Immunoglobulin Heavy Chain Constant Region Research Articles

Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology.

The CRISPR/Cas9 system, a defense mechanism naturally occurring in prokaryotes, has been recently repurposed as an RNA-guided DNA targeting platform and widely used as a powerful tool for genome editing. Here we describe how to modify the carboxy-terminal region, called Fragment crystallizable (Fc) region, of a murine monoclonal antibody by replacing the heavy chain constant exons with those from a teleost fish antibody by the CRISPR/Cas9 system. We outline optimal conditions for knockout and knockin mechanisms to edit the Immunoglobulin heavy chain (IgH) constant region gene locus in a murine hybridoma cell line. A chimeric mouse-fish monoclonal antibody can be successfully produced by hybridoma cell lines engineered according to this protocol.

Maturation of the Human Immunoglobulin Heavy Chain Repertoire With Age.

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced immunoglobulin heavy chain (IgH) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream IgH constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing IgH repertoires and stress the importance of using well-selected, age-appropriate controls in IgH studies.

SCIDOT-06. TOWARDS EXCLUSIVELY LOCAL THERAPY OF GLIOBLASTOMA - ENGINEERING IL-12Fc WITH SUPERIOR TISSUE RETENTION AND MINIMAL SYSTEMIC EXPOSURE AFTER CNS ADMINISTRATION

Abstract Intratumoral application of IL-12 overrides the prevailing glioblastoma (GB) associated immunosuppression and can trigger efficient anti-tumor responses. With clinical studies currently testing local expression of IL-12 in brain tumors, concerns on IL-12 systemic toxicity via leakage resurface. The fusion to an immunoglobulin heavy chain constant region (“Fc tag”) should increase tissue retention of IL-12. However, export of IL-12Fc into the blood via the neonatal Fc receptor (FcRn) could nevertheless lead to systemic exposure. Subsequent systemic recycling via FcRn could lead to gradually increasing and eventually toxic serum concentrations. We analysed the effect of the Fc-tag on IL-12 tissue retention and evaluated whether FcRn also is involved in brain export and systemic recycling of IL-12Fc upon local delivery. Human or murine IL-12Fc was delivered in GB-bearing or naïve wt or FcRn-humanized mice (mFcRn-/- hFcRn tg) continuously via mini osmotic pumps or as bolus via convection-enhanced delivery (CED). Brain and blood concentration levels were assessed via ELISA. FcRn affinity of IL-12Fc mutants was assessed via ELISA and surface plasmon resonance. We observed much higher tissue retention of IL-12Fc compared to unmodified IL-12, but also an FcRn-dependent gradual increase of IL-12Fc serum levels. Testing a battery of amino acid substitutions at the FcRn interaction interface, we discovered unique substitutions that largely abolish brain export and systemic accumulation while preserving IL-12Fc functionality, leading to an over 100-fold higher brain to blood ratio than unmodified IL-12. We currently test the impact on efficacy of local IL-12Fc and checkpoint brain tumor treatment in vivo. Achieving high local concentrations at low to absent systemic exposure is an important prerequisite for a large therapeutic window for local treatment of neurologic diseases. The novel Fc-modifications present a promising platform for reducing systemic leakage of Fc-containing therapeutics in the context of continuous or intermittent CNS delivery beyond brain cancer therapy.

Induction of t(11;14) IgH enhancer/promoter-cyclin D1 gene translocation using CRISPR/Cas9.

Chromosomal translocation is a key process in the oncogenic transformation of somatic cells. Previously, artificial induction of chromosomal translocation was performed using homologous recombination-mediated loxP labeling of target regions followed by Cre-mediated recombination. Recent progress in genome editing techniques has facilitated the easier induction of artificial translocation by cutting two targeted genome sequences from different chromosomes. The present study established a system to induce t(11;14)(q13;q32), which is observed primarily in multiple myeloma (MM) and involves the repositioning of the cyclin D1 (CCND1) gene downstream of the immunoglobulin heavy chain (IgH) constant region enhancers by translocation. The placing of tandem gRNAs designed to cut both the IgH Eµ and CCND1 15-kb upstream regions in lentiCRISPRv2 enabled the induction of chromosomal translocation in 293T cells, with confirmation by translocation-specific PCR and fluorescence in situ hybridization probing with IgH and CCND1. At the translocation junctions, small deletions and the addition of DNA sequences (indels) were observed in several clones. Cloned cells with t(11;14) exhibited slower growth and lower CCND1 mRNA expression compared to the parent cells, presenting the opposite phenomena induced by t(11;14) in MM cells, indicating that the silent IgH gene juxtaposed to CCND1 may negatively affect CCND1 gene expression and cell proliferation in the non-B lymphocyte lineage. Therefore, the present study achieved the induction of silent promoter/enhancer translocation in t(11;14)(q13;q32) as a preparatory experiment to study the role of IgH constant region enhancer-driven CCND1 overexpression in oncogenic transformation processes in B lymphocytes.

Genomic organization and adaptive evolution of IGHC genes in marine mammals

Immunoglobulins are important elements of the adaptive immune system that bind to an immense variety of microbial antigens to neutralize infectivity and specify effector functions. In the present study, the immunoglobulin heavy chain constant region (IGHC) genes from marine mammals were identified and compared with those of their terrestrial relatives to explore their genomic organization and evolutionary characteristics. The genomic organization of marine mammal IGHC genes was shown to be conservative with other eutherian mammals. Stronger signals of positive selection on IGHC were revealed in terrestrial mammals than that in marine mammals with the branch-site model, displaying different selective pressure, which might suggest their divergent adaptations to contrasted environments.

Age-associated distribution of normal B-cell and plasma cell subsets in peripheral blood

Humoral immunocompetence develops stepwise throughout life and contributes to individual susceptibility to infection, immunodeficiency, autoimmunity, and neoplasia. Immunoglobulin heavy chain (IgH) isotype serum levels can partly explain such age-related differences, but their relationship with the IgH isotype distribution within memory B-cell (MBC) and plasma cell (PCs) compartments remains to be investigated. We studied the age-related distribution of MBCs and PCs expressing different IgH isotypes in addition to the immature/transitional and naive B-cell compartments. B-cell and PC subsets and plasma IgH isotype levels were studied in cord blood (n=19) and peripheral blood (n=215) from healthy donors aged 0 to 90years by using flow cytometry and nephelometry, respectively. IgH-switched MBCs expressing IgG1, IgG2, IgG3, IgA1, and IgA2 were already detected in cord blood and newborns at very low counts, whereas CD27+IgM++IgD+ MBCs only became detectable at 1 to 5months and remained stable until 2 to 4years, and IgD MBCs peaked at 2 to 4years, with both populations decreasing thereafter. MBCs expressing IgH isotypes of the second immunoglobulin heavy chain constant region (IGHC) gene block (IgG1, IgG3, and IgA1) peaked later during childhood (2-4years), whereas MBCs expressing third IGHC gene block immunoglobulin isotypes (IgG2, IgG4, and IgA2) reached maximum values during adulthood. PCs were already detected in newborns, increasing in number until 6 to 11months for IgM, IgG1, IgG2, IgG3, IgA1, and IgA2; until 2 to 4years for IgD; and until 5 to 9years for IgG4 and decreasing thereafter. For most IgH isotypes (except IgD and IgG4), maximum plasma levels were reached after PC and MBC counts peaked. PC counts reach maximum values early in life, followed by MBC counts and plasma IgH isotypes. Importantly, IgH isotypes from different IGHC gene blocks show different patterns, probably reflecting consecutive cycles of IgH isotype switch recombination through life.

Molecular engineering of antibodies for site-specific covalent conjugation using CRISPR/Cas9

Site-specific modification of antibodies has become a critical aspect in the development of next-generation immunoconjugates meeting criteria of clinically acceptable homogeneity, reproducibility, efficacy, ease of manufacturability, and cost-effectiveness. Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line to manufacture antibodies capable of site-specific conjugation. This enabled an effective enzymatic site-controlled conjugation of fluorescent and radioactive cargoes to a genetically tagged mAb without impairment of antigen binding activity. After injection in mice, these immunoconjugates showed almost doubled specific targeting in the lung vs. chemically conjugated maternal mAb, and concomitant reduction in uptake in the liver and spleen. The approach outlined in this work provides a facile method for the development of more homogeneous, reproducible, effective, and scalable antibody conjugates for use as therapeutic and diagnostic tools.

Preparing for re-entry: is sequential switching the result of recurrent secondary responses?

Preparing for re-entry: is sequential switching the result of recurrent secondary responses?

The repetitive portion of the Xenopus IgH Mu switch region mediates orientation-dependent class switch recombination.

Vertebrates developed immunoglobulin heavy chain (IgH) class switch recombination (CSR) to express different IgH constant regions. Most double-strand breaks for Ig CSR occur within the repetitive portion of the switch regions located upstream of each set of constant domain exons for the Igγ, Igα or Igϵ heavy chain. Unlike mammalian switch regions, Xenopus switch regions do not have a high G-density on the non-template DNA strand. In previous studies, when Xenopus Sμ DNA was moved to the genome of mice, it is able to support substantial CSR when it is used to replace the murine Sγ1 region. Here, we tested both the 2kb repetitive portion and the 4.6 kb full-length portions of the Xenopus Sμ in both their natural (forward) orientation relative to the constant domain exons, as well as the opposite (reverse) orientation. Consistent with previous work, we find that the 4.6 kb full-length Sμ mediates similar levels of CSR in both the forward and reverse orientations. Whereas, the forward orientation of the 2kb portion can restore the majority of the CSR level of the 4.6 kb full-length Sμ, the reverse orientation poorly supports R-looping and no CSR. The forward orientation of the 2kb repetitive portion has more GG dinucleotides on the non-template strand than the reverse orientation. The correlation of R-loop formation with CSR efficiency, as demonstrated in the 2kb repetitive fragment of the Xenopus switch region, confirms a role played by R-looping in CSR that appears to be conserved through evolution.

Defining Risk of MGUS and AMM Progression to Myeloma By Ig Heavy-Chain FISH

Interphase FISH analysis using probes for the immunoglobulin heavy chain constant region (IGHC) and variable regions (IGHV) revealed that plasma cells from healthy individuals and from patients with plasma cell (PC) dyscrasias had different patterns of signal-combinations of the two probes. We hypothesized that these patterns indicate diagnostic and prognostic significance.Patients and Methods: Bone marrow specimens from 39 healthy donors, 111 patients with MGUS, 89 patients with AMM, and 96 patients with MM were examined using interphase FISH with probes for IGHC and IGHV simultaneously (Tian et al. Gene Chr Can 2014) and immune staining for cytoplasmic Ig isotypes. For each specimen, the combination patterns of IGHC and IGHV probes were scored separately for Kappa and Lambda expressing PCs. In addition, gene expression profiles (GEP) of CD138+-purified PCs were analyzed. FISH were also performed with the probes to chromosome 1q21 (CKS1B), 1p13 (AHCYL1), 17p13 (TP53), and 17q12 (ERBB2) loci.Results: 1.IGHC and IGHV pattern diversity of PCs was present in all specimens, including healthy individuals. A bivariate distribution of polyclonal diversities among healthy individuals was calculated to create an ellipse defining the normal population pattern. Applying the ellipsoid definition to the patient populations, two subgroups could be distinguished in MGUS/AMM cases: one within the ellipse, representing patients with polyclonal PCs (without a dominant clone), while the other, who had a dominant clone with identical IGHC/IGHV FISH pattern, fell outside of the ellipse and was termed asymptomatic monoclonal gammopathy (Figure). With 99% confidence, 54 of 111 (48.7%) of MGUS and 66 of 89 (74.2%) of AMM were classified as monoclonal. In MM, 80 of 96 (86.0%) were stratified outside of the ellipse.2.The FISH-defined monoclonal subgroup (outside the ellipse) among AMM patients was at high risk of progressing to symptomatic myeloma; one year progression to MM requiring therapy was 26%, 2-year progression was 37%, and the 5-year progression rate was estimated at 64%. The subgroup with polyclonal PCs in AMM had significantly lower risk of disease progression (<10% in 2 years, p<0.0001). In univariate Cox regression analysis, besides FISH-defined monoclonality (HR 2.72, p<0.004), serum albumin (< 3.5 g/dL), GEP70 risk score (> -0.26), t(4;14), excess lambda light chain, and gain of chromosome1q21 region (>2) were significant progression factors of FISH-defined monoclonal patients (Hazard Ratios >2.2; p<0.005). In multivariate analysis, FISH-defined monoclonality (HR 2.15), age (>65 yr, HR 2.05), serum albumin (< 3.5 g/dL, HR 3.21) and having AMM (HR 3.02) remained significant predictors of early disease progression with an R2 of 0.434.Conclusion:We established a FISH predictive model on the basis of healthy bone marrow. Based on normal distribution of PC clonal diversities, patient subgroups with polyclonal PC and with monoclonal PC were detected in MGUS and AMM patients. The presence of a dominant clone in the bone marrow at the time of diagnosis identifies, among patients with MGUS and AMM, a subgroup at high-risk of early disease progression to myeloma requiring therapy. We are currently developing a model to identify with high accuracy patients will progress within 1, and 2 years.Figure: A 100(1-α)% prediction ellipse based on clonal distribution of healthy donors (left) defines the polyclonal PC region. MGUS, AMM, and MM patients with monoclonal PCs are outside this region. DisclosuresMorgan:Celgene Corp: Membership on an entity’s Board of Directors or advisory committees; Novartis: Membership on an entity’s Board of Directors or advisory committees; Janssen: Membership on an entity’s Board of Directors or advisory committees; Myeloma UK: Membership on an entity’s Board of Directors or advisory committees; International Myeloma Foundation: Membership on an entity’s Board of Directors or advisory committees; The Binding Site: Membership on an entity’s Board of Directors or advisory committees; MMRF: Membership on an entity’s Board of Directors or advisory committees.

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.

Cytogenetic Features Of Patients With Monoclonal Gammopathy Of Undetermined Significance (MGUS) and Asymptomatic Multiple Myeloma (AMM)

Cytogenetic Features Of Patients With Monoclonal Gammopathy Of Undetermined Significance (MGUS) and Asymptomatic Multiple Myeloma (AMM)

Combinatorial H3K9acS10ph Histone Modification in IgH Locus S Regions Targets 14-3-3 Adaptors and AID to Specify Antibody Class-Switch DNA Recombination

Class-switch DNA recombination (CSR) is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH) constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID)-centered CSR machinery excises and rejoins DNA between an upstream (donor) and a downstream (acceptor) S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5'-AGCT-3' repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is "written" specifically in S regions and is "read" by 14-3-3 adaptors to target AID for CSR as an important biological outcome.

FISH and GEP Based Prediction of Chromosomal Translocation Identifies Myeloma Patients Who Do Not Benefit From Bortezomib

Abstract 1814Gene expression profiling (GEP) provides valuable information on molecular subclass and prognostic risk of multiple myeloma (MM). In addition to subclass designation, GEP also predicts many cytogenetic aberrations in MM (Zhou, Blood 119:e148). We re-examined GEP derived assignments of translocation-related molecular subgroups (CD1, CD2 MF, and MS) in correlation to FISH data. Focusing on 14q32 translocation, we used FISH probes to MMSET/FGFR3, CCND3, CCND1, MAF, and MAFB in combinations with probes specific to the immunoglobulin heavy chain (IGH) constant and viable regions (IGHC and IGHV) to identify translocations in myeloma cells from Total Therapy (TT) 4 and TT5 patients at baseline. Combining our FISH data with GEP expression of probe sets specific to the IgH translocation partners, we identified threshold expression levels; above this threshold the expression level was clearly indicative of genomic translocations of t(4;14)(p16;q32), t(6;14)(p21;q32), t(11;14)(q13;32), t(14;16)(q32;q23), or t(14;20)(q32;q11). Below these thresholds, increased signals reflected gene copy numbers. Overall, 42.2% (113/268) of patients were predicted and confirmed to have 14q translocations: 14.9% had t(4;14), 3.4% t(6;14), 19.4% t(11;14), 3.4% t(14;16), and 1.1% t(14;20). In cases of GEP indicated simultaneous spikes of several 14q translocation partners, we discovered that 14q translocation were indeed mutually exclusive - one 14q translocation per clone. The spikes of the other 14q partner gene(s) shown by GEP, reflected copy number driven increases, or in a few cases, the co-existence of distinct clones, each with a unique 14q translocation. Using threshold expression levels equivalent to those developed in the TT4 and TT5 training set, we predicted that 42.3% (335/792) of patients treated on TT2 and TT3 protocols had 14q translocations at baseline, as follows; 14.9% t(4;14), 1.4% t(6;14), 19.8% t(11;14), 4.4% (14;16), and 1.8% t(14;20). Next, we correlated the effects of 14q translocations with overall survival of patients treated with TT2 and TT3. Patients whose GEP spikes predicted MMSET/FGFR3 and CCND3 translocations significantly benefited from the inclusion of Bortezomib in TT3; and patients with CCND1 and MAFB translocations had improved median survivals comparing 8 years follow-ups of TT2 and TT3. In contrast, patients with high MAF expression clearly did not benefit from the inclusion of the proteasome inhibitor in TT3. In conclusion, by combining interphase FISH and GEP we established predictive threshold expression levels of 14q translocations in MM and identified a group of patients who do not benefit from Bortezomib. This single-gene approach to define molecular subgroups is expedient and provides important information of prognostic significance. Disclosures:Tian:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties. Sawyer:University of Arkansas for Medical Sciences: Employment, under panding process, under panding process Patents & Royalties. Epstein:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties. Barlogie:University of Arkansas for Medical Sciences: Employment, under pending process, under pending process Patents & Royalties.

Allelic polymorphism of Immunoglobulin heavy chain genes in the Antarctic teleost Trematomus bernacchii

IgM represents the main immunoglobulin isotype shared by all teleost fish. However, Antarctic fish IgM possess a peculiar hinge region, which connects the CH2 and CH3 domains, not seen in any other teleost species. In the present study allelic polymorphism of IgM gene of the Antarctic teleost Trematomus benacchii was investigated. By nucleotide sequencing the entire Immunoglobulin heavy chain constant region from ten T. bernacchii individuals, 47 positions were found to be polymorphic. The largest number of polymorphic positions, accounting for 51% of the total, was found to fall within the hinge region. This region not only displayed extensive nucleotide variation, but also length diversity; in fact several sequences were one amino acid shorter as resulting from the usage of a different splice acceptor site of the CH3 exon, as demonstrated by genomic DNA analysis. The Ka/Ks ratios of the polymorphic positions showed typical values higher than 1, indicative of positive selection acting to polymorphic codons to favor amino acid replacements and maintain allelic variants.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

New targets of pemphigus vulgaris antibodies identified by protein array technology

We performed partial evaluation of pemphigus vulgaris (PV) autoantibody profile using the protein array technology. The sera from seven patients with acute PV and five healthy donors were probed for the presence of autoantibodies characteristic of the organ-non-specific autoimmune disorders rheumatoid arthritis, lupus erythematosus, scleroderma, diabetes and some other autoimmune disorders, but not to desmosomal proteins. The array targeted 785 human genes amplified using Mammalian Gene Clone Collection with gene-specific primers containing 20-bp nucleotide extension complementary to ends of linear pXT7 vector. The array identified PV antibodies significantly (P<0.05) differentially reactive with 16 antigens, most of which were cell-surface proteins, such as CD2, CD31, CD33, CD36, CD37, CD40, CD54, CD66c and CD84 molecules, nicotinamide/nicotinic acid mononucleotide adenylyltransferase, immunoglobulin heavy chain constant region gamma 2 and others. Reactivity with Fc-IgG helps explain an ability of the chimeric desmoglein constructs to absorb out all disease-causing PV antibodies. Anti-M(1) muscarinic receptor antibody was also identified, consistent with the facts that while blockade of this receptor causes keratinocyte detachment, its activation is therapeutic in PV. Further proteomics analysis of PV antibodies should help elucidate the immunopathogenic mechanisms underlying keratinocyte detachment and blistering.

Alternative end-joining catalyzes robust IgH locus deletions and translocations in the combined absence of ligase 4 and Ku70

Class switch recombination (CSR) in B lymphocytes is initiated by introduction of multiple DNA double-strand breaks (DSBs) into switch (S) regions that flank immunoglobulin heavy chain (IgH) constant region exons. CSR is completed by joining a DSB in the donor S mu to a DSB in a downstream acceptor S region (e.g., S gamma1) by end-joining. In normal cells, many CSR junctions are mediated by classical nonhomologous end-joining (C-NHEJ), which employs the Ku70/80 complex for DSB recognition and XRCC4/DNA ligase 4 for ligation. Alternative end-joining (A-EJ) mediates CSR, at reduced levels, in the absence of C-NHEJ, even in combined absence of Ku70 and ligase 4, demonstrating an A-EJ pathway totally distinct from C-NHEJ. Multiple DSBs are introduced into S mu during CSR, with some being rejoined or joined to each other to generate internal switch deletions (ISDs). In addition, S-region DSBs can be joined to other chromosomes to generate translocations, the level of which is increased by absence of a single C-NHEJ component (e.g., XRCC4). We asked whether ISD and S-region translocations occur in the complete absence of C-NHEJ (e.g., in Ku70/ligase 4 double-deficient B cells). We found, unexpectedly, that B-cell activation for CSR generates substantial ISD in both S mu and S gamma1 and that ISD in both is greatly increased by the absence of C-NHEJ. IgH chromosomal translocations to the c-myc oncogene also are augmented in the combined absence of Ku70 and ligase 4. We discuss the implications of these findings for A-EJ in normal and abnormal DSB repair.

Replacement of Iμ-Cμ intron by NeoR gene alters Iμ germ-line expression but has no effect on V(D)J recombination

The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3′ regulatory region (3′RR). This polarized long-range effect of the 3′RR is disturbed upon insertion of NeoR gene in the IgH constant (CH) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Eμ enhancer or Eμ core enhancer (cEμ) by NeoR gene fully blocked V(D)J recombination and μ0 GL transcription which originates 5′ of DQ52 and severely diminished Iμ GL transcription derived from Eμ/Iμ promoter, suggesting a critical role for cEμ in the regulation of V(D)J recombination and of μ0 and Iμ expression. Here we focus on the effect of NeoR gene on μ0 and Iμ GL transcription in a mouse line in which the Iμ-Cμ intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Eμ. μ0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Iμ transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Iμ expression. Surprisingly, in stark contrast with the NeoR effect in the CH region, LPS-induced NeoR expression restored Iμ transcript levels to normal. The data suggest that Eμ enhancer may be the master control element that counteracts the down-regulatory “Neo effect” on Iμ expression upon LPS stimulation. More importantly, they reveal a complex and developmentally regulated interplay between IgH enhancers in the control of Iμ expression.

Syndecan-2 Regulates the Migratory Potential of Melanoma Cells

Syndecan-2, a transmembrane heparan sulfate proteoglycan, is a critical mediator in the tumorigenesis of colon carcinoma cells. We explored the function of syndecan-2 in melanoma, one of the most invasive types of cancers, and found that the expression of this protein was elevated in tissue samples from both nevus and malignant human melanomas but not in melanocytes of the normal human skin tissues. Similarly, elevated syndecan-2 expression was observed in various melanoma cell lines. Overexpression of syndecan-2 enhanced migration and invasion of melanoma cells, whereas the opposite was observed when syndecan-2 levels were knocked down using small inhibitory RNAs. Syndecan-2 expression was enhanced by fibroblast growth factor-2, which is known to stimulate melanoma cell migration; however, alpha-melanocyte-stimulating hormone decreased syndecan-2 expression and melanoma cell migration and invasion in a melanin synthesis-independent manner. Furthermore, syndecan-2 overexpression rescued the migration defects induced by alpha-melanocyte-stimulating hormone treatment. Together, these data strongly suggest that syndecan-2 plays a crucial role in the migratory potential of melanoma cells.