Evolution of human IgH3′EC duplicated structures: both enhancers HS1,2 are polymorphic with variation of transcription factor's consensus sites

Abstract

The enhancer complex regulatory region at the 3V of the immunoglobulin heavy cluster (IgH3VEC) is duplicated in apes along with four constant genes and the region is highly conserved throughout humans. Both human IgH3VECs consist of three loci high sensitive (HS) to DNAse I with enhancer activity. It is thus possible that the presence of structural divergences between the two IgH3VECs and of relative polymorphisms correspond to functional regulatory changes. To analyse the polymorphisms of these almost identical regions, it resulted mandatory to identify the presence of divergent sequences, in order to select distinctive primers for specific PCR genomic amplifications. To this aim, we first compared the two entire IgH3VECs in silicio, utilising the updated GenBank (GB) contigs, then we analysed the two IgH3'ECs by cloning and sequencing amplicons from independent genomes. In silicio analysis showed that several inversions, deletions and short insertions had occurred after the duplication. We analysed in detail, by sequencing specific regions, the polymorphisms occurring in enhancer HS1,2-A (which lies in IgH3VEC-1, 3V to the Ca-1 gene) and in enhancer HS1,2-B (which lies in IgH3VEC-2, 3V to Ca-2). Polymorphisms are due to the repetition (occurring one to four times) of a 38-bp sequence present at the 3V of the core of enhancers HS1,2. The structure of both human HS1,2 enhancers has revealed not yet described polymorphic features due to the presence of variable spacer elements separating the 38-bp repetitions and to variable external elements bordering the repetition cluster. We found that one of the external elements gave rise to a divergent allele 3 in the two clusters. The frequency of the different alleles of the two loci varies in the Italian population and allele 3 of both loci are very rare. The analysis of the Callicebus moloch, Gorilla gorilla and Pan troglodytes HS1,2 enhancers showed the transformation from the ancestral structure with the 31-to the 17-bp external element in hominids.The relevance of the polymorphisms in the HS1,2 enhancers is due to the variable number of binding sites for the transcription factors: NF-nB, CMYB, BSAP1/2, AP1/4, E47, MyoD and AE5 and thus to the possible influence of these variations on switch, production of Ig and on maturation of B cells.

Analysis of the functional relevance of a putative regulatory SNP of PDCD1, PD1.3, associated with systemic lupus erythematosus

“…[27][28][29] Some of them are polymorphic and affect transcriptional regulation, but in all the described examples the functional polymorphism affects the number of copies of the repeat. It is less likely that an SNP affecting a single TFBS in a repeated structure will have functional consequences, as it will be compensated by the same TF bound to the other repeats.…”

Allele *2 of the HS1,2A enhancer of the Ig regulatory region associates with rheumatoid arthritis

“…The nested second PCR was performed using the primers P3 forward 5′-GACTCATTCTGGGCAGACTTG-3′ and D3 reverse 5′-GTCCTGGTCCCAAAGATGG-3′ and with 1/25 of the volume of the first PCR, minimising the carry-over of the genomic DNA; control reactions were performed with 2 and 5 ng total genomic DNA, giving no visible amplification on gel agarose electrophoresis. Reaction, conditions and cycles were the same as described previously 9. Negative and positive controls, without a DNA template or with a heterozygote control DNA sample were always included.…”

“…In these diseases, the variation of allelic frequencies involves allele *2, which has one consensus site for the NF-κB transcription factor missing in the second more frequent allele *1 9. The IgH 3′ regulatory region is active in the transcription of the heavy constant genes for class switch recombination and in the Ig transcription 10…”

Allele *1 of HS1.2 Enhancer Associates with Selective IgA Deficiency and IgM Concentration

“…AJ544218, AJ544219, AJ544220, AJ544221), we conducted a selective PCR, which amplified the HS1.2-A region, but not the identical inverted HS1.2-B region (26). Genomic DNA was extracted from peripheral blood nucleated cells or from buccal mucosal swabs and amplified with the primers described previously (26). The cycle conditions were 94°C 2Ј for a first step, followed by 94°C 30ЈЈ, 61°C 30ЈЈ, 68°C 5Ј for 10 cycles and 94°C 30ЈЈ, 59°C 30ЈЈ, 68°C 5Ј for 20 cycles, ending with 72°C 10Ј.…”

“…Control reactions were performed with 1 and 5 ng of total genomic DNA and resulted in no visible amplification in those conditions on gel agarose electrophoresis. The primers for the PCRs are reported in Giambra et al (26). The second PCR was conducted with the same volumes and concentrations used in the first PCR, except for the use of 1 U of Platinum TaqDNA polymerase (Invitrogen).…”