How DNA loop extrusion mediated by cohesin enables V(D)J recombination

‘Structural maintenance of chromosomes’ (SMC) complexes are required for the folding of genomic DNA into loops. Theoretical considerations and single-molecule experiments performed with the SMC complexes cohesin and condensin indicate that DNA folding occurs via loop extrusion. Recent work indicates that this process is essential for the assembly of antigen receptor genes by V(D)J recombination in developing B and T cells of the vertebrate immune system. Here, I review how recent studies of the mouse immunoglobulin heavy chain locus Igh have provided evidence for this hypothesis and how the formation of chromatin loops by cohesin and regulation of this process by CTCF and Wapl might ensure that all variable gene segments in this locus (V_H segments) participate in recombination with a re-arranged DJ_H segment, to ensure generation of a maximally diverse repertoire of B-cell receptors and antibodies.     

Genetic Analysis of the Dumpy Complex Locus in DROSOPHILA MELANOGASTER: Complementation, Fine Structure and Function

An extensive genetic analysis of the dumpy locus is presented. This study includes complementation, fine structure mapping and allelic interaction. A number of complementing recessive lethals of the dp complex have been genetically mapped. Two alleles of the ol(v) type that complement l alleles map to the left portion of the locus. A number of olv alleles that complement both l and lv lethals map within the right portion of the locus.——Fine-structure analysis demonstrated that both olv and o alleles are distributed among various subloci. Evidence for spacer regions between subloci is presented.——An extensive discussion of the data considers whether the locus is unicistronic or multicistronic. The conclusion reached is that the locus is not a single functional cistron. The possibility of a single cistron encoding a multifunctional polypeptide is discussed.——The hypothesis is proposed that the left portion of the map and the l mutations function as regulatory sequences and that the right portion of the map encodes structural sequences.     

Differential reactivity of germ line allelic variants of a broadly neutralizing HIV-1 antibody to a gp41 fusion intermediate conformation

Genetic factors, as well as antigenic stimuli, can influence antibody repertoire formation. Moreover, the affinity of antigen for unmutated naïve B cell receptors determines the threshold for activation of germinal center antibody responses. The gp41 2F5 broadly neutralizing antibody (bNAb) uses the V_H2-5 gene, which has 10 distinct alleles that use either a heavy-chain complementarity-determining region 2 (HCDR2) aspartic acid (D_H54) or an HCDR2 asparagine (N_H54) residue. The 2F5 HCDR2 D_H54 residue has been shown to form a salt bridge with gp41 ⁶⁶⁵K; the V_H2-5 germ line allele variant containing N_H54 cannot do so and thus should bind less avidly to gp41. Thus, the induction of 2F5 bNAb is dependent on both genetic and structural factors that could affect antigen affinity of unmutated naïve B cell receptors. Here, we studied allelic variants of the V_H2-5 inferred germ line forms of the HIV-1 gp41 bNAb 2F5 for their antigen binding affinities to gp41 linear peptide and conformational protein antigens. Both V_H2-5 2F5 inferred germ line variants bound to gp41 peptides and protein, including the fusion intermediate protein mimic, although more weakly than the mature 2F5 antibody. As predicted, the affinity of the N_H54 variant for fusion-intermediate conformation was an order of magnitude lower than that of the D_H54 V_H2-5 germ line antibody, demonstrating that allelic variants of 2F5 germ line antibodies differentially bind to gp41. Thus, these data demonstrate a genetically determined trait that may affect host responses to HIV-1 envelope epitopes recognized by broadly neutralizing antibodies and has implications for unmutated ancestor-based immunogen design.     

Discriminating between allelic and interlocus differences among human immunoglobulin VH4 sequences by analyzing single spermatozoa

To address the challenging issue of distinguishing allelic and interlocus differences among repetitive sequences, human immunoglobulin V_H4 loci in the parental haplotypes of 13 donors were determined by analyzing single spermatozoa. V_H4 sequences detected among these donors were assigned to their corresponding loci based on the fact that allelic sequences usually segregate into different gametes. Four out of the ten V_H4 loci were shown to contain null alleles that are undetectable with diploid materials. The distribution of the allelic variation within the analyzed regions at the V_H4 loci is highly biased. Received: 17 January 1997 / Accepted: 10 March 1997     

A fine balance: epigenetic control of cellular quiescence by the tumor suppressor PRDM2/RIZ at a bivalent domain in the cyclin a gene

Adult stem cell quiescence is critical to ensure regeneration while minimizing tumorigenesis. Epigenetic regulation contributes to cell cycle control and differentiation, but few regulators of the chromatin state in quiescent cells are known. Here we report that the tumor suppressor PRDM2/RIZ, an H3K9 methyltransferase, is enriched in quiescent muscle stem cells in vivo and controls reversible quiescence in cultured myoblasts. We find that PRDM2 associates with >4400 promoters in G₀ myoblasts, 55% of which are also marked with H3K9me2 and enriched for myogenic, cell cycle and developmental regulators. Knockdown of PRDM2 alters histone methylation at key promoters such as Myogenin and CyclinA2 (CCNA2), and subverts the quiescence program via global de-repression of myogenesis, and hyper-repression of the cell cycle. Further, PRDM2 acts upstream of the repressive PRC2 complex in G₀. We identify a novel G₀-specific bivalent chromatin domain in the CCNA2 locus. PRDM2 protein interacts with the PRC2 protein EZH2 and regulates its association with the bivalent domain in the CCNA2 gene. Our results suggest that induction of PRDM2 in G₀ ensures that two antagonistic programs—myogenesis and the cell cycle—while stalled, are poised for reactivation. Together, these results indicate that epigenetic regulation by PRDM2 preserves key functions of the quiescent state, with implications for stem cell self-renewal.     

Genetic passportization and identification of Siberian cranes (Grus leucogeranus Pallas) in captivity

The genetic diversity of the founders of an artificial population of the Siberian crane Grus leucogeranus Pallas (rare species of cranes) was characterized using 10 microsatellite loci. It was established that the allelic diversity (on average, 5.9 alleles per locus) and genic (H _O = 0.739) diversity of the Siberian crane is rather high and comparable with the estimations for natural populations of different crane species. Genetic passportization of the birds (119 individuals) from the register of the Siberian crane International Studbook was carried out at the initial stage. The efficiency of genetic passportization for individual identification, identification of the origin, paternity analysis, and exclusion of inbreeding was demonstrated in Siberian cranes under natural mating and artificial insemination. Cases of natural reproduction in pairs of Siberian cranes imprinted to the human and continuous storage of spermatozoa in the female reproductive ducts were registered.     

Genetic Variation at a Locus (TAM-1) for Submaxillary Gland Protease in the Mouse and Its Location on Chromosome 7

Electrophoretic and activity variants for a testosterone-induced esteroprotease have been discovered in submaxillary glands from inbred strains of mice. The enzyme is tentatively designated tamase (TAM-1) and the variant genetic locus is Tam-1. The alleles Tam-1^a and Tam-1^b determine electrophoretically distinct zones of tamase activity, while Tam-1^c produces no detectable enzyme activity. Data from recombinant inbred strains and B6AF₁ x B6 and B6D2F₁ x B6 backcrosses established linkage of Tam-1 to glucose phosphate isomerase (Gpi-1), pink-eyed dilution (p) and β-hemoglobin (Hbb) on chromosome 7. The gene order is Gpi-1—Tam-1—p—Hbb. Analysis of congenic resistant strains indicates that Tam-1 is closely linked to the minor histocompatibility locus, H-4. TAM-1 was not cross-reactive with antisera to mouse nerve growth factor, submaxillary renin, or tamases A and D.     

Bipartite structure of the inactive mouse X chromosome

BackgroundIn mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems.ResultsWe find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary region. Comparison with the recently reported two-superdomain structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the boundary region is conserved and located near the Dxz4/DXZ4 locus. In mouse, the boundary region also contains a minisatellite, Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation compared with the active alleles and with genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele.ConclusionsBy applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0728-8) contains supplementary material, which is available to authorized users.     

The Serine/Threonine Phosphatase PP4 Is Required for Pro-B Cell Development through Its Promotion of Immunoglobulin VDJ Recombination

PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific pp4 knockout mice and have identified an essential role for PP4 in B cell development. Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJ_H recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM⁺ B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of V_HDJ_H recombination.     

Cell Penetrable Humanized-VH/VHH That Inhibit RNA Dependent RNA Polymerase (NS5B) of HCV

NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V_HH) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V_HH from a humanized-camel VH/V_HH display library. VH/V_HH from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3′di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V_HH hallmark and were designated V_HH6 and V_HH24; other clones were conventional VH, designated VH9 and VH13. All VH/V_HH were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V_HH6 and V_HH24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V_HH mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.     

Amylase Variation in the Salt Marsh Amphipod, GAMMARUS PALUSTRIS

There are two common alleles at the Amylase-2 locus in populations of Gammarus palustris, the salt marsh amphipod. Intensive sampling of individuals from two localities at Jamaica Bay revealed a consistent pattern of heterozygote deficiency.—Five possible sources of heterozygote deficiency were examined in this study. Four of them—selection against heterozygotes, null alleles at the locus, assortative mating for amylase genotype and inbreeding—are inconsistent with the evidence and are rejected. The fifth possibility, Wahlund effects due to genetic differentiation of the population, is tentatively accepted. Although there is no direct evidence for differentiation within this population, separate populations along the Eastern seaboard are highly differentiated in a nonclinal pattern. Furthermore, the Wahlund hypothesis is consistent with observations on differences in degree of deficiency exhibited among collections at Jamaica Bay.—Animals from this population exhibit feeding preferences correlated with genotype. Given the choice of two green algae, Enteromorpha or Ulva, the frequency of the slow allele among individuals choosing Enteromorpha was higher than among those choosing Ulva. This suggests that the animals assort themselves in the field into subpopulations with different allelic frequencies. This assortment could contribute to the maintenance of the polymorphism and to the observed heterozygote deficiency. We hypothesize that genotype influences behavior in this system through the action of enzyme on substrate, which determines the nature of the oligosaccharide pool liberated early in amylolysis.     

In Situ IgM Production and Clonal Expansion of B-1 Cells in Peritoneal Cavity Promote Elimination of C. albicans Infection in IgH Transgenic Mice with VH Derived from a Natural Antibody

B-1 cells are innate-like cells that play important roles in host defense against infection. However, the function of B-1 cells in fungi infection remains unclear. Previously we produced IgH transgenic mice TgV_H3B4 with V_H derived from a natural antibody 3B4 that can identify C. albicans, and found that TgV_H3B4 mice were resistant to intraperitoneal (i. p.) and intravenous C. albicans infection. Most of the peritoneal cavity (PEC) B-1 cells in TgV_H3B4 mice express transgenic BCR that binds C. albicans. In the present study, we explored the response of B-1 cells to C. albicans infection by applying i. p. inoculation of fungi in TgV_H3B4 mice. We found that C. albicans was cleared more efficiently in TgV_H3B4 mice after i. p. inoculation than that of littermate control. The level of C. albicans-reactive IgM in PEC of TgV_H3B4 mice was much higher than that of control, and the number of B-1a B cells was also elevated in TgV_H3B4 mice, which was mainly due to enhanced proliferation of B-1 cells. Additionally, numbers of C. albicans-specific B cells increased greatly in TgV_H3B4 mice after C. albicans inoculation. Our data suggested that in situ IgM production and clonal expansion of B-1 cells in PEC participate in host defense against C. albicans infection.     

Effects of soaking seeds in exogenous vitamins on active oxygen metabolism and seedling growth under low-temperature stress

This study investigated the influence of the exogenous application of vitamin B2 (V_B2), B12 (V_B12), biotin (V_H), and nicotinic acid (V_PP) on oxygen production in maize (Zea mays L.) seedlings at 5 °C for day 1, 3, 5 and 7. The seeds were soaked in V_B2, V_B12, V_H, and V_PP solutions for 24 h at the concentration of 100 mg/L, and control was soaked in distilled water. A total of 50 seeds were used for each treatment in germination boxes was repeated three times. The germination box was placed in a hypothermic incubator for 1, 3, 5, and 7 days in the dark at 5 °C, then moved to a plant growth room and kept for seven days. Compared with the V_H and V_PP treatments, the V_B2 and V_B12 treatments had higher thiobarbituric acid reactive substances, proline, and soluble sugars. The V_B2 and V_B12 treatments also increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) than other treatments. The V_B2 and V_B12 treatments reduced the contents of hydrogen peroxide (H₂O₂^–), superoxide anion (O₂^–), and the damage of reactive oxygen species (ROS) to cells, increased the stability of the cell membrane and the content of cell osmoregulation substances. Moreover, V_B2 and V_B12 had higher seedling growth, germination rate, and index. Treatments V_B2 and V_B12 could promote maize seed germination and growth under low-temperature stress. Exogenous vitamins in crop production can be a valuable tool for protecting plants against low-temperature stress.