Ahmed Amine Khamlichi

Immunoglobulin switch μ sequence causes RNA polymerase II accumulation and reduces dA hypermutation

Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung−/− and Aid−/−Ung−/− mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung−/− B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.

Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

ABSTRACT The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the Sμ locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The Sμ R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core Sμ repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core Sμ repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core Sμ repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.

Primary structure of a variable region of the VI subgroup (ISE) in light chain deposition disease

Although structural abnormalities of monoclonal immunoglobulin light chains (LC) are suspected to play a determinant role in non‐amyloid light chain deposition disease (LCDD), this condition is as yet poorly documented at the molecular level, since only three sequences have been reported to date. In a case of myeloma‐associated LCDD, the patients urine contained an unglycosylated κ Bence Jones protein made up of dimers and monomers with an apparent molecular mass of 25000 which was assigned to the VκI subgroup by N‐terminal amino acid sequencing. The complete variable region sequence of the monoclonal κ chain produced by the malignant plasma cells was amplified by polymerase chain reaction (PCR) using small amounts of material obtained by bone marrow aspiration. The sequence of three independently amplified cDNA clones derived from a normal‐sized κ messenger RNA was identical to that of the urinary κ chain. The κ mRNA had an overall normal structure made up of a VκI sequence rearranged to JκI‐ Several unusual features of the variable region (the first complete VκI sequence reported in LCDD) included three substitutions that introduced hydrophobic residues at spatially close positions. The strategy associating N‐terminal sequence determination and cDNA cloning by PCR could help in accumulating new sequence data and improving our understanding of LCDD pathogenesis.

Primary structure of a monoclonal κ chain in myeloma with light chain deposition disease

Previous data suggest that structural abnormalities of immunoglobulin light chains may be responsible for non‐amyloid light chain deposition disease (LCDD). We report on the complete primary sequence deduced from complementary (c)DNA analysis of a normal‐sized κ chain in a case of myeloma‐associated LCDD. The patients urine contained a κ type Bence‐Jones protein made of monomers and dimers of an unglycosylated κ chain. The bone marrow myeloma cells contained intracellular κ and γ chains by immunofluorescence. Biosynthesis experiments showed the production of normal‐sized γ chains and of κ chains with the same apparent molecular mass (Mr) in SDS gels as the urinary κ chain (26000–27000). These κ chains were secreted as assembled IgG molecules and as a large excess of free monomers and dimers. The complete sequence of two identical cDNA clones derived from a normal‐sized κ messenger RNA indicated that this K chain belonged to the rare VκIV subgroup. The κ mRNA had an overall normal structure made up of the VκIV sequence rearranged to JκIV and followed by a normal constant exon of the Km(3) allotype. The variable region differed from the VκIV‐JκI germline sequence by 17 amino acid substitutions. The peculiar sequence of the variable region of this κ chain of a rare subgroup might relate lo its tissue deposition.

Germ-line transcription occurs on both the functional and the non-functional alleles of immunoglobulin constant heavy chain genes.

In B cells undergoing class switching, recombination is usually directed to the same switch sequences on both homologous chromosomes and is generally preceded by germ‐line transcription initiated from I promoters upstream of immunoglobulin constant heavy chain genes. Whether germ‐line transcription occurs on both alleles or is restricted to the productive one has not been directly addressed before. To clearly distinguish between these two possibilities, a system is needed in which one might detect allele‐specific germ‐line transcripts. We used homologous recombination to set up twoin vivo systems in which one γ3 allele was marked by a loxP sequence whereas the other allele was silenced by inhibiting μ heavy chain production. Here, we provide evidence that germ‐line transcription occurs on both the functional and the non‐functional alleles.

Downstream class switching leads to IgE antibody production by B lymphocytes lacking IgM switch regions

Ig heavy chain (IgH) class-switch recombination (CSR) replaces the IgH Cμ constant region exons with one of several sets of downstream IgH constant region exons (e.g., Cγ, Cε, or Cα), which affects switching from IgM to another IgH class (e.g., IgG, IgE, or IgA). Activation-induced cytidine deaminase (AID) initiates CSR by promoting DNA double-strand breaks (DSBs) within switch (S) regions flanking the donor Cμ (Sμ) and a downstream acceptor CH (e.g., Sγ, Sε, Sα) that are then joined to complete CSR. DSBs generated in Sμ frequently are joined within Sμ to form internal switch region deletions (ISD). AID-induced ISD and mutations have been considered rare in downstream S regions, suggesting that AID targeting to these S regions requires its prior recruitment to Sμ. We have now assayed for CSR and ISD in B cells lacking Sμ (Sμ−/− B cells). In Sμ−/− B cells activated for CSR to IgG1 and IgE, CSR to IgG1 was greatly reduced; but, surprisingly, CSR to IgE occurred at nearly normal levels. Moreover, normal B cells had substantial Sγ1 ISD and increased mutations in and near Sγ1, and levels of both were greatly increased in Sμ−/− B cells. Finally, Sμ−/− B cells underwent downstream CSR between Sγ1 and Sε on alleles that lacked Sμ CSR to these sequences. Our findings show that AID targets downstream S regions independently of CSR with Sμ and implicate an alternative pathway for IgE class switching that involves generation and joining of DSBs within two different downstream S regions before Sμ joining.

Sense transcription through the S region is essential for immunoglobulin class switch recombination.

Class switch recombination (CSR) occurs between highly repetitive sequences called switch (S) regions and is initiated by activation‐induced cytidine deaminase (AID). CSR is preceded by a bidirectional transcription of S regions but the relative importance of sense and antisense transcription for CSR in vivo is unknown. We generated three mouse lines in which we attempted a premature termination of transcriptional elongation by inserting bidirectional transcription terminators upstream of Sμ, upstream of Sγ3 or downstream of Sγ3 sequences. The data show, at least for Sγ3, that sense transcriptional elongation across S region is absolutely required for CSR whereas its antisense counterpart is largely dispensable, strongly suggesting that sense transcription is sufficient for AID targeting to both DNA strands.

The effect of intron sequences on expression levels of Ig cDNAs

Several cDNA expression vectors were constructed and tested by stable transfection into a murine lymphoid cell line in order to compare secretion rates of a human immunoglobulin (Ig) light chain (LC). When the cDNA was under transcriptional control of the SV40 promoter and enhancer and preceded by the SV40 19S late mRNA intron, a weak LC production was detected. Secretion rate was not improved by replacing the SV40 promoter and enhancer by a combination of a murine Ig heavy chain (HC) gene promoter and enhancer even with insertion of additional Ig enhancers. In contrast, replacement of the 19S intron by a large intron derived from a human Ig HC gene and containing the intronic enhancer dramatically increased the secretion rate. High-level production was also obtained with the same enhancer-containing intron placed downstream from the LC cDNA. Stable transfectants were obtained that secreted the human LC in amounts comparable to those obtained with Ig genes. Our results suggest that the SV40 19S late mRNA intron used in several expression vectors is not appropriate when the purpose is to produce large amounts of antibody molecules. By providing transcriptional, splicing and polyadenylation signals, the presently described vectors will be useful for easy cloning and high-level expression in lymphoid cells of cDNAs or PCR products encoding antibody molecules.

Replacement of Iγ3 germ-line promoter by Iγ1 inhibits class-switch recombination to IgG3

Class-switch recombination (CSR) enables IgM-producing B cells to switch to the production of IgG, IgE, and IgA. The process requires germ-line (GL) transcription that initiates from promoters upstream of switch (S) sequences and is regulated by the 3′ regulatory region (3′RR) located downstream of the Ig heavy chain (IgH) locus. How the 3′RR effect its long-range activation is presently unclear. We generated a mouse line in which Iγ3 GL promoter was replaced by Iγ1. We found that GL transcription could initiate from the inserted Iγ1 promoter and was induced by increased concentrations of IL-4 and that the transcripts were normally spliced. However, when compared with GL transcripts derived from the endogenous Iγ1 promoter in the same stimulation conditions, those from the inserted Iγ1 promoter were less abundant. CSR to Cγ3 was abrogated both in vivo and in vitro. The results strongly suggest that the endogenous Iγ1 promoter insulates the inserted Iγ1 from the long-range activating effect of the 3′RR. The implications of our findings are discussed in light of the prominent models of long-distance activation in complex loci.

Developmental Switch in the Transcriptional Activity of a Long-Range Regulatory Element

ABSTRACT Eukaryotic gene expression is often controlled by distant regulatory elements. In developing B lymphocytes, transcription is associated with V(D)J recombination at immunoglobulin loci. This process is regulated by remote cis-acting elements. At the immunoglobulin heavy chain (IgH) locus, the 3′ regulatory region (3′RR) promotes transcription in mature B cells. This led to the notion that the 3′RR orchestrates the IgH locus activity at late stages of B cell maturation only. However, long-range interactions involving the 3′RR were detected in early B cells, but the functional consequences of these interactions were unknown. Here we show that not only does the 3′RR affect transcription at distant sites within the IgH variable region but also it conveys a transcriptional silencing activity on both sense and antisense transcription. The 3′RR-mediated silencing activity is switched off upon completion of VH-DJH recombination. Our findings reveal a developmentally controlled, stage-dependent shift in the transcriptional activity of a master regulatory element.