## 3 Discussion
The loss of Hsp sensitivity to CO\({}_{2}\) in NR4A2-depleted cells (Supplementary Figure S7D), as well as the loss of CO\({}_{2}\)-responsive GO terms associated with protein folding and ribosome biogenesis (Supplementary Figure S8), indicates that _NR4A2_ is responsible, at least in part, for the regulation of certain genes in hypercapnia. Taken together, our data provide evidence of selective regulation of CO\({}_{2}\)-dependent transcripts in shNR4A2 and shNR4A3 cells with regard to mitochondrial genes and Hsp-related genes, respectively. In addition, our data provides evidence of many CO\({}_{2}\)-sensitive genes being relatively insensitive to depletion of _NR4A2_ and _NR4A3_, indicating that other conserved transcription factors are involved in the transcriptional response to buffered hypercapnia in this model. There is a known redundancy among NR4A family members [10], meaning it is possible that knockdown of one isoform can be compensated for by the remaining other isoforms. To gain insight into other potential CO\({}_{2}\)-sensitive transcription factors involved in the transcriptional response to buffered hypercapnia, we next focused on transcriptional signatures that were common to shNT, shNR4A2, and shNR4A3 cells. We reasoned that transcripts that were consistently CO\({}_{2}\)-sensitive across the three different cell lines represent particularly robustly CO\({}_{2}\) sensitive transcripts. Data in Figure 4 reveals a striking overlap of CO\({}_{2}\)-sensitive genes in cells that have quite different basal transcriptional profiles [15]. Indeed, nine genes were CO\({}_{2}\)-sensitive in all three cell lines, both with and without LPS stimulation. The consistency of the response is evident in Figure 4D and Supplementary Figure S10, with four transcripts consistently upregulated in hypercapnia (_RRP9, DHP5, SERPINHI_, and _POLRIA_) and five transcripts consistently downregulated (_SRSF5, CYTH2, A4 GALT, DPP8_, and _SLC6A6_). These genes are associated with RNA metabolism (_SRSF5, POLRIA_), metabolite conversion (_A4 GALT_), protein modification (_DPP8_), and protein-binding activity (_SERPINHI_, CYTH2_), with no obvious initial common link apparent. Taken together, these data speak to the responsiveness of multiple important, diverse pathways to buffered hypercapnia. In order to gain further insight into CO\({}_{2}\)-sensitive transcriptional regulators, we sought to exploit our knowledge regarding genes commonly regulated by CO\({}_{2}\) across our three cell lines. Metacore transcription factor analysis revealed a notable conservation of interactions between several transcription factors, including AML1, SOX17, TAL1, ETS1, and LMO2 (Supplementary Table S5). Transcription factors previously linked with hypercapnia were also identified in our list but were not among the most prominent hits in our model, e.g., Transcription Factor P65 (ReIA) [29], NFkB Subunit 2 (NFKB2) [22], Hypoxia Inducible Factor 1 Alpha (HIF1\(\alpha\)) [37], Heat Shock Transcription Factor 1 (HSF1) [35], Cyclic AMP-Responsive Element-Binding Protein 1 (CREB) [38], Forkhead Box O3 (FOXO3a) [39], Early Growth Response 2 (EGR2) [40], Sterol Regulatory Element Binding Transcription Factor 2 (SREBP2) [41]. AML1 is a leukaemia-associated transcription factor linked with hematopoiesis [42]. It has been identified as an overexpressed gene in the lung of COPD patients [43]. SOX17 is a key transcription factor involved in development that affects the cardiovascular system and several endoderm-derived organs [44]. SOX17 deficiency is linked to pulmonary arterial hypertension (PAH) risk via interactions with Hypoxia Inducible Factor 2 Alpha (HIF-2\(\alpha\)) [45]. TAL1 is also a leukaemia associated transcription factor associated with hematopoiesis [45], which can act in complex with LMO2, a LIM-domain protein [46]. ETS-1 is a transcription factor that recognises conserved ETS DNA-binding domains and contributes to cytokine and chemokine regulation. ETS transcription factors are associated with a number of cancers, including breast and leukaemia.