Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

Transmembrane region of N-methyl-D-aspartate receptor (NMDAR) subunit is required for receptor subunit assembly

N-Methyl-D-aspartate receptors (NMDARs), one of three main classes of ionotropic glutamate receptors, play major roles in synaptic plasticity, synaptogenesis, and excitotoxicity. Unlike non-NMDA receptors, NMDARs are thought to comprise obligatory heterotetrameric complexes mainly composed of GluN1 and GluN2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, most of the NMDAR subunits can neither leave the endoplasmic reticulum (ER) nor be expressed in the cell membrane because of the ER retention signals. Only when NMDARs are heteromerically assembled can the ER retention signals be masked and NMDARs be expressed in the surface membrane. However, the mechanisms underlying NMDAR assembly remain poorly understood. To identify regions in subunits that mediate this assembly, we made a series of truncated or chimeric cDNA constructs. Using FRET measurement in living cells combined with immunostaining and coimmunoprecipitation analysis, we examined the assembly-determining domains of NMDAR subunits. Our results indicate that the transmembrane region of subunits is necessary for the assembly of NMDAR subunits, both for the homodimer and the heteromer.     

Flanking V and J sequences of complementary determining region 3 of T cell receptor (TCR) δ1 (CDR3δ1) determine the structure and function of TCRγ4δ1

The γδ T cell receptor (TCR) differs from immunoglobulin and αβ TCR in its overall binding mode. In human, genes δ1, δ2, and δ3 are used for TCRδ chains. Previously, we have studied antigen binding determinants of TCRδ2 derived from dominant γδ T cells residing in peripheral blood. In this study we have investigated the critical determinants for antigen recognition and TCR function in TCRδ1 originated from gastric tumor-infiltrating γδ T lymphocytes using three independent experimental strategies including complementary determining region 3 (CDR3) of TCRδ1 (CDR3δ1)-peptide mediated binding, CDR3δ1-grafted TCR fusion protein-mediated binding, and TCRγ4δ1- and mutant-expressing cell-mediated binding. All three approaches consistently showed that the conserved flanking V and J sequences but not the diverse D segment in CDR3δ1 determine the antigen binding. Most importantly, we found that mutations in the V and J regions of CDR3δ1 also abolish the assembly of TCR and TCR-CD3 complexes in TCRγ4δ1-transduced J.RT3-T3.5 cells. Together with our previous studies on CDR3δ2 binding, our finding suggests that both human TCRδ1 and TCRδ2 recognize antigen predominately via flanking V and J regions. These results indicate that TCRγδ recognizes antigens using conserved parts in their CDR3, which provides an explanation for a diverse repertoire of γδTCRs only recognizing a limited number of antigens.     

The functional VNTR of IGH enhancer HS1.2 associates with human longevity and interacts with TNFA promoter diplotype in a population of Central Italy

The dysregulation of both immune and inflammatory responses occurring with aging is believed to substantially contribute to morbidity and mortality in humans. We have already reported the association of the functional Variable Number of Tandem Repeat (VNTR) at the Immunoglobulin heavy chain (IGH) enhancer HS1.2 with Immunoglobulin levels and with several autoimmune diseases. Herein we tested the association of the VNTR at the HS1.2 enhancer with human longevity, also evaluating the possible modulatory effect of TNFA promoter diplotype (rs361525/rs1800629). HS1.2 enhancer genotypes have been determined for 193 unrelated healthy individuals from Central Italy divided into two groups: Group 1 (18–84 yrs, mean age 56.8 ± 19.4) and Group 2 (85–100 yrs, mean age 93.0 ± 3.5). Homozygous subjects for *2 allele were significantly disadvantaged in reaching higher life-expectancy (OR = 0.457, p = 0.021). A significant interaction between TNFA promoter diplotype status, HS1.2 2/2 genotype and the two Groups was found (p = 0.014). Of note, TNFA − 308A allele seems to exert a protective effect in HS1.2 2/2 carriers. These results support the hypothesis of an important role of HS1.2 VNTR in the puzzle of the immune-system regulation, evidenced also by the potential interaction with TNFA. Moreover, the previous results showing the association of HS1.2 *2 allele with inflammatory phenomena are consistent with the hypothesis that this allele is a detrimental factor in reaching advanced age.     

北京市方庄地区儿童全血微量营养素含量检测结果分析

目的:通过分析北京市方庄地区儿童全血微量营养素的含量及影响因素,研究探讨微量营养素缺乏的防治措施。方法:2011年1月至12月北京中医药大学东方医院儿科门诊1887例健康体检儿童,收集末梢血,采用原子吸收光谱仪检测全血5种微量营养素铜、锌、钙、镁、铁的含量,并从性别、年龄、季节(月份)方面进行比较分析。结果:全血5种微量营养素含量无性别差异(P0.05),但存在季节差异,4-6月铜、锌含量降低;7-9月钙、镁含量降低;10-12月铁含量降低(P0.05)。全血锌、铁含量存在年龄差异,婴幼儿最低(P0.05)。1887例儿童中,缺乏较多的元素依次为铁(38.8%)、钙(22.3%)、锌(12.8%),其中,4-7岁(17.0%)、7-12岁(21.5%)年龄段组以锌缺乏居多。1岁以内(51.2%)、1-3岁(44.6%)年龄段组以铁缺乏居多。全血铜、锌、钙、铁缺乏无性别差异(P0.05)。全血5种微量营养素缺乏存在季节差异,1-3月镁缺乏明显,4-6月铜、锌缺乏明显,10-12月钙、铁缺乏明显(P0.05)。结论:北京市方庄地区儿童主要存在锌、钙、铁的缺乏,以秋冬季及初春明显。其中婴幼儿以铁缺乏为主,学龄前期及学龄期儿童以锌缺乏为主。应加强对此地区儿童的营养宣教和饮食指导。     

Comparative genomics of methicillin-resistant Staphylococcus aureus ST239: distinct geographical variants in Beijing and Hong Kong

Background

The ST239 lineage is a globally disseminated, multiply drug-resistant hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA). We performed whole-genome sequencing of representative HA-MRSA isolates of the ST239 lineage from bacteremic patients in hospitals in Hong Kong (HK) and Beijing (BJ) and compared them with three published complete genomes of ST239, namely T0131, TW20 and JKD6008. Orthologous gene group (OGG) analyses of the Hong Kong and Beijing cluster strains were also undertaken.

Results

Homology analysis, based on highest-percentage nucleotide identity, indicated that HK isolates were closely related to TW20, whereas BJ isolates were more closely related to T0131 from Tianjin. Phylogenetic analysis, incorporating a total of 30 isolates from different continents, revealed that strains from HK clustered with TW20 into the ‘Asian clade’, whereas BJ isolates and T0131 clustered closely with strains of the ‘Turkish clade’ from Eastern Europe. HK isolates contained the typical φSPβ-like prophage with the SasX gene similar to TW20. In contrast, BJ isolates contained a unique 15 kb PT1028-like prophage but lacked φSPβ-like and φSA1 prophages. Besides distinct mobile genetic elements (MGE) in the two clusters, OGG analyses and whole-genome alignment of these clusters highlighted differences in genes located in the core genome, including the identification of single nucleotide deletions in several genes, resulting in frameshift mutations and the subsequent predicted truncation of encoded proteins involved in metabolism and antimicrobial resistance.

Conclusions

Comparative genomics, based on de novo assembly and deep sequencing of HK and BJ strains, revealed different origins of the ST239 lineage in northern and southern China and identified differences between the two clades at single nucleotide polymorphism (SNP), core gene and MGE levels. The results suggest that ST239 strains isolated in Hong Kong since the 1990s belong to the Asian clade, present mainly in southern Asia, whereas those that emerged in northern China were of a distinct origin, reflecting the complexity of dissemination and the dynamic evolution of this ST239 lineage.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-529) contains supplementary material, which is available to authorized users.     

Transmission of Stability Information through the N-domain of Tropomyosin Is Interrupted by a Stabilizing Mutation (A109L) in the Hydrophobic Core of the Stability Control Region (Residues 97–118)

Tropomyosin (Tm) is an actin-binding, thin filament, two-stranded α-helical coiled-coil critical for muscle contraction and cytoskeletal function. We made the first identification of a stability control region (SCR), residues 97–118, in the Tm sequence that controls overall protein stability but is not required for folding. We also showed that the individual α-helical strands of the coiled-coil are stabilized by Leu-110, whereas the hydrophobic core is destabilized in the SCR by Ala residues at three consecutive d positions. Our hypothesis is that the stabilization of the individual α-helices provides an optimum stability and allows functionally beneficial dynamic motion between the α-helices that is critical for the transmission of stabilizing information along the coiled-coil from the SCR. We prepared three recombinant (rat) Tm(1–131) proteins, including the wild type sequence, a destabilizing mutation L110A, and a stabilizing mutation A109L. These proteins were evaluated by circular dichroism (CD) and differential scanning calorimetry. The single mutation L110A destabilizes the entire Tm(1–131) molecule, showing that the effect of this mutation is transmitted 165 Å along the coiled-coil in the N-terminal direction. The single mutation A109L prevents the SCR from transmitting stabilizing information and separates the coiled-coil into two domains, one that is ∼9 °C more stable than wild type and one that is ∼16 °C less stable. We know of no other example of the substitution of a stabilizing Leu residue in a coiled-coil hydrophobic core position d that causes this dramatic effect. We demonstrate the importance of the SCR in controlling and transmitting the stability signal along this rodlike molecule.