SUMMARY A major challenge in biology is determining how evolutionarily novel characters originate; however, mechanistic explanations for the origin of new characters are almost completely unknown. The evolution of pregnancy is an excellent system in which to study the origin of novelties because mammals preserve stages in the transition from egg laying to live birth. To determine the molecular bases of this transition, we characterized the pregnant/gravid uterine transcriptome from tetrapods to trace the evolutionary history of uterine gene expression. We show that thousands of genes evolved endometrial expression during the origins of mammalian pregnancy, including genes that mediate maternal-fetal communication and immunotolerance. Furthermore, thousands of cis-regulatory elements that mediate decidualization and cell-type identity in decidualized stromal cells are derived from ancient mammalian transposable elements (TEs). Our results indicate that one of the defining mammalian novelties evolved from DNA sequences derived from ancient mammalian TEs coopted into hormone-responsive regulatory elements distributed throughout the genome.

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Adenoid Cystic Carcinoma (ACC), the second most common malignancy of salivary glands, is a rare tumor with bleak prognosis for which therapeutic targets are unavailable. We used RNA-sequencing (RNA-seq) to analyze low-quality RNA from archival, formaldehyde-fixed, paraffin-embedded samples. In addition to detecting the most common ACC translocation, t(6;9) fusing the MYB proto-oncogene to NFIB, we also detected previously unknown t(8;9) and t(8;14) translocations fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively. RNA-seq provided information about gene fusions, alternative RNA splicing and gene expression signatures. Interestingly, tumors with MYB and MYBL1 translocations displayed similar gene expression profiles, and the combined MYB and MYBL1 expression correlated with outcome, suggesting that the related Myb proteins are interchangeable oncogenic drivers in ACC. Our results provide important details about the biology of ACC and illustrate how archival tissue samples can be used for detailed molecular analyses of rare tumors.

To address the dual needs for improved methods to assess potential health risks associated with chemical exposure in aquatic environments and for new models for in vivo mutagenesis studies, we developed transgenic fish that carry multiple copies of a bacteriophage vector that harbors the cII gene as a mutational target. We adapted a forward mutation assay, originally developed for transgenic rodents, to recover cII mutants efficiently from fish genomic DNA by in vitro packaging. After infecting and plating phage on a hfl؊ bacterial host, cII mutants were detected under selective conditions. We demonstrated that many fundamental features of mutation analyses based on transgenic rodents are shared by transgenic fish. Spontaneous mutant frequencies, ranging from 4.3 ؋ 10 ؊5 in liver, 2.9 ؋ 10 ؊5 in whole fish, to 1.8 ؋ 10 ؊5 in testes, were comparable to ranges in transgenic rodents. Treatment with ethylnitrosourea resulted in concentration-dependent, tissue-specific, and time-dependent mutation inductions consistent with known mechanisms of action. Frequencies of mutants in liver increased insignificantly 5 days after ethylnitrosourea exposure, but increased 3.5-, 5.7-and 6.7-fold above background at 15, 20, and 30 days, respectively. Mutants were induced 5-fold in testes at 5 days, attaining a peak 10-fold induction 15 days after treatment. Spontaneous and induced mutational spectra in the fish were also consistent with those of transgenic rodent models. Our results demonstrate the feasibility of in vivo mutation analyses using transgenic fish and illustrate the potential value of fish as important comparative animal models. medaka ͉ ethylnitrosourea A major challenge to the detection of spontaneous and induced mutations is the difficulty with which mutant genes can be efficiently recovered and accurately identified in vivo. Considering that mutations must be detected at low frequencies (e.g., Ϸ1 spontaneous mutation͞10 5 -10 7 loci), and that sufficient DNA sequence information must be available to distinguish mutant from nonmutant genes, the problem of efficiently detecting and quantifying mutations in whole animals can be formidable. Transgenic animals that carry specific genes for quantitation of spontaneous and induced mutations have been developed to assist in improving in vivo mutation analyses (1). In this approach, a transgenic animal carries a prokaryotic vector that harbors a gene that serves as a mutational target. After mutagen exposure, the vector is separated from the animal's genomic DNA and shuttled into indicator bacteria where mutant and nonmutant genes are readily quantified (2, 3). Transgenic mutation assays offer numerous benefits for in vivo mutation detection not available by using other approaches. Benefits include the ability to screen rapidly statistically meaningful numbers of genetically neutral mutational targets in a variety of tissues and the ability to characterize mutations to aid in disclosing possible mechanisms of mutagen action. † A significant additional attribute is the pot...

BackgroundDuring the menstrual cycle, the ovarian steroid hormones estrogen and progesterone control a dramatic transcriptional reprogramming of endometrial stromal cells (ESCs) leading to a receptive state for blastocyst implantation and the establishment of pregnancy. A key marker gene of this decidualization process is the prolactin gene. Several transcriptional regulators have been identified that are essential for decidualization of ESCs, including the Hox genes HoxA-10 and HoxA-11, and the forkhead box gene FOXO1A. While previous studies have identified downstream target genes for HoxA-10 and FOXO1A, the role of HoxA-11 in decidualization has not been investigated. Here, we show that HoxA-11 is required for prolactin expression in decidualized ESC. While HoxA-11 alone is a repressor on the decidual prolactin promoter, it turns into an activator when combined with FOXO1A. Conversely, HoxA-10, which has been previously shown to associate with FOXO1A to upregulate decidual IGFBP-1 expression, is unable to upregulate PRL expression when co-expressed with FOXO1A. By co-immunoprecipitation and chromatin immunoprecipitation, we demonstrate physical association of HoxA-11 and FOXO1A, and binding of both factors to an enhancer region (−395 to −148 relative to the PRL transcriptional start site) of the decidual prolactin promoter. Because FOXO1A is induced upon decidualization, it serves to assemble a decidual-specific transcriptional complex including HoxA-11. These data highlight cooperativity between numerous transcription factors to upregulate PRL in differentiating ESC, and suggest that this core set of transcription factors physically and functionally interact to drive the expression of a gene battery upregulated in differentiated ESC. In addition, the functional non-equivalence of HoxA-11 and HoxA-10 with respect to PRL regulation suggests that these transcription factors regulate distinct sets of target genes during decidualization.

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Current models of developmental evolution suggest changes in gene regulation underlie the evolution of morphology. Despite the fact that protein complexes regulate gene expression, the evolution of regulatory protein complexes is rarely studied. Here, we investigate the evolution of a protein-protein interaction (PPI) between Homeobox A11 (HoxA11) and Forkhead box 01A (Foxo1a). Using extant and "resurrected" ancestral proteins, we show that the physical interaction between HoxA11 and Foxo1a originated in the mammalian stem lineage. Functional divergence tests and coimmunoprecipitation with heterologous protein pairs indicate that the evolution of interaction was attributable to changes in HoxA11, and deletion studies demonstrate that the interaction interface is located in the homeodomain region of HoxA11. However, there are no changes in amino acid sequence in the homeodomain region during this time period, indicating that the origin of the derived PPI was attributable to changes outside the binding interface. We infer that the amino acid substitutions in HoxA11 altered Foxo1a's access to the conserved binding interface at the HoxA11 homeodomain. We also found an expansion in the number of paired Hox/Fox binding sites in the genomes of mammalian lineage species suggesting the complex has a biological function. Our data indicate that the physical interaction between HoxA11 and Foxo1a evolved through noninterface changes that facilitate the PPI, which prevents inappropriate interactions, rather than through the evolution of a novel binding interface. We speculate that evolutionary changes of intramolecular regulation have limited pleiotropic effects compared with changes to interaction domains themselves.protein-protein interaction evolution | transcription factor evolution C hanges in gene regulation are the driving force in the origin and evolution of novel phenotypes. Gene expression is coordinated by the formation of multiprotein complexes that bind to cis-regulatory promoter and enhancer regions for target genes and activate or repress transcription in a signal-dependent fashion (1-3). There is strong evidence that changes in both cisregulatory elements and regulatory proteins, such as transcription factors, have led to gene regulatory evolution (4-12). However, the mechanisms by which transcription factor evolution affects gene regulation are poorly understood. For example, it has been suggested that the evolution of novel protein-protein interactions (PPIs), posttranslational modifications, and DNA-and ligandbinding specificities may all contribute to the origin of regulatory activities in transcription factors; however, to date, few studies have carefully dissected potential mechanisms.Here, we address this question by investigating the evolution of the physical interaction between two transcription factors, Homeobox A11 (HoxA11) and Forkhead box 01A (Foxo1a), which play a major role in regulating gene expression in endometrial stromal cells during pregnancy in placental mammals (8,13). By examining the a...

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3 (MAP2K), and a MAPK kinase kinase (MAP3K). Regulation of the MAP3K, presumably by phosphorylation, provides the impetus for activation of the three-kinase module. Once activated, the MAP3Ks activate MAP2Ks by phosphorylation of two residues within the activation loop. Phosphorylation of MAP2Ks activates these dual specificity kinases to phosphorylate MAPKs on a conserved threonine and tyrosine motif, TXY, also within the activation loop. Once phosphorylated, the MAPKs phosphorylate protein substrates and regulate cellular processes like growth, protein synthesis, gene expression, and nucleotide synthesis (2).Over the last decade, a large body of work has characterized events that occur downstream of the MAPKs. However, little is known regarding the regulatory mechanisms that modulate the MEKK proteins to ultimately regulate the MAPKs. For example, it is known that overexpression of MEKK3 activates the ERK (3, 4), JNK (3-5), p38 (5, 6), ERK5 (7), and NF-B pathways (8 -10). Typically, MEKK3-dependent regulation of these pathways is studied by using transfection studies, and activation of these pathways rarely requires an agonist. Therefore, it appears that some process intrinsic to MEKK3 is critical and sufficient for activation of these pathways.The activation loop of some protein kinase families, such as the arginine-aspartate family, is positioned between subdomains VII and VIII and is phosphorylated by other protein kinases or through autophosphorylation of the kinase itself (11). Phosphorylation within the activation loop of protein kinases results in conformational changes in the protein structure that (i) enhance substrate binding, (ii) correctly position amino acids involved in catalysis, and (iii) relieve steric hindrance within the catalytic domain. Regardless of how the activation loop is phosphorylated, regulation of catalytic activity frequently correlates with phosphorylation of the activation loop.Given that phosphorylation plays a critical role in regulating the MAPKs and the MAP2Ks, we investigated how phosphorylation of MEKK3 might affect its catalytic activity. Since phosphorylation sites within the activation loop of MEKK3 have not been reported, we systematically mutated serine and threonine residues within the activation loop to alanine and monitored MEKK3-dependent activities in HEK293 EBNA cells. Two key amino acids were identified at positions 526 and 530 using a luciferase-based reporter gene assay as well as assays that measure the ERK, JNK, and p38 MAP...