Phylogenomic analyses across land plants reveals motifs and coexpression patterns useful for functional prediction in the BAHD acyltransferase family.

The BAHD acyltransferase family is one of the largest enzyme families in flowering plants, containing dozens to hundreds of genes in individual genomes. Highly prevalent in angiosperm genomes, members of this family contribute to several pathways in primary and specialized metabolism. In this study, we performed a phylogenomic analysis of the family using 52 genomes across the plant kingdom to gain deeper insights into its functional evolution and enable function prediction. We found that BAHD expansion in land plants was associated with significant changes in various gene features. Using pre-defined BAHD clades, we identified clade expansions in different plant groups. In some groups, these expansions coincided with the prominence of metabolite classes such as anthocyanins (flowering plants) and hydroxycinnamic acid amides (monocots). Clade-wise motif-enrichment analysis revealed that some clades have novel motifs fixed on either the acceptor or the donor side, potentially reflecting historical routes of functional evolution. Co-expression analysis in rice and Arabidopsis further identified BAHDs with similar expression patterns, however, most co-expressed BAHDs belonged to different clades. Comparing BAHD paralogs, we found that gene expression diverges rapidly after duplication, suggesting that sub/neo-functionalization of duplicate genes occurs quickly via expression diversification. Analyzing co-expression patterns in Arabidopsis in conjunction with orthology-based substrate class predictions and metabolic pathway models led to the recovery of metabolic processes of most of the already-characterized BAHDs as well as definition of novel functional predictions for some uncharacterized BAHDs. Overall, this study provides new insights into the evolution of BAHD acyltransferases and sets up a foundation for their functional characterization.

Orthology-based analysis helps map evolutionary diversification and predict substrate class use of BAHD acyltransferases.

Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast-evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG-based analysis enabled identification of signature motifs in anthocyanin-acylating BAHDs, whose importance was validated via molecular dynamic simulations, site-directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration-enabled approach for improved functional annotation of plant enzyme families.

Mosaic cis-regulatory evolution drives transcriptional partitioning of HERVH endogenous retrovirus in the human embryo.

The human endogenous retrovirus type-H (HERVH) family is expressed in the preimplantation embryo. A subset of these elements are specifically transcribed in pluripotent stem cells where they appear to exert regulatory activities promoting self-renewal and pluripotency. How HERVH elements achieve such transcriptional specificity remains poorly understood. To uncover the sequence features underlying HERVH transcriptional activity, we performed a phyloregulatory analysis of the long terminal repeats (LTR7) of the HERVH family, which harbor its promoter, using a wealth of regulatory genomics data. We found that the family includes at least eight previously unrecognized subfamilies that have been active at different timepoints in primate evolution and display distinct expression patterns during human embryonic development. Notably, nearly all HERVH elements transcribed in ESCs belong to one of the youngest subfamilies we dubbed LTR7up. LTR7 sequence evolution was driven by a mixture of mutational processes, including point mutations, duplications, and multiple recombination events between subfamilies, that led to transcription factor binding motif modules characteristic of each subfamily. Using a reporter assay, we show that one such motif, a predicted SOX2/3 binding site unique to LTR7up, is essential for robust promoter activity in induced pluripotent stem cells. Together these findings illuminate the mechanisms by which HERVH diversified its expression pattern during evolution to colonize distinct cellular niches within the human embryo.