Molecular regulation of vasopressin receptor among (mostly) monogamous prairie voles

Intraspecific variation in social behavior is common and often dramatic, but little is known about its underlying mechanisms. We use the prairie vole (Microtus ochrogaster) to examine how intraspecific variation in brain and behavior emerges as a result of genetic, epigenetic and environmental variation. Although prairie voles are socially monogamous, they vary in sexual fidelity; faithful prairie voles are described as intra-pair fertilizing (IPF), while unfaithful voles are extra-pair fertilizing (EPF). EPF males have large home-ranges and frequently mate with other females, but do so at the cost of being cuckolded. IPF males however, form small exclusive home-ranges, rarely intrude and are better at mate-guarding. These behavioral differences are predicted by abundance of the vasopressin receptor 1a (V1aR) in the retrosplenial cortex (RSC), a brain region implicated in spatial memory. We find that variation in RSC-V1aR and associated behaviors are predicted by two alternative avpr1a alleles. These “HI” and “LO” alleles are defined by four linked single nucleotide polymorphisms, one of which is a polymorphic CpG site (polyCpG) located within a putative intron enhancer. This polyCpG is weakly linked to several other polyCpGs in the enhancer. Since CpGs are targets for DNA methylation, polyCpGs may cause individual differences in DNA methylation, gene regulation and environmental sensitivity. The unusually high number of polyCpGs within the intron enhancer drives avpr1a genotype differences in CpG density and methylation, which predict avpr1a expression and RSC-V1aR. Examination of avpr1a methylation among wild-caught voles also showed that RSC-V1aR correlated with enhancer methylation, possibly due to genotype differences in enhancer silencing or affinity for transcription factors, but not with promoter methylation. We also found that genotype differences in RSC-V1aR emerge in the first postnatal week, along with changes in enhancer methylation. Before this neurodevelopmental stage, the LO allele, which has more enhancer CpGs, is more sensitive to environmentally-induced changes in RSC-V1aR. These changes however, are not caused by alteration of enhancer methylation, suggesting additional regulatory elements contribute to genotype differences in RSC-V1aR regulation and its environmental sensitivity. Our findings show how genetic and epigenetic variation at a critical gene can shape intraspecific variation in brain and behavior.Ecology, Evolution and Behavio

An intronic SNP in the thyroid hormone receptor β gene is associated with pituitary cell-specific over-expression of a mutant thyroid hormone receptor β2 (R338W) in the index case of pituitary-selective resistance to thyroid hormone

<p>Abstract</p> <p>Background</p> <p>The syndrome of resistance to thyroid hormone (RTH) is caused by mutations in the thyroid hormone receptor β gene (<it>THRB</it>). The syndrome varies from asymptomatic to diffuse hypothyroidism, to pituitary-selective resistance with predominance of hyperthyroid signs and symptoms. The wide spectrum of clinical presentation is not completely attributable to specific <it>THRB </it>mutations. The <it>THRB </it>gene encodes two main isoforms, TR β1 which is widely distributed, and TR β2, whose expression is limited to the cochlea, retina, hypothalamus, and pituitary. Recent data demonstrated that in mice an intron enhancer region plays a critical role in the pituitary expression of the β2 isoform of the receptor. We thus hypothesized that polymorphisms in the human homologous region could modulate the pituitary expression of the mutated gene contributing to the clinical presentation of RTH.</p> <p>Methods</p> <p>Screening and <it>in vitro </it>characterization of polymorphisms of the intron enhancer region of the <it>THRB </it>gene in the index case of pituitary-selective RTH.</p> <p>Results</p> <p>The index case of pituitary-selective resistance is characterized by the missense R338W exon 9 mutation in <it>cis </it>with two common SNPs, rs2596623T and rs2596622C, located in the intron enhancer region of the <it>THRB </it>gene. Reporter gene assay experiments in GH3 pituitary-derived cells indicate that rs2596623T generates an increased pituitary cell-specific activity of the TR β2 promoter suggesting that rs2596623T leads to pituitary over-expression of the mutant allele.</p> <p>Conclusions</p> <p>The combined coding mutation and non-coding SNP therefore generate a tissue-specific dominant-negative condition recapitulating the patient's peculiar phenotype. This case illustrates the role of regulatory regions in modifying the clinical presentation of genetic diseases.</p

Somatic hypermutation of immunoglobulin ĸ light chain genes

Somatic hypermutation of immunoglobulin genes is a tightly controlled, site directed process. The nucleotide changes are targeted in the region surrounding the rearranged V gene segment of the heavy and light chain loci. The targeting of somatic mutation does neither require V region including the Vĸ promoter specific features nor a specific distance between the C ĸ and the 3' enhancer. On the other hand, removal of either the ĸ 3' enhancer or the ĸ intron enhancer/M AR causes a drastic reduction in hypermutation, which is particularly marked in the latter case. In the work described in this dissertation I have used transgenic mice carrying immunoglobulin ĸ transgenes with deleted regulatory elements, to make a tighter delineation of which DNA regions within the ĸ locus are important for hypermutation. In addition I have used transgenic mice to study the role of the primary sequence and the DNA secondary structure in this process. This thesis describes the generation of artificial Ig genes constructs to make transgenic lines. ĸ transgenes were characterized by Southern blot and by ELISA in the seven mouse lines obtained. The role of the 3' enhancer core in the transcription of the ĸ chain was also studied in stable transfected S I07 mouse myelom a cells. The analysis of the transgenes carrying deletions of regulatory elements revealed that hypermutation was impaired (but not severely affected) when the intron enhancer was deleted. This also was the case when the flanking regions of the 3' enhancer core were deleted. Hypermutation was severely affected (but not abolished) when the core of the 3' enhancer was removed. This reflects the relative roles of both components of the 3' enhancer region in gene expression. The matrix attachment region seems to be essential, since its deletion abolished hypermutation, but not expression, since abundant ĸ mRNA was still produced. Furthermore these studies showed that the 3' enhancer can drive hypermutation from a position upstream of the Vĸ region.The major role of the primary sequence in the formation of hotspots and the mutational machinery preferences for AGY encoded Ser and the bias against TCA encoded Ser as target for hypermutation were disclosed with the analysis of the transgenes carrying VĸOx1 gene with modifications in the CDR1 sequence. However, whilst hairpin loops do not define which base is going to be targeted, they seem to have a role in the accessibility of the 'favoured' nucleotides for hypermutation

Bypass of lethality with mosaic mice generated by Cre–loxP-mediated recombination

AbstractBackground The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre–loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments.Results We generated nestin–cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the γ-chain of the interleukin-2 receptor (IL-2Rγ); in these animals, the IL-2Rγ-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase β die perinatally, we studied the effects of DNA polymerase β deficiency in mosaic animals. We found that some of the mosaic polymerase β-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase β-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage.Conclusions The nestin–cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase β, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types

Complete analysis of the B-cell response to a protein antigen, from in vivo germinal centre formation to 3-D modelling of affinity maturation

Somatic hypermutation of immunoglobulin variable region genes occurs within germinal centres (GCs) and is the process responsible for affinity maturation of antibodies during an immune response. Previous studies have focused almost exclusively on the immune response to haptens, which may be unrepresentative of epitopes on protein antigens. In this study, we have exploited a model system that uses transgenic B and CD4&lt;sup&gt;+&lt;/sup&gt; T cells specific for hen egg lysozyme (HEL) and a chicken ovalbumin peptide, respectively, to investigate a tightly synchronized immune response to protein antigens of widely differing affinities, thus allowing us to track many facets of the development of an antibody response at the antigen-specific B cell level in an integrated system &lt;i&gt;in&lt;/i&gt; &lt;i&gt;vivo&lt;/i&gt;. Somatic hypermutation of immunoglobulin variable genes was analysed in clones of transgenic B cells proliferating in individual GCs in response to HEL or the cross-reactive low-affinity antigen, duck egg lysozyme (DEL). Molecular modelling of the antibody–antigen interface demonstrates that recurring mutations in the antigen-binding site, selected in GCs, enhance interactions of the antibody with DEL. The effects of these mutations on affinity maturation are demonstrated by a shift of transgenic serum antibodies towards higher affinity for DEL in DEL-cOVA immunized mice. The results show that B cells with high affinity antigen receptors can revise their specificity by somatic hypermutation and antigen selection in response to a low-affinity, cross-reactive antigen. These observations shed further light on the nature of the immune response to pathogens and autoimmunity and demonstrate the utility of this novel model for studies of the mechanisms of somatic hypermutation

V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element

The immunoglobulin heavy chain intron enhancer (Eμ) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Eμ, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Eμ, the Eμ core as well as its flanking 5′ and 3′ matrix associated regions (5′ and 3′ MARs) upregulate V(D)J recombination while the downstream section of the 3′ MAR of Eμ does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3′ MAR of Eμ stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is not mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5′ and 3′ MARs of Eµ and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Eµ cor

Stringent doxycycline-dependent control of gene activities using an episomal one-vector system

Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1

E2 regulates epigenetic signature on neuroglobin enhancer-promoter in neuronal cells

Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB) is one of the estrogen target genes involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERβ) that bind to specific regulatory genomic regions. We focused our attention on 17β-estradiol (E2)-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2). Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ERα) binding suggesting that ERα may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERα binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative diseases