bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453775; this version posted July 27, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 1 Title: Cell-specific expression buffering promotes cell survival and cancer robustness 2 3 Hao-Kuen Lina,b,1, Jen-Hao Chenga,c,1, Chia-Chou Wua,1, Feng-Shu Hsieha, Carolyn Dunlapa, 4 and Sheng-hong Chena,c* 5 6 a Lab for Cell Dynamics, Institute of Molecular Biology, Academia Sinica, Taipei 115, 7 Taiwan 8 b College of Medicine, National Taiwan University, Taipei 106, Taiwan 9 c Genome and Systems Biology Degree Program, Academia Sinica and National Taiwan 10 University, Taipei, Taiwan 11 12 1 equal contribution 13 * Correspondence: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453775; this version posted July 27, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 14 Summary blurb 15 This study explores a genome-wide functional buffering mechanism, Cell-specific 16 Expression Buffering (CEBU), where gene expression contributes to functional buffering in 17 specific cell types and tissues. CEBU is prevalent in humans, linking to genetic interactions, 18 tissue homeostasis and cancer robustness. bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453775; this version posted July 27, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 4.0 International license. 19 Abstract 20 Functional buffering ensures biological robustness critical for cell survival and physiological 21 homeostasis in response to environmental challenges. However, in multicellular organisms, 22 the mechanism underlying cell- and tissue-specific buffering and its implications for cancer 23 development remain elusive. Here, we propose a Cell-specific Expression-BUffering (CEBU) 24 mechanism, whereby a gene’s function is buffered by cell-specific expression of a buffering 25 gene, to describe functional buffering in humans. The likelihood of CEBU between gene 26 pairs is quantified using a C-score index. By computing C-scores using genome-wide 27 CRISPR screens and transcriptomic RNA-seq of 684 human cell lines, we report that C- 28 score-identified putative buffering gene pairs are enriched for members of the same pathway, 29 protein complex and duplicated gene family. Furthermore, these buffering gene pairs 30 contribute to cell-specific genetic interactions and are indicative of tissue-specific robustness. 31 C-score derived buffering capacities can help predict patient survival in multiple cancers. Our 32 results reveal CEBU as a critical mechanism of functional buffering contributing to cell 33 survival and cancer robustness in humans. 34 35 Running title: Expression buffering for cancer robustness 36 Keywords: functional buffering, expression buffering, buffering capacity, genetic interaction, 37 tissue homeostasis, cancer robustness 38 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453775; this version posted July 27, 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 4.0 International license. 39 Introduction 40 Robustness in biological systems is critical for organisms to carry out vital functions in the 41 face of environmental challenges [1, 2]. A fundamental requirement for achieving biological 42 robustness is functional buffering, whereby the biological functions performed by one gene 43 can also be attained via other buffering genes. These buffering genes thereby enhance the 44 robustness of the biological system. Multiple mechanisms underlie this functional buffering, 45 either via the responsive or intrinsic activities of buffering genes. Responsive buffering 46 mechanisms activate buffering genes only if the function of a buffered gene is compromised. 47 Therefore, the mechanism incorporates a control system that senses a function has been 48 compromised and then activates the buffering genes, usually by up-regulating their gene 49 expression [3]. One classical responsive buffering mechanism is genetic compensation 50 among duplicated genes, whereby functional compensation is achieved by activating 51 expression of paralogs when the functions of the original duplicated genes is compromised 52 [4]. Genetic analyses of duplicated genes in Saccharomyces cerevisiae have revealed up- 53 regulated gene expression in ~10% of paralogs when cell growth is compromised due to 54 deletions of their duplicated genes [5-7]. Apart from duplicated genes, non- 55 orthologous/analogous genes can also be activated for functional buffering [8]. For example, 56 multiple growth signaling pathways coordinate cellular growth in parallel, and inactivation of 57 one pathway can lead to activation of others for better survival [3, 4]. In cases of both 58 duplicated and analogous genes, expression of their buffering genes is not activated unless 59 their buffered functions are compromised [3, 7], so responsiveness follows a “need-based” 60 principal. This responsive buffering mechanism has been observed for stress responses of 61 both unicellular and multicellular organisms [3]. 62 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453775; this version posted July 27, 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 4.0 International license. 63 Although numerous studies over recent decades have focused on investigating such 64 responsive buffering mechanisms, a few recent studies have revealed another less- 65 characterized type of buffering mechanism in multicellular organisms, i.e. intrinsic buffering 66 through cell-specific gene expression. In Caenorhabditis elegans, essentiality of a gene can 67 depend on the natural variation in expression of other genes in the same pathway, suggesting 68 that functionally analogous genes in the same pathway can buffer each other [9]. The 69 essentiality of duplicated genes across different human cells can be associated with the 70 expression levels of their paralogs [10-12]. Specifically, higher expression of the buffering 71 paralogs is associated with diminished essentiality of their corresponding buffered duplicated 72 genes, indicating that cell-specific expression of paralogs can contribute to different levels of 73 functional buffering. Thus, expression of buffering genes, either non-duplicated or duplicated, 74 is constitutively active even if the cell does not experience compromised function. 75 Accordingly, this scenario represents an intrinsic buffering mechanism whereby expression 76 of the buffering genes is constitutive in specific cells and tissue types. It is likely that this 77 intrinsic buffering mechanism is controlled by cell- and tissue-specific transcriptional or 78 epigenetic regulators, leading to variable essentiality across different cells and tissues. 79 80 In this study, we directly investigated if cell-specific gene expression can act as an intrinsic 81 buffering mechanism (which we have termed “Cell-specific Expression-BUffering” or CEBU, 82 Fig. 1A) to buffer functionally related genes in the genome, thereby strengthening cellular 83 plasticity for cell- and tissue-specific tasks. To estimate buffering capability, we quantified 84 the proposed CEBU mechanism using a quantitative index, the C-score. This index calculates 85 the adjusted correlation between expression of a buffering gene and the essentiality of the 86 buffered gene (Fig. 1B), utilizing transcriptomics data [13] and gene dependency data from 87 the DepMap project [13, 14] across 684 human cell lines. Our results suggest that intrinsic bioRxiv preprint doi: https://doi.org/10.1101/2021.07.26.453775; this version posted July 27, 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 4.0 International license. 88 buffering by CEBU plays a critical role in cell-specific survival, tissue homeostasis, and 89 cancer robustness. 90 91 Results 92 Development of the C-score to infer cell-specific expression buffering 93 In seeking an index to infer cell-specific expression buffering, we postulated a buffering 94 relationship where the essentiality of a buffered gene (G1) increases when expression of its 95 buffering gene (G2) decreases. Given that G2 expression differs among cells, the strength of 96 functional buffering varies across cells, thereby conferring on G1 cell-specific essentiality 97 (Fig. 1A). This cell-specific expression buffering mechanism, here named CEBU, is the basis 98 for our development of C-score. The C-score of a gene pair is derived from the correlation 99 between the essentiality of a buffered gene (G1) and expression of its buffering gene (G2) 100 (see C-score plot, Fig. 1B), and is formulated