Functional annotation of novel lineage-specific genes using co-expression and promoter analysis

Abstract

Background: The diversity of placental architectures within and among mammalian orders is believed to be the result of adaptive evolution. Although, the genetic basis for these differences is unknown, some may arise from rapidly diverging and lineage-specific genes. Previously, we identified 91 novel lineage-specific transcripts (LSTs) from a cow term-placenta cDNA library, which are excellent candidates for adaptive placental functions acquired by the ruminant lineage. The aim of the present study was to infer functions of previously uncharacterized lineage-specific genes (LSGs) using co-expression, promoter, pathway and network analysis.

Results: Clusters of co-expressed genes preferentially expressed in liver, placenta and thymus were found using 49 previously uncharacterized LSTs as seeds. Over-represented composite transcription factor binding sites (TFBS) in promoters of clustered LSGs and known genes were then identified computationally. Functions were inferred for nine previously uncharacterized LSGs using co-expression analysis and pathway analysis tools. Our results predict that these LSGs may function in cell signaling, glycerophospholipid/fatty acid metabolism, protein trafficking, regulatory processes in the nucleus, and processes that initiate parturition and immune system development.

Conclusions: The placenta is a rich source of lineage-specific genes that function in the adaptive evolution of placental architecture and functions. We have shown that co-expression, promoter, and gene network analyses are useful methods to infer functions of LSGs with heretofore unknown functions. Our results indicate that many LSGs are involved in cellular recognition and developmental processes. Furthermore, they provide guidance for experimental approaches to validate the functions of LSGs and to study their evolution.

Figure 1

Schema for inferring functions of LSGs.

Figure 2

Co-expression of LSTs and other genes in LIVR cluster. The cluster of 212 genes (LIVR) shows a significant (P < 0.0001) difference in expression (~1.8-fold) between ad-libitum (AD; left panel) and energy-restricted (RS; right panel) diets at +1 and +14 days post-partum.

Figure 3

Co-expression of genes in PLAC and THYM clusters. The average pairwise Pearson correlation (r) within each cluster was r ≥ 0.75. The correlation between any one of the LSTs and any known gene was r ≥ 0.90: A) Co-expression of LSTs 22JE, 34FL, and 104JE with 113 other genes using expression data from 18 cattle tissues. A cluster of 116 genes (PLAC) shows preferential expression in placentome, with each gene having ≥ 2-fold higher expression in placentome as compared to any other tissue: B) Co-expression of LSTs 383NG and 21PW with 30 other genes using expression data from 18 cattle tissues. A cluster of 32 genes (THYM) shows preferential expression in thymus with each gene having ≥ 2 fold higher expression in thymus as compared to any other tissue. L_Intestine, large intestine; M_L_Node, mesenteric lymph node; S_Intestine, small intestine.

Figure 4

Clustering of 28 LSTs significantly expressed (P < 0.05) at one or more time-point(s) and by diet. Gray cells indicate that the gene is either not expressed at that time-point/diet, had missing data, or did not meet the filtering criteria (see Methods). The numbers at the intersection of branches indicate the branch-correlation. Green cells indicate under-expression, orange/red/pink cells indicate over-expression, and yellow cells indicate no change in expression compared to the reference sample.

Figure 5

Interaction network for co-expressed genes in liver and the LIVR cluster showing roles in glycerophospholipid metabolism, protein transport, and signaling. LIVR cluster genes were analyzed using GeneGo MetaCore [32] and its human-specific interaction database. A sub-network was built starting with PLCE1, NGLY1, MX1, TRIP10 and ARF5, which are genes that are predicted to be co-regulated with 237NG and 266NG. Hub genes (IL22RA1, GGA1) predicted by WGCNA were then added to this network. Only interactions known to occur in liver tissue are shown as determined using a liver tissue trace in GeneGO. Majority of these are common to placenta tissue as determined using a placenta tissue trace. Those interactions that are specific to liver only are marked with the letter L. Genes that are expressed as part of the LIVR cluster are indicated with a red circle. A legend explaining the symbols is provided at http://portal.genego.com/legends/legend_6.png.