Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.     

Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin

Sex hormone-binding globulin (SHBG) binds steroids in the blood but is also present in the extravascular compartments of some tissues. Mice expressing a human SHBG transgene in the liver have human SHBG in their blood. In these animals, human SHBG accumulates within the stromal matrix of the endometrium and epididymis. This is remarkable because these tissues do not express the transgene. Human SHBG administered intravenously to wild-type mice in the presence of estradiol is rapidly sequestered within the endometrial stroma, and this prompted us to search for SHBG interacting proteins. Yeast two-hybrid screens revealed that fibulin-1D and fibulin-2 interact with the amino-terminal laminin G domain of SHBG. These interactions were verified in GST-pull down assays in which human SHBG bound the carboxyl-terminal domains of fibulin-1D and fibulin-2 in a steroid-dependent manner, with estradiol being the most effective ligand, and were enhanced by reducing the N-glycosylation of human SHBG. Like human SHBG, fibulin-1 and fibulin-2 concentrate within the endometrial stroma. In addition, SHBG co-immunoprecipitates with these fibulins in a proestrus uterine extract. These matrix-associated proteins may therefore sequester plasma SHBG within uterine stroma where it can control sex-steroid access to target cells. Given the interplay between fibulins and numerous proteins within the basal lamina, interactions between SHBG and matrix proteins may exert novel biological effects.     

A targeted proteomic approach for the identification of tumor-associated membrane antigens using the ProteomeLab PF-2D in tandem with mass spectrometry

Mapping differential expression of soluble proteins has become fairly routine using chromatofocusing in combination with the reversed-phase HPLC (ProteomeLab PF-2D by Beckman Coulter Inc.); however, identification of membrane antigens has not been reported thus far. In this report, we demonstrate a targeted proteomic approach employing immunoprecipitation, prior to 2D-LC separation, in tandem with MS/MS that can be used to identify tumor-associated membrane antigens. This system is very sensitive and reproducible in that only 1/4th the amount of starting material is required for analysis as compared to gel-based analysis, and permits a focused environment for eliminating non-specific interactions leading to an accurate resolution of the cognate antigen. This system also circumvents the well-known limitations associated with gel-based approaches. This approach has been validated in the identification of ErB2/HER-2 and was subsequently used to identify CD44E as the cognate antigen for VB1-008, one of our fully human, tumor-specific, monoclonal antibodies.     

Effects of progressive and maximal exercise on plasma levels of adhesion molecules in athletes with sickle cell trait with or without alpha-thalassemia.

The aim of the study was to examine the effects of exercise on soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) in sickle cell trait (SCT) athletes with or without alpha-thalassemia. Six athletes with SCT, seven athletes with both SCT and alpha-thalassemia (SCTAT), and seven control athletes (Cont) performed an incremental and maximal test on cycloergometer. Levels of sICAM-1 and sVCAM-1 were assessed at rest, immediately after the end of exercise, and 1, 2, and 24 h after exercise. Although Cont and SCTAT groups exhibited similar basal plasma levels of inflammatory and adhesion molecules, the SCT group had higher sVCAM-1 basal concentrations. Incremental exercise resulted in a significant increase of sVCAM-1 in all subjects, which remained elevated only in the SCT group during the recovery period. In conclusion, as sVCAM-1 increased with exercise and during the recovery period, our findings support the concept that SCT athletes might be at risk for microcirculatory disturbances and adhesive phenomena developing at rest and several hours after exercise. alpha-Thalassemia might be considered protective among exercising SCT subjects.     

La surexpression de l'androgen-binding protein (ABP) provoque des modifications morphologiques et fonctionnelles dans le testicule de souris

The Sertoti cells (SC) of many species produce an androgen-binding protein (ABP) which is secreted into both the blood and lumen of the seminiferous tubule. In the latter, it is transported to the epididymis where is taken up by epithelial cells, and is thought to play a role in sperm maturation. In view of the importance of ABP, we thought it would be pertinent to make several transgenic mice (TM) lines bearing the rABP gene to unravel its role in male reproductive physiology. A 5.5 Kb rat genomic DNA clone was microinjected into the pronucleus of fertilized mouse ova which were subsequently implanted into the oviduct of pseudopregnant CD-1 female mice. Detection of TM was performed by Southern Blot and PCR analysis using respectively, a 32p labeled rABP cDNA probe and oligonucleotides recognizing exons 1 and 7 of rABP gene. Chromosomic localization of the transgene was carried out by fluorescent in situ hybridization (FISH) in metaphasic cells obtained from bone marrow of TM. rABP expression was analyzed by Northern blot and RT-PCR techniques in most tissues of heterozigote TM. In the testis, specific cell expression was determined by in situ hybridization (ISH) and protein localization by immunohistochemistry. ABP-binding activity was performed by the carbon dextran method and analysis of protein internalization by autohistoradiographic detection of a (3H) testosterone-ABP complex. DNA fragmentation was investigated by the TUNEL technique and by electrophoresis of total genomic DNA. Testicular and epididymal morphology was studied by light and electron microscopy. Two offspring carrying the rABP gene were identified by Southern blotting, and two lines of mice (designated ABP7 and ABP24) were generated by selective breeding of the male founders with normal B6D2F1 females. FISH analysis demonstrated a different chromosomal localization of the transgene in both lines. Both rABP transgenic pedigrees presented reduced fertility. Northern blot and RT-PCR studies showed overexpression of rABP mRNA in the testis. ovary and uterus in ABP24 and ABP7 transgenic lines. rABP mRNA was appropriately expressed in SC as demonstrated by ISH. DHT-sH binding of testicular homogenates was increased 10 fold in TM compared to controls. In adult testis of TM, some seminiferous tubules showed disorganization of the epithelium, increased number of SC, presence of vacuoles, germ cell meiotic arrest and germ cell degeneration. DNA fragmentation was demonstrated in germ cells during meiosis. rABP protein was localized in the intersticial space, and into some tubules, in SC and germ cells at different steps of maturation. rABP internalization was strongly increased in both germ and epididymal cells in TM. The present results reinforce the increasing evidence of the role of ABP during spermatogenesis, even though further experiments are required to unravel its definitive implication in testicular and epidididymal homeostasis.