Rapid evolution of promoters from germline-specifically expressed genes including transposon silencing factors.

BACKGROUND: The piRNA pathway in animal gonads functions as an 'RNA-based immune system', serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23-28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways. RESULTS: By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, we demonstrate that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. Our findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, we observe that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression. CONCLUSION: Our results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons.

Regulation of THI and PDC genes by Pdc2 in Nakaseomyces glabratus (Candida glabrata) is complex.

Thiamine (vitamin B1) is essential for glucose catabolism. In the yeast species Nakaseomyces glabratus (formerly Candida glabrata) and Saccharomyces cerevisiae, the transcription factor Pdc2 (with Thi3 and Thi2) upregulates pyruvate decarboxylase (PDC) genes and thiamine biosynthetic and acquisition (THI) genes during starvation. There have not been genome-wide analyses of Pdc2 binding. Previously, we identified small regions of Pdc2 regulated genes sufficient to confer thiamine regulation. Here, we performed deletion analyses on these regions. We observed that when the S. cerevisiae PDC5 promoter is introduced into N. glabratus, it is thiamine starvation inducible but does not require the Thi3 coregulator. The ScPDC5 promoter contains a 22 bp duplication with an AT-rich spacer between the two repeats, which are important for regulation. Loss of the first 22 bp element does not eliminate regulation, but the promoter becomes Thi3-dependent, suggesting cis architecture can generate a Thi3-independent, thiamine starvation inducible response. Whereas many THI promoters only have one copy of this element, addition of the first 22 bp element to a Thi3-dependent promoter confers Thi3-independence. Finally, we performed fluorescence anisotropy and ChIP-seq. Pdc2 and Thi3 bind to regions that share similarity to the 22 bp element in the ScPDC5 promoter and previously identified cis elements in N. glabratus promoters. Also, while Pdc2 binds to THI and PDC promoters, neither Pdc2 nor Thi3 appear to bind the evolutionarily new NgPMU3 promoter that is regulated by Pdc2. Further study is warranted because PMU3 is required for cells to acquire thiamine from environments where thiamine is phosphorylated, such as in the human bloodstream.

Promoter generation for the chimeric sex-determining gene dm-W in Xenopus frogs.

Many sex-determining genes (SDGs) were generated as neofunctionalized genes through duplication and/or mutation of gonadal formation-related genes. We previously identified dm-W as an SDG in the African clawed frog Xenopus laevis and found that a partial duplication of the masculinization gene dmrt1 created the neofunctionalized dm-W after allotetraploidization by interspecific hybridization. The allotetraploid Xenopus species have two dmrt1 genes, dmrt1.L and dmrt1.S. Xenopus laevis dm-W has four exons: two dmrt1.S-derived exons (exons 2 and 3) and two other exons (noncoding exon 1 and exon 4). Our recent work revealed that exon 4 originated from a DNA transposon, hAT-10. Here, to clarify when and how the noncoding exon 1 and its coexisting promoter evolved during the establishment of dm-W after allotetraploidization, we newly determined nucleotide sequences of the dm-W promoter region from two other allotetraploid species, X. largeni and X. petersii, and performed an evolutionary analysis. We found that dm-W acquired a new exon 1 and TATA-type promoter in the common ancestor of the three allotetraploid Xenopus species, resulting in the deletion of the dmrt1.S-derived TATA-less promoter. In addition, we demonstrated that the TATA box contributes to dm-W promoter activity in cultured cells. Collectively, these findings suggest that this novel TATA-type promoter was important for the establishment of dm-W as a sex-determining gene, followed by the degeneration of the preexisting promoter.

Promoter evolution of mammalian gene duplicates.

BACKGROUND: Gene duplication is thought to be a central process in evolution to gain new functions. The factors that dictate gene retention following duplication as well paralog gene divergence in sequence, expression and function have been extensively studied. However, relatively little is known about the evolution of promoter regions of gene duplicates and how they influence gene duplicate divergence. Here, we focus on promoters of paralog genes, comparing their similarity in sequence, in the sets of transcription factors (TFs) that bind them, and in their overall promoter architecture. RESULTS: We observe that promoters of recent duplications display higher sequence similarity between them and that sequence similarity rapidly declines between promoters of more ancient paralogs. In contrast, similarity in cis-regulation, as measured by the set of TFs that bind promoters of both paralogs, does not simply decrease with time from duplication and is instead related to promoter architecture-paralogs with CpG Islands (CGIs) in their promoters share a greater fraction of TFs, while CGI-less paralogs are more divergent in their TF binding set. Focusing on recent duplication events and partitioning them by their duplication mechanism enables us to uncover promoter properties associated with gene retention, as well as to characterize the evolution of promoters of newly born genes: In recent retrotransposition-mediated duplications, we observe asymmetry in cis-regulation of paralog pairs: Retrocopy genes are lowly expressed and their promoters are bound by fewer TFs and are depleted of CGIs, in comparison with the original gene copy. Furthermore, looking at recent segmental duplication regions in primates enable us to compare successful retentions versus loss of duplicates, showing that duplicate retention is associated with fewer TFs and with CGI-less promoter architecture. CONCLUSIONS: In this work, we profiled promoters of gene duplicates and their inter-paralog divergence. We also studied how their characteristics are associated with duplication time and duplication mechanism, as well as with the fate of these duplicates. These results underline the importance of cis-regulatory mechanisms in shaping the evolution of new genes and their fate following duplication.

Adaptation dynamics between copy-number and point mutations.

Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of genomic data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic reporter system that can distinguish between copy-number and point mutations, we study their early and transient adaptive dynamics in real time in Escherichia coli. We find two qualitatively different routes of adaptation, depending on the level of functional improvement needed. In conditions of high gene expression demand, the two mutation types occur as a combination. However, under low gene expression demand, copy-number and point mutations are mutually exclusive; here, owing to their higher frequency, adaptation is dominated by copy-number mutations, in a process we term amplification hindrance. Ultimately, due to high reversal rates and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation, but also constrain sequence divergence over evolutionary time scales.

Early Evolution of Transcription Systems and Divergence of Archaea and Bacteria.

DNA template-dependent multi-subunit RNA polymerases (RNAPs) found in all three domains of life and some viruses are of the two-double-Ψ-ß-barrel (DPBB) type. The 2-DPBB protein format is also found in some RNA template-dependent RNAPs and a major replicative DNA template-dependent DNA polymerase (DNAP) from Archaea (PolD). The 2-DPBB family of RNAPs and DNAPs probably evolved prior to the last universal common cellular ancestor (LUCA). Archaeal Transcription Factor B (TFB) and bacterial σ factors include homologous strings of helix-turn-helix units. The consequences of TFB-σ homology are discussed in terms of the evolution of archaeal and bacterial core promoters. Domain-specific DPBB loop inserts functionally connect general transcription factors to the RNAP active site. Archaea appear to be more similar to LUCA than Bacteria. Evolution of bacterial σ factors from TFB appears to have driven divergence of Bacteria from Archaea, splitting the prokaryotic domains.

Multi-omic analysis suggests tumor suppressor genes evolved specific promoter features to optimize cancer resistance.

Tumor suppressor genes (TSGs) exhibit distinct evolutionary features. We speculated that TSG promoters could have evolved specific features that facilitate their tumor-suppressing functions. We found that the promoter CpG dinucleotide frequencies of TSGs are significantly higher than that of non-cancer genes across vertebrate genomes, and positively correlated with gene expression across tissue types. The promoter CpG dinucleotide frequencies of all genes gradually increase with gene age, for which young TSGs have been subject to a stronger evolutionary pressure. Transcription-related features, namely chromatin accessibility, methylation and ZNF263-, SP1-, E2F4- and SP2-binding elements, are associated with gene expression. Moreover, higher promoter CpG dinucleotide frequencies and chromatin accessibility are positively associated with the ability of TSGs to resist downregulation during tumorigenesis. These results were successfully validated with independent datasets. In conclusion, TSGs evolved specific promoter features that optimized cancer resistance through achieving high expression in normal tissues and resistance to downregulation during tumorigenesis.

Kozak Sequence Acts as a Negative Regulator for De Novo Transcription Initiation of Newborn Coding Sequences in the Plant Genome.

The manner in which newborn coding sequences and their transcriptional competency emerge during the process of gene evolution remains unclear. Here, we experimentally simulated eukaryotic gene origination processes by mimicking horizontal gene transfer events in the plant genome. We mapped the precise position of the transcription start sites (TSSs) of hundreds of newly introduced promoterless firefly luciferase (LUC) coding sequences in the genome of Arabidopsis thaliana cultured cells. The systematic characterization of the LUC-TSSs revealed that 80% of them occurred under the influence of endogenous promoters, while the remainder underwent de novo activation in the intergenic regions, starting from pyrimidine-purine dinucleotides. These de novo TSSs obeyed unexpected rules; they predominantly occurred ∼100 bp upstream of the LUC inserts and did not overlap with Kozak-containing putative open reading frames (ORFs). These features were the output of the immediate responses to the sequence insertions, rather than a bias in the screening of the LUC gene function. Regarding the wild-type genic TSSs, they appeared to have evolved to lack any ORFs in their vicinities. Therefore, the repulsion by the de novo TSSs of Kozak-containing ORFs described above might be the first selection gate for the occurrence and evolution of TSSs in the plant genome. Based on these results, we characterized the de novo type of TSS identified in the plant genome and discuss its significance in genome evolution.

Promoter Length Affects the Initiation of T7 RNA Polymerase In Vitro: New Insights into Promoter/Polymerase Co-evolution.

Polymerases are integral factors of gene expression and are essential for the maintenance and transmission of genetic information. RNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate transcription de novo and this promoter recognition requires the presence of specific DNA binding domains in the polymerase. Bacteriophage T7 RNA polymerase (T7RNAP) is the prototype for single subunit RNA polymerases which include bacteriophage and mitochondrial RNAPs, and the structure and mechanistic aspects of transcription by T7 RNAP are well characterized. Here, we describe experiments to determine whether the prototype T7 RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. These assays have not only defined the limits of conventional de novo initiation on truncated promoters, but have identified novel activities of initiation of RNA synthesis. We propose that these novel activities may be vestigial activities surviving from the transition of single subunit polymerase initiation using primers to de novo initiation using promoters.

Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish.

BACKGROUND: In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (nonfunctionalization), acquire a new function (neofunctionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (subfunctionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter. RESULTS: In this study, we describe the PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters and compare them to the PPAR regulation of the spotted gar fabp1 promoter, representative of the ancestral fabp1 gene. Evolution of the fabp1 promoter was inferred by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine explant cells, and in HEK293A cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. The promoter activity of spotted gar fabp1, representative of the ancestral fabp1, was induced by both PPARα- and PPARγ-specific agonists, but displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, and fabp1b.2 was not regulated by PPAR. CONCLUSIONS: The zebrafish fabp1 promoters underwent two successive rounds of subfunctionalization with respect to PPAR regulation leading to retention of three zebrafish fabp1 genes with stimuli-specific regulation. Using a pharmacological approach, we demonstrated here the divergent regulation of the zebrafish fabp1a, fabp1b.1, and fabp1b.2 with regard to subfunctionalization of PPAR regulation following two rounds of gene duplication.

Measuring transcription factor-binding site turnover: a maximum likelihood approach using phylogenies.

A major mode of gene expression evolution is based on changes in cis-regulatory elements (CREs) whose function critically depends on the presence of transcription factor-binding sites (TFBS). Because CREs experience extensive TFBS turnover even with conserved function, alignment-based studies of CRE sequence evolution are limited to very closely related species. Here, we propose an alternative approach based on a stochastic model of TFBS turnover. We implemented a maximum likelihood model that permits variable turnover rates in different parts of the species tree. This model can be used to detect changes in turnover rate as a proxy for differences in the selective pressures acting on TFBS in different clades. We applied this method to five TFBS in the fungi methionine biosynthesis pathway and three TFBS in the HoxA clusters of vertebrates. We find that the estimated turnover rate is generally high, with half-life ranging between approximately 5 and 150 My and a mode around tens of millions of years. This rate is consistent with the finding that even functionally conserved enhancers can show very low sequence similarity. We also detect statistically significant differences in the equilibrium densities of estrogen- and progesterone-response elements in the HoxA clusters between mammal and nonmammal vertebrates. Even more extreme clade-specific differences were found in the fungal data. We conclude that stochastic models of TFBS turnover enable the detection of shifts in the selective pressures acting on CREs in different organisms. The analysis tool, called CRETO (Cis-Regulatory Element Turn-Over) can be downloaded from http://www.bioinf.uni-leipzig.de/Software/creto/.

Rapid sequence divergence rates in the 5 prime regulatory regions of young Drosophila melanogaster duplicate gene pairs

While it remains a matter of some debate, rapid sequence evolution of the coding sequences of duplicate genes is characteristic for early phases past duplication, but long established duplicates generally evolve under constraint, much like the rest of the coding genome. As for coding sequences, it may be possible to infer evolutionary rate, selection, and constraint via contrasts between duplicate gene divergence in the 5 prime regions and in the corresponding synonymous site divergence in the coding regions. Finding elevated rates for the 5 prime regions of duplicated genes, in addition to the coding regions, would enable statements regarding the early processes of duplicate gene evolution. Here, 1 kb of each of the 5 prime regulatory regions of Drosophila melanogaster duplicate gene pairs were mapped onto one another to isolate shared sequence blocks. Genetic distances within shared sequence blocks (d5’) were found to increase as a function of synonymous (dS), and to a lesser extend, amino-acid (dA) site divergence between duplicates. The rate d5’/dS was found to rapidly decay from values > 1 in young duplicate pairs (dS < 0.3) to 0.28 or less in older duplicates (dS > 0.8). Such rapid rates of 5 prime evolution exceeding 1 (~neutral) predominantly were found to occur in duplicate pairs with low amino-acid site divergence and that tended to be co-regulated when assayed on microarrays. Conceivably, functional redundancy and relaxation of selective constraint facilitates subsequent positive selection on the 5 prime regions of young duplicate genes. This might promote the evolution of new functions (neofunctionalization) or division of labor among duplicate genes (subfunctionalization). In contrast, similar to the vast portion of the non-coding genome, the 5 prime regions of long-established gene duplicates appear to evolve under selective constraint, indicating that these long-established gene duplicates have assumed critical functions.