What are housekeeping genes?

Abstract

The concept of "housekeeping gene" has been used for four decades but remains loosely defined. Housekeeping genes are commonly described as "essential for cellular existence regardless of their specific function in the tissue or organism", and "stably expressed irrespective of tissue type, developmental stage, cell cycle state, or external signal". However, experimental support for the tenet that gene essentiality is linked to stable expression across cell types, conditions, and organisms has been limited. Here we use genome-scale functional genomic screens together with bulk and single-cell sequencing technologies to test this link and optimize a quantitative and experimentally validated definition of housekeeping gene. Using the optimized definition, we identify, characterize, and provide as resources, housekeeping gene lists extracted from several human datasets, and 10 other animal species that include primates, chicken, and C. elegans. We find that stably expressed genes are not necessarily essential, and that the individual genes that are essential and stably expressed can considerably differ across organisms; yet the pathways enriched among these genes are conserved. Further, the level of conservation of housekeeping genes across the analyzed organisms captures their taxonomic groups, showing evolutionary relevance for our definition. Therefore, we present a quantitative and experimentally supported definition of housekeeping genes that can contribute to better understanding of their unique biological and evolutionary characteristics.

Fig 1. The relationship between the axiomatically assigned four properties of housekeeping genes remains unexplored.

Fig 2. Gini coefficient captures the first property of housekeeping genes: stable expression.

(A) Coverage of the 3688 Eisenberg and Levanon housekeeping genes [1] among the 3688 genes with lowest Gini coefficients within each dataset. (B) Gini coefficients of individual genes are highly correlated across human datasets regardless of sample type; however, spearman correlations within sample types are tighter. Dots represent unique genes. Red specifically depicts housekeeping genes identified by Eisenberg and Levanon [1].

Fig 3. Housekeeping genes are mostly expressed at relatively low levels.

(A-B) Comparison of median expression levels and Gini coefficients calculated for the (C) HPA and (B) GTEx datasets. The spearman correlations between Gini coefficients and median expression values were -0.6545 (p = 0) and -0.7244 (p = 0) for HPA and GTEx, respectively. Dots represent unique genes. Red specifically depicts housekeeping genes identified by Eisenberg and Levanon [1].

Fig 4. Gini genes are essential.

Gini coefficients of essential genes (black dots) compared to the housekeeping genes from Eisenberg and Levanon when using (A) CellMiner (Wilcoxon p-value = 8.373 x 10⁻³⁸; KS p-value = 2.48 x 10⁻²⁶) and (B) Klijn et al. [37] (Wilcoxon p-value = 5.96 x 10⁻¹⁸; KS p-value = 4.44 x 10⁻²³) cancer datasets, and (C) CHO datasets. The 2800 genes essential in the 20 cancer cell lines were extracted from DepMap [40,41], and the 338 CHO essential genes were extracted from Xiong et al. [39] (D) GO term coverage of essential genes and that of Gini genes from CellMiner (blue, 0.8557), Klijn et al. [37] (yellow, 0.8907), and CHO (green, 0.7055) are correlated. The left-tailed Wilcoxon p-values when comparing essential genes with the overall datasets are 2.35 x 10⁻¹⁴⁶ for CellMiner and 9.16 x 10⁻³¹³ for Klijn et al. [37] This suggests that essential genes and housekeeping genes are coming from populations with equal medians and similar cumulative distribution functions. The slightly lower correlation in CHO cells is likely due to the fewer number of essential genes reported in the CHO screen. (E) High- and medium-confidence essential genes in healthy C. elegans have significantly lower G_C than non-essential genes (p = 4.53 x 10⁻⁵).

Fig 5. Gini coefficients accurately capture organism-specific differences.

(A-B) Spearman correlations of Gini coefficients of Gini genes (bottom 20^th percentile of G_C in at least one organism) identified using organism-specific transcriptomes capture cluster containing primates. The number of Gini genes with 1:1 orthologs in all organisms is shown using the bar plot on the right of the dendrogram. Please see S10 Fig. for p-values. (C) Principal component 1 (PC1) also captures the cluster containing primates. Also shown are the top 20 primate Gini genes (pink), the top 20 shared Gini genes (green), and top 20 Gini genes in all non-primates (blue) using the principal component coefficients of the first principal components. (D) Correlation among Gini coefficients across different organisms reproduce cluster containing primates (left panel). The Gini coefficients of genes belonging to top 20, middle 20, and bottom 20 coefficients of PC1 are shown (right panel). Left 20 Gini genes are specific to non-primates, middle 20 Gini genes are shared, and right 20 Gini genes are specific to primates. (E) GO term coverage is highly correlated across different datasets, also shown are the GO term correlations with human datasets used in S6 Fig.