Regulation of the Sox3 Gene in an X0/X0 Mammal without Sry, the Amami Spiny Rat, Tokudaia osimensis

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.

An Allosteric Mechanism for Epigenetic Activation of the V(D)J Recombinase

V(D)J recombination, the process by which antigen receptor genes are assembled from discrete DNA segments during lymphoid development, is responsible for the generation of the primary immune repertoire. Errors in V(D)J recombination have been implicated in the pathogenesis of lymphoid malignancies, including follicular lymphoma, MALT lymphoma and mantle cell lymphoma. V(D)J recombination is initiated by a specialized transposase, RAG, consisting of RAG-1 and RAG-2 subunits. RAG mobilizes participating gene segments in a site-specific fashion by cleaving DNA at conserved recombination signal sequences. The accessibility of these sequences to RAG is subject to locus- and developmental stage-specific control by mechanisms that are as yet poorly understood. Elucidation of these mechanisms is fundamental to our understanding of the off-target events linking RAG activity to tumorigenesis. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). RAG-2 contains a plant homeodomain (PHD) finger that binds specifically to H3K4me3. Disruption of this PHD finger impairs V(D)J recombination in vivo. Peptides bearing H3K4me3 stimulate substrate binding and catalysis of DNA cleavage by RAG. This stimulation is dependent on an intact PHD finger, suggesting that H3K4me3 is an allosteric activator of the V(D)J recombinase. Indeed, binding of H3K4me3 to the RAG-2 PHD induces dynamic conformational changes in RAG-1. Because substrate binding and catalysis are functions of RAG-1, information regarding occupancy of the RAG-2 PHD must be transmitted to the RAG-1 subunit. To understand how the recognition of active chromatin is coupled to the binding and cleavage of recombination signal sequences, we sought to trace the path of allostery from the RAG-2 PHD finger to RAG-1. Our strategy has been: (1) to generate chimeric RAG-2 proteins in which the mouse PHD finger is replaced by the PHD finger of a phylogenetically distant RAG-2; (2) to identify chimeric RAG-2 proteins that are capable of binding H3K4me3 but incapable of allosteric activation; (3) to systematically back-mutate residues in the foreign PHD to the mouse sequence; and (4) to identify back-mutations that rescue allosteric activation. A chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the bamboo shark, C. punctatum, fails to support V(D)J recombination in vivo. This chimeric protein retains the ability to bind H3K4me3 but engagement of H3K4me3 does not result in allosteric activation, suggesting that the allosteric interface of the PHD finger is disrupted. The amino acid sequence differences between mouse and C. punctatum form several clusters, located on the opposite side of the PHD from the H3K4me3 binding site. Each of these clusters in the C. punctatum PHD finger was mutated to the mouse sequence and the corresponding back-mutated chimeric RAG-2 proteins were tested for their ability to support V(D)J recombination. Strikingly, mutation of one such cluster, corresponding to residues 425 - 429, 431 and 433 of mouse RAG-2, was sufficient to rescue recombination activity to the level of wild-type. Taken together, our observations indicate that the binding of H3K4me3 by RAG-2 is itself insufficient to support recombination; rather, information regarding the engagement of H3K4me3 must be transmitted allosterically. Moreover, our mutational analysis has identified a putative allosteric surface within the PHD finger and distinct from the H3K4me3 binding site that is responsible for transmitting the allosteric signal. The requirement for allosteric activation by H3K4me3 may play a role in defining patterns of RAG-mediated DNA cleavage during normal development and in the generation of lymphoid malignancies. Disclosures Desiderio: Genentech: Consultancy; AbbVie: Consultancy.

Learning and recognition of tactile temporal sequences by mice and humans

The world around us is replete with stimuli that unfold over time. When we hear an auditory stream like music or speech or scan a texture with our fingertip, physical features in the stimulus are concatenated in a particular order. This temporal patterning is critical to interpreting the stimulus. To explore the capacity of mice and humans to learn tactile sequences, we developed a task in which subjects had to recognise a continuous modulated noise sequence delivered to whiskers or fingertips, defined by its temporal patterning over hundreds of milliseconds. GO and NO-GO sequences differed only in that the order of their constituent noise modulation segments was temporally scrambled. Both mice and humans efficiently learned tactile sequences. Mouse sequence recognition depended on detecting transitions in noise amplitude; animals could base their decision on the earliest information available. Humans appeared to use additional cues, including the duration of noise modulation segments.

Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus

The molecular events leading to the development of the bat wing remain largely unknown, and are thought to be caused, in part, by changes in gene expression during limb development. These expression changes could be instigated by variations in gene regulatory enhancers. Here, we used a comparative genomics approach to identify regions that evolved rapidly in the bat ancestor but are highly conserved in other vertebrates. We discovered 166 bat accelerated regions (BARs) that overlap H3K27ac and p300 ChIP-seq peaks in developing mouse limbs. Using a mouse enhancer assay, we show that five Myotis lucifugus BARs drive gene expression in the developing mouse limb, with the majority showing differential enhancer activity compared to the mouse orthologous BAR sequences. These include BAR116, which is located telomeric to the HoxD cluster and had robust forelimb expression for the M. lucifugus sequence and no activity for the mouse sequence at embryonic day 12.5. Developing limb expression analysis of Hoxd10-Hoxd13 in Miniopterus natalensis bats showed a high-forelimb weak-hindlimb expression for Hoxd10-Hoxd11, similar to the expression trend observed for M. lucifugus BAR116 in mice, suggesting that it could be involved in the regulation of the bat HoxD complex. Combined, our results highlight novel regulatory regions that could be instrumental for the morphological differences leading to the development of the bat wing.

Antibodies Specific For Human Neutrophil Antigen 3a (HNA-3a) Recognize Complex Epitopes On Choline Transporter Protein 2 (CTL2): Implications For HNA-3a Antibody Detection

Transfusion-related acute lung injury (TRALI), the leading cause of mortality associated with blood transfusion, usually results from passive transfer of antibodies present in donated blood to a patient. TRALI can be triggered by antibodies specific for Class I or Class II HLA antigens, human neutrophil antigens (HNA) and possibly other targets. For reasons not well understood, antibodies specific for the leukocyte antigen HNA-3a cause particularly severe, often fatal TRALI. It would be highly desirable therefore to be able to screen blood donors routinely for HNA-3a antibodies. HNA-3a/b antigens are carried on choline transporter-like protein 2 (CTL2), an apparent 10 membrane-spanning protein with 5 extracellular loops and N and C intracellular termini. The HNA-3a/b polymorphism is created by an R/Q substitution at position 154 in the first of these extracellular loops (Loop 1). In solid phase assays, about one-half of HNA-3a antibodies implicated in TRALI recognize Loop 1 peptides containing R154 (Type 1 antibodies). The remaining antibodies (Type 2) are non-reactive with peptides despite reacting well against full length CTL2. We studied reactions of Type 1 and Type 2 HNA-3a antibodies against soluble recombinant CTL2 fragments, human CTL2, mouse CTL2, and human/mouse CTL2 chimeras expressed in HEK293 cells to characterize the basis for HNA-3a antibody heterogeneity. The following observations were made:1) Only Type 1 antibodies react with detergent-solublized CTL2 in solid phase assays.2) A soluble recombinant fragment derived from the first extracellular (EC) loop (R154) of human CTL2 reacts only with Type 1 antibodies.3) Mouse CTL2 is 91% identical to human CTL2 and contains the R154 residue critical for HNA-3a expression. Type 1 antibodies recognize mouse CTL2, but Type 2 antibodies do not.4) Chimeric CTL2 containing human sequence in EC Loop 1 and mouse sequence in Loops 2-5 (H1M) reacts only with Type 1 antibodies. The reciprocal construct with mouse sequence in EC loop 1 and human sequence in Loops 2-5 (M1H) reacts with both Type 1 and Type 2 antibodies.5) Chimeric CTL2 containing human sequence in EC Loops 1-2 and mouse sequence in Loops 3-5 (H2M) reacts only with Type 1 antibodies.6) Chimeric CTL2 containing human sequence in EC Loops 1-3 and mouse sequence in Loops 4-5 (H3M) reacts with both Type 1 and Type 2 antibodies. These findings show that Loop 1 peptides containing R154 are sufficient for Type 1 antibodies to recognize CTL2 and the Type 1 epitope survives detergent solubilization of the protein. However, Type 2 antibodies require human sequence in EC Loops 1-3 for binding and the epitope they recognize does not survive detergent treatment. Moreover both Type 1 and Type 2 recognize the M1H chimera with the entire EC loop 1 sequence derived from mouse. The simplest explanation for these observations is that Type 2 HNA-3a antibodies need to contact human amino acid residues in EC Loop 3 in addition to Loop 1 for tight binding to CTL2. An alternative possibility is both Type 1 and 2 antibodies recognize only Loop1, but EC Loops 2 and 3 are required to hold Loop 1 in a configuration suitable for Type 2 antibody binding. In either case, it appears that at least the first 3 EC loops of CTL2 (R154) need to be in a configuration that closely mimics their natural state in the cell membrane in order to be recognized by Type 2 HNA-3a antibodies. Considerable ingenuity will be required to engineer a target capable of detecting both Type 1 and Type 2 HNA-3a antibodies in a format suitable for large-scale blood donor screening. Disclosures: No relevant conflicts of interest to declare.

Differential androgen and estrogen substrates specificity in the mouse and primates type 12 17β-hydroxysteroid dehydrogenase

Recently, we have shown that human and monkey type 12 17β-hydroxysteroid dehydrogenases (17β-HSD12) are estrogen-specific enzymes catalyzing the transformation of estrone (E1) into estradiol (E2). To further characterize this novel steroidogenic enzyme in an animal model, we have isolated a cDNA fragment encoding mouse 17β-HSD12 and characterized its enzymatic activity. Using human embryonic kidney cells (HEK)-293 cells stably expressing mouse 17β-HSD12, we found that in contrast with the human and monkey enzymes, which are specific for the transformation of E1 to E2, mouse 17β-HSD12 also catalyzes the transformation of 4-androstenedione into testosterone (T), dehydroepiandroster-one (DHEA) into 5-androstene-3β,17β-diol (5-diol), as well as androsterone into 5α-androstane-3α,17β-diol (3α-diol). Previously, we have shown that the specificity of human and monkey 17β-HSD12s for C18-steroid is due to the presence of a bulky phenylalanine (F) at position 234 creating steric hindrance, preventing the entrance of C19-steroids into the active site. To determine whether the smaller size of the corresponding leucine (L) in the mouse sequence is responsible for the entrance of androgenic substrates, we performed site-directed mutagenesis to substitute Leu 234 for Phe in the mouse enzyme. In agreement with our hypothesis, the mutated enzyme has a highly reduced ability to metabolize androgens. mRNA quantification in several mouse tissues using real-time PCR shows that mouse 17β-HSD12 mRNA is highly expressed in the female clitoral gland, male preputial gland, as well as in retroperitoneal fat and adrenal of both sexes. The differential androgenic/estrogenic substrate specificity of type 12 17β-HSD in the mouse and primates seems to agree with the observation that androgen and estrogen in the mouse are provided almost exclusively by gonads, while in primates an important part of these steroid hormones are produced locally from adrenal precursors.

266 IDENTIFICATION OF Oct-4 AND Nanog, THE TWO PLURIPOTENCY MARKER GENES IN RABBIT PRE-IMPLANTATION-STAGE EMBRYOS

Several genes, including Oct-4 and Nanog, coordinate the embryogenesis of mammalian embryos. Whereas Oct-4 has an activator effect, the Nanog protein blocks the transcription of several genes in early stages; however, the product of these two genes appears parallel and directs early embryogenesis. The goal of this work was to isolate the Oct-4 and Nanog genes from rabbit, based on the sequences of other species published so far. The sequence of known genes has been analyzed, and primers have been designed based on similarity of sequences. Oocyte-to-blastocyt-stage embryos were collected from superovulated rabbits in RNase-free water. Embryonic mRNA was isolated by using the Dynal mRNA isolation KIT (Dynal, Biotech, Oslo, Norway). Real-time PCR was performed in a ABI PRISM� 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). The reaction mixture consisted SYBR� Green PCR Master Mix (Applied Biosystems), 300 nM of each primer, and an 1/8 aliquot of the embryo cDNA in a 25-�L final volume. The cDNA template was denatured by heating to 95�C for 10 min and amplified by 45 cycles of 95�C for 15 s and 60�C for 1 min, with a single fluorescence measurement at each cycle. When the reaction was finished, melting curves were plotted to confirm product purity. The reaction mix was electrophoretically separated to facilitate fragment isolation. In the case of Nanog, a 131-bp fragment was cloned. Sequence analysis revealed significant homology with the mouse sequence. Based on this finding, new primers were designed in order to isolate a larger fragment as well as the genomic copy of the gene. In the case of Oct-4, several combinations of primers were tested, because of the rather conservative sequence of the Oct-4 transcription factor family. As a result, an optimal primer pair was found that yielded a 450 bp fragment, expected according to known sequences. After direct sequencing, there was a high similarity to Oct-4 genes, indicating that the isolated cDNA is probably part of the rabbit Oct-4 cDNA. For further analysis, the cDNA fragments of both genes were isolated (MinElute Gel Extraction Kit; Qiagen, Valencia, CA, USA) and cloned into bacterial plasmids (TOP 10 Cloning KIT; Invitrogen, Carlsbad, CA, USA). At this point, it can be stated that: (1) both genes have been successfully identified in rabbit genome, (2) a method has been developed to detect expression of genes, and (3) gene-specific primers have been produced. Our further goal is to clone the whole coding region of the genes and to identify the sequence. The cloned fragments will be used for in situ hybridization in implanted stage embryo sections. This research was supported by EU-FP6-MEXT-CT-2003-59582, Wellcome Trust (Grant No. 070246), OTKA T046171, and NKTH BIO-00017/2002.

220 AN EXPRESSION PROFILE OF GENES CRUCIAL FOR PLACENTAL DEVELOPMENT IN SINGLE IN VIVO, IN VITRO AND CLONED BOVINE BLASTOCYSTS

Abnormalities of the placenta are a major factor contributing to early death in cloned bovine conceptuses. This is primarily due to incomplete chromatin remodeling and reprogramming of the donor nucleus. It is unknown whether genetic aberrations of genes crucial for placental development can be detected in pre-implantation cloned bovine embryos. This study looked at the expression profile of four genes in single bovine blastocysts derived from in vivo, in vitro produced (IVP), or cloning techniques, including handmade cloning (HMC) and serial HMC (SHMC). The genes studied included acrogranin, caudal type homeobox 2 (cdx2), estrogen-receptor-related receptor beta (essrb), and the mammalian relative of DnaJ (MRJ). These genes play a role in trophoblast regulation and placental development. Messenger RNA expression was analyzed by using PCR following cDNA amplification by means of SMART cDNA synthesis (Clontech, Palo alto, CA, USA). Primers were designed from homologous human and mouse sequence. PCR products were sequenced for verification. Five single blastocysts were analyzed from each of the following treatments: in vivo, IVP, HMC, and SHMC. Pooled (n = 10) IVP blastocyst cDNA produced by standard RT was used as a positive control. Grade 1 Day 7 blastocysts were selected for all treatments. Amplified cDNA was tested using control genes polyA, IFN-τ and GDF9. In vitro-produced embryos were matured, fertilized and cultured as published by Ruddock et al. (2004 Biol. Reprod. 70, 1131). Cloned HMC embryos were produced as described by Tecirlioglu et al. (2003 Reprod. Fertil. Dev. 15, 361). Serial HMC embryos were produced as per the HMC embryos, followed by a second round of nuclear transfer at the pronuclear stage. The pooled IVP, in vivo, and IVP blastocysts expressed all four genes of interest. In the HMC-cloned embryos, all four genes were expressed. However, in the SHMC cloned embryos, although MRJ was found to be expressed in all blastocysts, three of the five blastocysts did not express acrogranin. Similarly, two SHMC embryos did not express cdx2, and essrb was weakly expressed in three of the five embryos analyzed. Initial pregnancy rates of HMC and SHMC embryo transfers are similar. Further pregnancy results are pending. These results indicate that aberrations of genes crucial for placental development can be detected in single cloned blastocysts. It also suggests that failed implantation and/or placental defects may stem from patterned genetic defects in the pre-implantation embryo. An increase in the number of embryos analyzed would further strengthen results. These genes could act as markers to identify cloning techniques that produce more embryos with normal genetic profiles. The benefits of developing a screening tool to assess abnormalities in single pre-implantation embryos would be significant.