DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSα/hMutSβ ratio and reduces the efficiency of base–base mismatch repair

The level and fate of hMSH3 (human MutS homolog 3) were examined in the promyelocytic leukemia cell line HL-60 and its methotrexate-resistant derivative HL-60R, which is drug resistant by virtue of an amplification event that spans the dihydrofolate reductase (DHFR) and MSH3 genes. Nuclear extracts from HL-60 and HL-60R cells were subjected to an identical, rapid purification protocol that efficiently captures heterodimeric hMutSα (hMSH2hMSH6) and hMutSβ (hMSH2hMSH3). In HL-60 extracts the hMutSα to hMutSβ ratio is roughly 6:1, whereas in methotrexate-resistant HL-60R cells the ratio is less than 1:100, due to overproduction of hMSH3 and heterodimer formation of this protein with virtually all the nuclear hMSH2. This shift is associated with marked reduction in the efficiency of base–base mismatch and hypermutability at the hypoxanthine phosphoribosyltransferase (HPRT) locus. Purified hMutSα and hMutSβ display partial overlap in mismatch repair specificity: both participate in repair of a dinucleotide insertion–deletion heterology, but only hMutSα restores base–base mismatch repair to extracts of HL-60R cells or hMSH2-deficient LoVo colorectal tumor cells.     

IκBζ is essential for natural killer cell activation in response to IL-12 and IL-18

IκBζ, encoded by Nfibiz, is a nuclear IκB-like protein harboring ankyrin repeats. IκBζ has been shown to regulate IL-6 production in macrophages and Th17 development in T cells. However, the role of IκBζ in natural killer (NK) cells has not be understood. In the present study, we found that the expression of IκBζ was rapidly induced in response to IL-18 in NK cells, but not in T cells. Analysis of Nfkbiz^−/− mice revealed that IκBζ was essential for the production of IFN-γ production and cytotoxic activity in NK cells in response to IL-12 and/or IL-18 stimulation. IL-12/IL-18–mediated gene induction was profoundly impaired in Nfkbiz^−/− NK cells. Whereas the phosphorylation of STAT4 was normally induced by IL-12 stimulation, STAT4 was not recruited to the Ifng gene regions in Nfkbiz^−/− NK cells. Acetylation of histone 3 K9 on Ifng regions was also abrogated in Nfkbiz^−/− NK cells. IκBζ was recruited on the proximal promoter region of the Ifng gene, and overexpression of IκBζ together with IL-12 activated the Ifng promoter. Furthermore, Nfkbiz^−/− mice were highly susceptible to mouse MCMV infection. Taken together, these results demonstrate that IκBζ is essential for the activation of NK cells and antiviral host defense responses.     

Expression in yeast of binding regions of karyopherins α and β inhibits nuclear import and cell growth

Using truncated forms of recombinant yeast karyopherins α and β in in vitro binding assays, we mapped the regions of karyopherin α that bind to karyopherin β and the regions of karyopherin β that interact with karyopherin α and with Ran-GTP. Karyopherin α’s binding region for karyopherin β was localized to its N-terminal domain, which contains several clusters of basic residues, whereas karyopherin β’s binding region for karyopherin α was localized to an internal region containing two clusters of acidic residues. Karyopherin β’s binding region for Ran-GTP overlaps with that for karyopherin α and comprises at least one of the two acidic clusters required for karyopherin α binding in addition to further downstream determinants not required for karyopherin α binding. Overexpression in yeast of fragments containing either karyopherin β’s binding region for α and Ran-GTP or karyopherin α’s binding region for β resulted in sequestration of most of the cytosolic karyopherin α or karyopherin β, respectively, in complexes containing the truncated proteins. As these binding region-containing fragments lack other domains required for function of the corresponding protein, the overexpression of either fragment also inhibited in vivo nuclear import of a model reporter protein as well as cell growth.     

Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLα endonuclease activation

MutLα endonuclease can be activated on covalently continuous DNA that contains a MutSα- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)_n/(CTG)_n triplet repeat sequences have implicated both MutSβ and MutLα and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)_n or (CTG)_n element is sufficient to trigger MutLα activation. (CAG)_n and (CTG)_n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLα endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLα activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLα-endonuclease-dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-Pereira M, Fortune MT, Ingram L, McAbney JP, Monckton DG (2004) Hum Mol Genet 13(16):1815–1825]. They may also have implications for triplet repeat processing at a replication fork.     

Genome-wide model for the normal eukaryotic DNA replication fork

To investigate DNA replication enzymology across the nuclear genome of budding yeast, deep sequencing was used to establish the pattern of uncorrected replication errors generated by an asymmetric mutator variant of DNA polymerase δ (Pol δ). Sequencing of 16 genomes identified 1,206-bp substitutions generated over 33 generations by L612M Pol δ in a mismatch repair defective strain. Alignment of sequences flanking these substitutions identified “hotspot” motifs for Pol δ replication errors. The substitutions were distributed evenly across all 16 chromosomes. The vast majority were transitions that occurred with a strand bias that varied in a predictable manner relative to known functional origins of replication. This strand bias strongly supports the idea that Pol δ is primarily a lagging strand polymerase during replication across the entire nuclear genome.     

Interaction of voltage-gated sodium channels with the extracellular matrix molecules tenascin-C and tenascin-R

The type IIA rat brain sodium channel is composed of three subunits: a large pore-forming α subunit and two smaller auxiliary subunits, β1 and β2. The β subunits are single membrane-spanning glycoproteins with one Ig-like motif in their extracellular domains. The Ig motif of the β2 subunit has close structural similarity to one of the six Ig motifs in the extracellular domain of the cell adhesion molecule contactin (also called F3 or F11), which binds to the extracellular matrix molecules tenascin-C and tenascin-R. We investigated the binding of the purified sodium channel and the extracellular domain of the β2 subunit to tenascin-C and tenascin-R in vitro. Incubation of purified sodium channels on microtiter plates coated with tenascin-C revealed saturable and specific binding with an apparent K_d of ≈15 nM. Glutathione S-transferase-tagged fusion proteins containing various segments of tenascin-C and tenascin-R were purified, digested with thrombin to remove the epitope tag, immobilized on microtiter dishes, and tested for their ability to bind purified sodium channel or the epitope-tagged extracellular domain of β2 subunits. Both purified sodium channels and the extracellular domain of the β2 subunit bound specifically to fibronectin type III repeats 1–2, A, B, and 6–8 of tenascin-C and fibronectin type III repeats 1–2 and 6–8 of tenascin-R but not to the epidermal growth factor-like domain or the fibrinogen-like domain of these molecules. The binding of neuronal sodium channels to extracellular matrix molecules such as tenascin-C and tenascin-R may play a crucial role in localizing sodium channels in high density at axon initial segments and nodes of Ranvier or in regulating the activity of immobilized sodium channels in these locations.     

Temperature dependence of protein motions in a thermophilic dihydrofolate reductase and its relationship to catalytic efficiency

We report hydrogen deuterium exchange by mass spectrometry (HDX-MS) as a function of temperature in a thermophilic dihydrofolate reductase from Bacillus stearothermophilus (Bs-DHFR). Protein stability, probed with circular dichroism, established an accessible temperature range of 10 °C to 55 °C for the interrogation of HDX-MS. Although both the rate and extent of HDX are sensitive to temperature, the majority of peptides showed rapid kinetics of exchange, allowing us to focus on plateau values for the maximal extent of exchange at each temperature. Arrhenius plots of the ratio of hydrogens exchanged at 5 h normalized to the number of exchangeable hydrogens vs. 1/T provides an estimate for the apparent enthalpic change of local unfolding, ΔH°_unf(avg). Most regions in the enzyme show ΔH°_unf(avg) ≤ 2.0 kcal/mol, close to the value of kT; by contrast, significantly elevated values for ΔH°_unf(avg) are observed in regions within the core of protein that contain the cofactor and substrate-binding sites. Our technique introduces a new strategy for probing the temperature dependence of local protein unfolding within native proteins. These findings are discussed in the context of the demonstrated role for nuclear tunneling in hydride transfer from NADPH to dihydrofolate, and relate the observed enthalpic changes to two classes of motion, preorganization and reorganization, that have been proposed to control the efficiency of hydrogenic wave function overlap. Our findings suggest that the enthalpic contribution to the heavy atom environmental reorganizations controlling the hydrogenic wave function overlap will be dominated by regions of the protein proximal to the bound cofactor and substrate.     

The matrix attachment region in the Chinese hamster dihydrofolate reductase origin of replication may be required for local chromatid separation

Centered in the Chinese hamster dihydrofolate reductase origin of replication is a prominent nuclear matrix attachment region (MAR). Indirect lines of evidence suggested that this MAR might be required for origin activation in early S phase. To test this possibility, we have deleted the MAR from a Chinese hamster ovary variant harboring a single copy of the dihydrofolate reductase locus. However, 2D gel replicon mapping shows that removal of the MAR has no significant effect either on the frequency or timing of initiation in this locus. Rather, fluorescence in situ hybridization studies on cells swollen under either neutral or alkaline conditions show that deletion of the MAR interferes with local separation of daughter chromatids. This surprising result provides direct genetic evidence that at least a subset of MARs performs an important biological function, possibly related to chromatid cohesion and separation.     

Increased expression of integrins on fibroblast-like synoviocytes from rheumatoid arthritis in vitro correlates with enhanced binding to extracellular matrix proteins

OBJECTIVE—To compare in vitro expression of β1, β3, and β4 integrins in normal fibroblast-like synoviocytes (FBS) and in FBS from rheumatoid arthritis (RA) synovium and to investigate the adhesion of normal FBS and RA-FBS to the integrin binding extracellular matrix (ECM) proteins: collagen type IV, fibronectin, laminin, and tenascin.
METHODS—Expression of integrin receptors of cultured FBS was detected by flow cytometry. Attachment of FBS to ECM proteins was quantified by adhesion assays. Inhibition studies were performed using monoclonal antibodies to the integrin subunits.
RESULTS—Compared with normal FBS, RA-FBS showed increased expression of α1 to α6, β1, and β4 integrin subunits and enhanced binding of ECM proteins. Binding to ECM proteins was partly or completely blocked by an anti-β1 integrin antibody and antibodies to α3, α5, and α6 integrin subunits. The blocking efficiency was significantly (P < 0.05) higher in RA-FBS than in normal FBS.
CONCLUSIONS—The enhanced expression of the β1 integrin receptors on cultured RA-FBS correlated with increased attachment to ECM proteins. Adhesion of normal and RA-FBS to ECM proteins is mediated through β1 integrin receptors. Therefore, the tight binding of rheumatoid FBS to the matrix via β1 integrins might play a role in ECM remodelling in the rheumatoid process in vivo.

     

PPARβ/δ selectively regulates phenotypic features of age-related macular degeneration

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is a nuclear receptor that regulates differentiation, inflammation, lipid metabolism, extracellular matrix remodeling, and angiogenesis in multiple tissues. These pathways are also central to the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss globally. With the goal of identifying signaling pathways that may be important in the development of AMD, we investigated the impact of PPARβ/δ activation on ocular tissues affected in the disease. PPARβ/δ is expressed and can be activated in AMD vulnerable cells, including retinal pigment epithelial (RPE) and choroidal endothelial cells. Further, PPARβ/δ knockdown modulates AMD-related pathways selectively. Specifically, genetic ablation of Pparβ/δ in aged mice resulted in exacerbation of several phenotypic features of early dry AMD, but attenuation of experimentally induced choroidal neovascular (CNV) lesions. Antagonizing PPARβ/δ in both in vitro angiogenesis assays and in the in vivo experimentally induced CNV model, inhibited angiogenesis and angiogenic pathways, while ligand activation of PPARβ/δ, in vitro, decreased RPE lipid accumulation, characteristic of dry AMD. This study demonstrates for the first time, selective regulation of a nuclear receptor in the eye and establishes that selective targeting of PPARβ/δ may be a suitable strategy for treatment of different clinical sub-types of AMD.     

Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4⁻CD8⁻ double-negative thymocytes and Tcra gene segments in CD4⁺CD8⁺ double-positive thymocytes. Initial V_α-to-J_α recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E_α) interacts directly with V_α and J_α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E_α promotes interactions between these V_α and J_α segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E_α and to many locus promoters, biases E_α to interact with these promoters, and is required for efficient V_α–J_α recombination. Our data indicate that E_α and CTCF cooperate to create a developmentally regulated chromatin hub that supports V_α–J_α synapsis and recombination.     

Heterozygosity for the Y701C STAT1 mutation in a multiplex kindred with multifocal osteomyelitis

Heterozygosity for dominant-negative STAT1 mutations underlies autosomal dominant Mendelian susceptibility to mycobacterial diseases. Mutations conferring Mendelian susceptibility to mycobacterial diseases have been identified in the regions of the STAT1 gene encoding the tail segment, DNA-binding domain and SH2 domain. We describe here a new heterozygous mutation, Y701C, in a Japanese two-generation multiplex kindred with autosomal dominant Mendelian susceptibility to mycobacterial diseases. This mutation affects precisely the canonical STAT1 tyrosine phosphorylation site. The Y701C STAT1 protein is produced normally, but its phosphorylation is abolished, resulting in a loss-of-function for STAT1-dependent cellular responses to interferon-γ or interferon-α. In the patients’ cells, the allele is dominant-negative for γ-activated factor-mediated responses to interferon-γ, but not for interferon-stimulated gene factor-3-mediated responses to interferon-α/β, accounting for the clinical phenotype of Mendelian susceptibility to mycobacterial diseases without severe viral diseases. Interestingly, both patients displayed multifocal osteomyelitis, which is often seen in patients with Mendelian susceptibility to mycobacterial diseases with autosomal dominant partial IFN-γR1 deficiency. Multifocal osteomyelitis should thus prompt investigations of both STAT1 and IFN-γR1. This experiment of nature also confirms the essential role of tyrosine 701 in human STAT1 activity in natura.     

CD8+ T-cell-derived soluble factor(s), but not β-chemokines RANTES, MIP-1α, and MIP-1β, suppress HIV-1 replication in monocyte/macrophages

It has been demonstrated that CD8⁺ T cells produce a soluble factor(s) that suppresses human immunodeficiency virus (HIV) replication in CD4⁺ T cells. The role of soluble factors in the suppression of HIV replication in monocyte/macrophages (M/M) has not been fully delineated. To investigate whether a CD8⁺ T-cell-derived soluble factor(s) can also suppress HIV infection in the M/M system, primary macrophages were infected with the macrophage tropic HIV-1 strain Ba-L. CD8⁺ T-cell-depleted peripheral blood mononuclear cells were also infected with HIV-1 IIIB or Ba-L. HIV expression from the chronically infected macrophage cell line U1 was also determined in the presence of CD8⁺ T-cell supernatants or β-chemokines. We demonstrate that: (i) CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV replication in the M/M system; (ii) antibodies to regulated on activation normal T-cell expressed and Secreted (RANTES), macrophage inflammatory protein 1α (MIP-1α) and MIP-1β did not, whereas antibodies to interleukin 10, interleukin 13, interferon α, or interferon γ modestly reduced anti-HIV activity of the CD8⁺ T-cell supernatants; and (iii) the CD8⁺ T-cell supernatants did, but β-chemokines did not, suppress HIV-1 IIIB replication in peripheral blood mononuclear cells as well as HIV expression in U1 cells. These results suggest that HIV-suppressor activity of CD8⁺ T cells is a multifactorial phenomenon, and that RANTES, MIP-1α, and MIP-1β do not account for the entire scope of CD8⁺ T-cell-derived HIV-suppressor factors.     

DNA polymerase δ isolated from Schizosaccharomyces pombe contains five subunits

DNA polymerase δ (pol δ) plays an essential role in DNA replication, repair, and recombination. We have purified pol δ from Schizosaccharomyces pombe more than 10³-fold and demonstrated that the polymerase activity of purified S. pombe pol δ is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol δ complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.     

Targeting E2F1–DNA complexes with microgonotropen DNA binding agents

Microgonotropen (MGT) DNA binding drugs, which consist of an A+T-selective DNA minor groove binding tripyrrole peptide and polyamine chains attached to a central pyrrole that extend drug contact into the DNA major groove, were found to be extraordinarily effective inhibitors of E2 factor 1 (E2F1) association with its DNA promoter element (5′-TTTCGCGCCAAA). The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 μM. A relationship was found between the measured equilibrium constants for binding of MGTs to the A+T region of d(GGCGA₃T₃GGC)/d(CCGCT₃A₃CCG) and their inhibition of complex formation between E2F1 and the DNA promoter element. A representative of the potent MGT inhibitors was significantly more active on inhibition of E2F1–DNA complex formation compared with disruption of a preexisting complex.