The Role of AtMUS81 in Interference-Insensitive Crossovers in A. thaliana

MUS81 is conserved among plants, animals, and fungi and is known to be involved in mitotic DNA damage repair and meiotic recombination. Here we present a functional characterization of the Arabidopsis thaliana homolog AtMUS81, which has a role in both mitotic and meiotic cells. The AtMUS81 transcript is produced in all tissues, but is elevated greater than 9-fold in the anthers and its levels are increased in response to gamma radiation and methyl methanesulfonate treatment. An Atmus81 transfer-DNA insertion mutant shows increased sensitivity to a wide range of DNA-damaging agents, confirming its role in mitotically proliferating cells. To examine its role in meiosis, we employed a pollen tetrad–based visual assay. Data from genetic intervals on Chromosomes 1 and 3 show that Atmus81 mutants have a moderate decrease in meiotic recombination. Importantly, measurements of recombination in a pair of adjacent intervals on Chromosome 5 demonstrate that the remaining crossovers in Atmus81 are interference sensitive, and that interference levels in the Atmus81 mutant are significantly greater than those in wild type. These data are consistent with the hypothesis that AtMUS81 is involved in a secondary subset of meiotic crossovers that are interference insensitive

Altered mGluR5-Homer scaffolds and corticostriatal connectivity in a Shank3 complete knockout model of autism

Human neuroimaging studies suggest that aberrant neural connectivity underlies behavioural deficits in autism spectrum disorders (ASDs), but the molecular and neural circuit mechanisms underlying ASDs remain elusive. Here, we describe a complete knockout mouse model of the autism-associated Shank3 gene, with a deletion of exons 4–22 (Δe4–22). Both mGluR5-Homer scaffolds and mGluR5-mediated signalling are selectively altered in striatal neurons. These changes are associated with perturbed function at striatal synapses, abnormal brain morphology, aberrant structural connectivity and ASD-like behaviour. In vivo recording reveals that the cortico-striatal-thalamic circuit is tonically hyperactive in mutants, but becomes hypoactive during social behaviour. Manipulation of mGluR5 activity attenuates excessive grooming and instrumental learning differentially, and rescues impaired striatal synaptic plasticity in Δe4–22−/− mice. These findings show that deficiency of Shank3 can impair mGluR5-Homer scaffolding, resulting in cortico-striatal circuit abnormalities that underlie deficits in learning and ASD-like behaviours. These data suggest causal links between genetic, molecular, and circuit mechanisms underlying the pathophysiology of ASDs

Engineering HIV-Resistant Human CD4+ T Cells with CXCR4-Specific Zinc-Finger Nucleases

HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5) virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4) in place of or in addition to CCR5 (R5X4) remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals

Therapeutic approaches for shankopathies

Despite recent advances in understanding the molecular mechanisms of autism spectrum disorders (ASD), the current treatments for these disorders are mostly focused on behavioral and educational approaches. The considerable clinical and molecular heterogeneity of ASD present a significant challenge to the development of an effective treatment targeting underlying molecular defects. Deficiency of SHANK family genes causing ASD represent an exciting opportunity for developing molecular therapies because of strong genetic evidence for SHANK as causative genes in ASD and the availability of a panel of Shank mutant mouse models. In this article, we review the literature suggesting the potential for developing therapies based on molecular characteristics and discuss several exciting themes that are emerging from studying Shank mutant mice at the molecular level and in terms of synaptic function

Pollen tetrad-based visual assay for meiotic recombination in Arabidopsis

Recombination, in the form of cross-overs (COs) and gene conversion (GC), is a highly conserved feature of meiosis from fungi to mammals. Recombination helps ensure chromosome segregation and promotes allelic diversity. Lesions in the recombination machinery are often catastrophic for meiosis, resulting in sterility. We have developed a visual assay capable of detecting Cos and GCs and measuring CO interference in Arabidopsis thaliana. This flexible assay utilizes transgene constructs encoding pollen-expressed fluorescent proteins of three different colors in the qrt1 mutant background. By observing the segregation of the fluorescent alleles in 92,489 pollen tetrads, we demonstrate (i) a correlation between developmental position and CO frequency, (ii) a temperature dependence for CO frequency, (iii) the ability to detect meiotic GC events, and (iv) the ability to rapidly assess CO interference

Expression and genetic loss of function analysis of the HAT/DESC cluster proteases TMPRSS11A and HAT.

Genome mining at the turn of the millennium uncovered a new family of type II transmembrane serine proteases (TTSPs) that comprises 17 members in humans and 19 in mice. TTSPs phylogenetically belong to one of four subfamilies: matriptase, hepsin/TMPRSS, corin and HAT/DESC. Whereas a wealth of information now has been gathered as to the physiological functions of members of the hepsin/TMPRSS, matriptase, and corin subfamilies of TTSPs, comparatively little is known about the functions of the HAT/DESC subfamily of proteases. Here we perform a combined expression and functional analysis of this TTSP subfamily. We show that the five human and seven murine HAT/DESC proteases are coordinately expressed, suggesting a level of functional redundancy. We also perform a comprehensive phenotypic analysis of mice deficient in two of the most widely expressed HAT/DESC proteases, TMPRSS11A and HAT, and show that the two proteases are dispensable for development, health, and long-term survival in the absence of external challenges or additional genetic deficits. Our comprehensive expression analysis and generation of TMPRSS11A- and HAT-deficient mutant mouse strains provide a valuable resource for the scientific community for further exploration of the HAT/DESC subfamily proteases in physiological and pathological processes

Development, growth, and survival of TMPRSS11A- and HAT-deficient mice.

<p>A and B. Genotype distribution of weaning-age offspring of interbred <i>Tmprss11a^+/−</i> (A) and <i>Tmprss11d^+/−</i> (B) mice. C–F. Post-weaning weight gain of cohorts of littermate <i>Tmprss11a^+/+</i> (golden triangles, N = 16), <i>Tmprss11a^+/−</i> (black squares, N = 15), and <i>Tmprss11a^−/−</i> (red triangles, N = 15) females (C), <i>Tmprss11a^+/+</i> (golden triangles, N = 15), <i>Tmprss11a^+/−</i> (black squares, N = 15), and <i>Tmprss11a^−/−</i> (red triangles, N = 15) males (E), <i>Tmprss11d^+/+</i> (golden triangles, N = 12), <i>Tmprss11d^+/−</i> (black squares, N = 15), and <i>Tmprss11d^−/−</i> (red triangles, N = 6) females (D), <i>Tmprss11d^+/+</i> (golden triangles, N = 15), <i>Tmprss11d^+/−</i> (black squares, N = 15), and <i>Tmprss11d^−/−</i> (red triangles, N = 7) males (F). G and H. Survival of prospective cohorts of littermate <i>Tmprss11a^+/+</i> (golden lines, N = 30), <i>Tmprss11a^+/−</i> (black lines, N = 30), and <i>Tmprss11a^−/−</i> (red lines, N = 30) mice (G) and <i>Tmprss11d^+/+</i> (golden lines, N = 27), <i>Tmprss11a^+/−</i> (black lines, N = 30), and <i>Tmprss11a^−/−</i> (red lines, N = 13) (H) mice that were followed for at least 500 days.</p

Generation of TMPRSS11A-deficient mice.

<p>A. Schematic structure of the gene targeting replacement vector (top), wildtype <i>Tmprss11a</i> gene (middle), and targeted <i>Tmprss11a</i> gene (bottom). Position of Eco RI restriction enzyme cleavage sites used for digestion of genomic DNA for Southern blot analysis, position of Southern blot probe (bar), and positions of primers used for analysis of wildtype and mutant <i>Tmprss11a</i> alleles (arrows) and transcripts (arrowheads) are indicated. B. Southern blot hybridization of Eco-RI digested DNA from a control (lane 1) and a targeted (lane 2) embryonic stem cell clone. The positions of wildtype (14.8 kb) and mutant <i>Tmprss11a</i> (6.5 kb) alleles are indicated on the right. Positions of molecular weight markers (kb) are indicated at left. C. PCR analysis of tail biopsy DNA of offspring from interbred <i>Tmprss11a^+/−</i> mice. The position of wildtype (496 bp) and mutant <i>Tmprss11a</i> (266 bp) alleles are indicated. D. RT-PCR analysis of <i>Tmprss11a</i> mRNA transcripts from tongue of <i>Tmprss11a^+/+</i> (lane 1) and <i>Tmprss11a^−/−</i> (lane 2) mice using the exon 7-flanking primer pair indicated with arrowheads in A. Lanes 3 and 4, no reverse transcriptase and no RNA added to the reactions, respectively. Bottom panel. Amplification of <i>Gapdh</i> mRNA demonstrating the integrity of the cDNA preparation. E. RT-PCR amplication of <i>Tmprss11a</i> mRNA transcripts from tongue of <i>Tmprss11a^+/+</i> (lanes 1, 6, 11, and 16) and <i>Tmprss11a^−/−</i> (lane 2, 7, 12, and 17) mice using primer pairs capable of amplifying exons 2–9 (lanes 1–5), 2–5 (lanes 6–10), 8–9 (lanes 11–15), and <i>Gapdh</i> (lanes 16–20). Reverse transcriptase was omitted from the reactions in lanes 3, 4, 8, 9, 13, 14, 18, and 19. No RNA was added to reactions in lanes 5, 10, 15 and 20. Transcript size is indicated left.</p

Pathological findings in tissues from aging TMPRSS11A-deficient mice.

<p>Pathological findings in tissues from aging TMPRSS11A-deficient mice.</p