (in what is often referred to as \histone code" ). Results Nucleomorphs appear to be one of the few 19,21 excep- tions to this general rule. Inside nucleomorph genomes, Chromatin accessibility in nucleomorphs in both chlorarachniophytes and cryptophytes, only two histone genes are encoded, one for H3 and for H4, with To study the chromatin structure of the B. natans nucleo- H2A and H2B encoded by the host nuclear genome and im- morph genome, we carried out ATAC-seq experiments in B. ported from the host's cytoplasm 23. Sequence analyses of natans grown under standard conditions (see Methods). As the H3 and H4 proteins show remarkable divergence from B. natans has four different genomic compartments (Figure the typical amino acid sequence in eukaryotes; specifically, 1A) { nucleus, nucleomorph, mitochondrion and plastid { the chlorarachniophyte histones have lost nearly all key hi- we first examined the fragment length distribution