Human HS1 Research Articles

Aryl hydrocarbon receptor-induced activation of the human IGH hs1.2 enhancer: Mutational analysis of putative regulatory binding motifs

The human hs1.2 enhancer within the Ig heavy chain gene (IGH) is polymorphic and associated with a number of autoimmune diseases. The polymorphic region is characterized by tandem repeats of an ∼53-bp invariant sequence containing possible binding sites for several transcription factors. Our previous studies suggest the human hs1.2 enhancer is sensitive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant and high affinity ligand of the aryl hydrocarbon receptor (AhR). TCDD induced hs1.2 enhancer activity in an AhR-dependent manner and the number of invariant sequences influenced the magnitude of activity. To better understand the regulation of human hs1.2 enhancer activity, the objective of the current study was to utilize mutational analysis and luciferase reporter constructs to evaluate the contribution of putative transcription factor binding sites to overall hs1.2 enhancer activity and modulation by TCDD. Basal and LPS-induced activity of the hs1.2 enhancer appeared to be most affected by mutation of sites outside of the invariant sequence or deletion of the entire invariant sequence; whereas sites influencing the effect of TCDD were dependent on the cellular activation state (i.e. unstimulated vs. LPS stimulation) and relatively independent of the putative AhR binding site within the invariant sequence. These results suggest that AhR activation affects human hs1.2 activity through an as yet undetermined non-canonical pathway. A better understanding regarding the role of the hs1.2 enhancer in human Ig expression and how AhR ligands modulate its activity may lead to insights into overall Ig regulation and mechanisms of dysfunction.

The change in Ig regulation from children to adults disconnects the correlation with the 3'RR hs1.2 polymorphism.

BackgroundIn the immune system, the serum levels of immunoglobulin (Ig) increase gradually during ageing. Through B cell development, the Ig heavy chain expression is modulated by a regulatory region at the 3’ of the constant alpha gene (3’RR), in single copy in rodents and, due to a large duplication, in two copies in apes. The human 3’RR1 and 3’RR2 are both characterized by three enhancers, the central of which, namely hs1.2, is highly polymorphic. Human hs1.2 has four different variants with unique binding sites for transcription factors (e.g. NF-kB and SP1) and shows variable allelic frequencies in populations with immune disorders. In previous works, we have reported that in several autoimmune diseases the *2 allele of hs1.2 is genetically associated to high level of IgM in peripheral blood. In subjects with altered levels of circulating Ig, an increased level was associated to *2 allele of hs1.2 and low levels corresponded to high frequency of *1 allele.During ageing there is a physiological increase of Ig concentrations in the serum. Therefore, for this study, we hypothesized that the hs1.2 variants may impact differently the levels of secreted Ig during the growth.ResultsWe have correlated the allelic frequencies of hs1.2 with IgM, IgG and IgA serum concentrations in two cohorts of healthy people of different age and after three years follow-up in children homozygous for the allele. Here we show that when the expression levels of Ig in children are low and medium, the frequencies of *1 and *2 alleles are the same. Instead, when the Ig expression levels are high, there is a significantly higher frequency of the allele *2. The follow-up of children homozygous for *1 and *2 alleles showed that the increase or decrease of circulating Ig was not dependent on the number of circulating mature B cells.ConclusionsThese data support the idea that under physiologic condition there is a switch of regulative pathways involved in the maturation of Ig during ageing. This mechanism is evidenced by hs1.2 variants that in children but not in adults participate to Ig production, coordinating the three class levels.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0045-0) contains supplementary material, which is available to authorized users.

Transcriptional regulation by 2,3,7,8-tetrachlorodibenzo-ρ-dioxin within the human polymorphic hs1,2 enhancer (42.7)

Abstract The environmental contaminant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) inhibits Ig expression and secretion. Within the IgH gene, the 3’IgH regulatory region (3’IgHRR) has been identified as a transcriptional target of TCDD. TCDD inhibits mouse 3’IgHRR and induces aryl hydrocarbon receptor (AhR) binding to dioxin response elements (DREs) within the hs1,2 and hs4 enhancers. The human hs1,2 enhancer is polymorphic due to the presence of one to four invariant sequences (IS) which have been correlated with several autoimmune disorders. Interestingly, TCDD inhibits the transcriptional activity of the mouse hs1,2 enhancer, while the human polymorphic hs1,2 enhancer activity is increased. Several transcription factor binding sites are located within both the mouse and human hs1,2 enhancers, while others are only in the mouse or human. Thus, we hypothesize that the specific transcription factor within the mouse and the human enhancers lead to differential effects by TCDD. Our data show that insertion of Pax5, deletion of the whole IS, or mutation of the AP-1/Ets site outside the IS decreases overall transcriptional activity. However, mutation of the transcription factor binding sites within the IS or the Oct site outside the IS increase the transcriptional activity. These results underscore the complexity of the transcriptional regulation of the hs1,2 enhancer and suggest an interaction between multiple transcription factors, many of which have been shown to be modulated by TCDD.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Induces Transcriptional Activity of the Human Polymorphic hs1,2 Enhancer of the 3′Igh Regulatory Region

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant known to inhibit Ab secretion and Ig expression. Inhibition of Ig expression may be partially mediated through repression of the 3'Igh regulatory region (3'IghRR). TCDD inhibits mouse 3'IghRR activation and induces aryl hydrocarbon receptor binding to dioxin response elements within the 3'IghRR enhancers hs1,2 and hs4. The human hs1,2 enhancer (hu-hs1,2) is polymorphic as the result of the presence of one to four invariant sequences (ISs), which have been correlated with several autoimmune diseases. The IS also contains a dioxin response element core motif. Therefore, the objective was to determine whether hu-hs1,2 activity is sensitive to TCDD. Using a mouse B cell line (CH12.LX), we compared the effects of TCDD on mouse hs1,2 versus hu-hs1,2 activity. TCDD inhibited mouse hs1,2 similarly to the mouse 3'IghRR. In contrast, hu-hs1,2 was activated by TCDD, and antagonist studies supported an aryl hydrocarbon receptor-dependent activation, which was replicated in a human B cell line (IM-9). Absence of Pax5 binding sites is a major difference between the human and mouse hs1,2 sequence. Insertion of the high-affinity Pax5 site in hu-hs1,2 markedly blunted reporter activity but did not alter TCDD's effect (i.e., no shift from activation to inhibition). Additionally, deletional analysis demonstrated a significant IS contribution to hu-hs1,2 basal activity, but TCDD-induced activity was not strictly IS number dependent. Taken together, our results suggest that hu-hs1,2 is a significant target of TCDD and support species differences in hs1,2 regulation. Therefore, sensitivity of hu-hs1,2 to chemical-induced modulation may influence the occurrence and/or severity of human diseases associated with hu-hs1,2.

Modulation of the human polymorphic hs1,2 enhancer within the 3′IgH regulatory region by TCDD

2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) is a potent environmental toxin known to inhibit immunoglobulin (Ig) gene expression. Transcriptional regulation of the Ig heavy chain (IgH) involves the 3′IgH regulatory region (3′IgHRR) and its enhancers (hs3, hs1,2, and hs4) which contain DNA binding sites for several transcription factors including NF‐κB and dioxin response elements (DRE). TCDD induces binding of the aryl hydrocarbon receptor complex to DREs within the 3′IgHRR and inhibits murine 3′IgHRR activation in a well characterized mouse B‐cell line (CH12.LX). In humans, a polymorphism of the hs1,2 enhancer resulting in varying numbers of a 53 bp sequence has been correlated with autoimmune diseases like IgA nephropathy and Celiac disease. The repeated sequence contains κB and DRE binding sites. The objective of this study was to comparatively evaluate the effect of TCDD and LPS on human and mouse hs1,2 enhancers in the CH12.LX model. In transient luciferase studies, an increased number of repeats in the human hs1,2 enhancer increased the sensitivity to LPS. Interestingly, TCDD also markedly enhanced human hs1,2 activity and even augmented LPS‐induced activation. This starkly contrasted with TCDD‐induced inhibition of the mouse hs1,2 and 3′IgHRR in LPS‐stimulated CH12.LX cells suggesting a species difference in IgH transcriptional regulation.(Supported by NIEHS R01ES014676)

Immunoglobulin Enhancer HS1,2 polymorphism: a new powerful anthropogenetic marker

The human HS1,2 enhancer of the immunoglobulin (Ig) heavy chain 3' enhancer complex plays a central role in the regulation of Ig maturation and production. Four common alleles HS1,2-A*1, *2, *3, *4 are directly implicated with the transcription level and at least one of them, HS1, 2-A*2, seems to be related to immune disorders, such as coeliac disease, herpetiform dermatitis and Berger syndrome. Given their clinical significance it is of interest to know the distribution of HS1,2-A variants in populations from different continents, as well as to determine whether the polymorphism is associated to specific evolutionary factors. In this paper we report the distribution of the HS1,2-A polymorphism in 1098 individuals from various African, Asian and European populations. HS1,2-A*3 and HS1,2-A*4 alleles are at their highest frequencies among Africans, and HS1,2-A*2 is significantly lower in Africans in comparison with both Europeans and, to a lesser extent, Asians. Analysis of molecular variance of the allele frequencies indicates that the HS1,2-A polymorphism can be considered as a reliable anthropogenetic marker.

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

The enhancer complex regulatory region at the 3′ of the immunoglobulin heavy cluster (IgH3′EC) is duplicated in apes along with four constant genes and the region is highly conserved throughout humans. Both human IgH3′ECs 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 IgH3′ECs 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 IgH3′ECs 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 IgH3′EC-1, 3′ to the Cα-1 gene) and in enhancer HS1,2-B (which lies in IgH3′EC-2, 3′ to Cα-2). Polymorphisms are due to the repetition (occurring one to four times) of a 38-bp sequence present at the 3′ 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-κB, CMYB, BSAP1/2, AP1/4, E47, MyoD and μE5 and thus to the possible influence of these variations on switch, production of Ig and on maturation of B cells.

The HoxC4 Homeodomain Protein Mediates Activation of the Immunoglobulin Heavy Chain 3′ hs1,2 Enhancer in Human B Cells: RELEVANCE TO CLASS SWITCH DNA RECOMBINATION

The immunoglobulin heavy chain (IgH) 3' regulatory region modulates IgH locus transcription, upon induction by specific trans-acting factors, and plays a significant role in class switch DNA recombination (CSR) and, perhaps, somatic hypermutation (SHM). CSR and SHM are central to the maturation of the antibody response. In contrast to the single 5'-hs3a-hs1,2-hs3b-hs4-3 ' mouse IgH 3 ' regulatory region, the human IgH 3 ' regulatory region exists as a 5'-hs3-hs1,2-hs4-3' cluster duplicated 3 ' of Calpha1 and Calpha2. We show here that the human hs1,2 element is the strongest enhancer of transcription, as directed by a V(H)1 or the ECS-Igamma3 promoter, thereby suggesting a dominant role for hs1,2 over hs3 and hs4 in the overall activity of the 3 ' regulatory region. Within hs1,2, we identified three regions (1, 2, and 3) that are all necessary, but individually not sufficient, for enhancement of transcription. In region 2, a HoxC4 site and a HoxC4/embedded octamer (HoxC4/Oct) site are conserved across human, mouse, rat, and rabbit. These two sites recruit HoxC4 and Oct-1/Oct-2, which act synergistically with the Oca-B coactivator to effect the full hs1,2-enhancing activity. HoxC4, Oct-1/Oct-2, and Oca-B recruitment is negligible in pro-B cells, moderate in pre-B cells, and maximal in germinal center B cells and plasma cells, where HoxC4, Oct-2, and Oca-B expression correlates with hs1,2 activation and ongoing CSR. The hs1,2mediated enhancement of V(H) and C(H) promoter-driven transcription as induced by HoxC4 and Oct-1/Oct-2 suggests an important role of these homeodomain proteins in the overall regulation of the IgH locus expression.

The human HCLS1 gene maps to chromosome 3q13 by fluorescence in situ hybridization.

The human HS1 gene (HCLS1, hematopoietic cell-specific Lyn substrate 1) expressed in human hematopoietic cells encodes a major substrate of protein-tyrosine kinase, p75HS1. This intracellular protein is involved in the signal transduction pathways that initiate at the antigen receptors of both B and T lymphocytes. Fluorescence in situ hybridization using a 2.0-kb cDNA and an 8.0-kb genomic DNA clone of HCLS1 as probes revealed that the gene maps to 3q13.

Restoration of surface IgM-mediated apoptosis in an anti-IgM-resistant variant of WEHI-231 lymphoma cells by HS1, a protein-tyrosine kinase substrate.

The HS1 protein is one of the major substrates of non-receptor-type protein-tyrosine kinases and is phosphorylated immediately after crosslinking of the surface IgM on B cells. The mouse B-lymphoma cell line WEHI-231 is known to undergo apoptosis upon crosslinking of surface IgM by anti-IgM antibodies. Variants of WEHI-231 that were resistant to anti-IgM-induced apoptosis expressed dramatically reduced levels of HS1 protein. Expression of the human HS1 protein from an expression vector introduced into one of the variant cell lines restored the sensitivity of the cells to apoptosis induced by surface IgM crosslinking. These results suggest that HS1 protein plays a crucial role in the B-cell antigen receptor-mediated signal transduction pathway that leads to apoptosis.

Molecular Cloning and Characterization of Mouse HS1

Here we report the cloning of a cDNA and a genomic DNA encoding the mouse homolog of human HS1, a hematopoietic cell-specific protein-substrate of nonreceptor type protein-tyrosine kinase(s). Sequence analysis of the mouse HS1 cDNA revealed that it is highly homologous to human HS1 (total percent match = 84%) especially in the amino-terminal half, which contains unique repeating motifs, and in the carboxyl-terminal Src-homology 3 domain. As is the human counterpart, the mouse HS1 gene is expressed exclusively in hematopoietic cells. Genomic fragments covering most of the HS1 gene were isolated and used to map this gene to mouse chromosome 16.

Identification of a protein, SPY75, with repetitive helix-turn-helix motifs and an SH3 domain as a major substrate for protein tyrosine kinase(s) activated by Fc epsilon RI cross-linking.

Cross-linking of the high affinity receptor for IgE (Fc epsilon RI) initiates various biochemical and morphologic changes leading to degranulation and synthesis and release of cytokines and lipid mediators. Tyrosine phosphorylation of several cellular proteins was previously reported as the earliest signaling event for the Fc epsilon RI signal transduction pathway. By amino acid sequence determination and cDNA cloning analysis, a 75-kDa protein, termed SPY75, was identified as a major tyrosine-phosphorylated protein in activated mouse mast cells. SPY75, barely tyrosine phosphorylated in resting cells, was rapidly and transiently tyrosine phosphorylated on Fc epsilon RI cross-linking in an Ag concentration-dependent manner. Similar SPY75 tyrosine phosphorylation was observed when Ag receptors on B and T lymphocytes were cross-linked by appropriate antibodies. However, IL-3, granulocyte macrophage-CSF, or stem cell factor did not induce tyrosine phosphorylation of SPY75 in PT-18 mast cells, despite their responsiveness to these cytokines. SPY75 was not physically associated with the receptor or other known signaling molecules. This protein, the mouse homologue of the human HS1 gene product, has putative repetitive helix-turn-helix motifs found in many DNA-binding proteins and a putative nuclear transport signal. It also has a Src homology 3 domain, which is found in many signaling molecules and cytoskeletal proteins. These structural features and the rapid tyrosine phosphorylation on Fc epsilon RI cross-linking suggest that the signal generated by Fc epsilon RI cross-linking is transmitted through tyrosine phosphorylation of SPY75.

Identification of HS1 protein as a major substrate of protein-tyrosine kinase(s) upon B-cell antigen receptor-mediated signaling.

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins and rapid activation of Src-like kinases. The amino acid sequences of five proteolytic peptides of p75, a major substrate of protein-tyrosine(s) in the signaling, showed that p75 is the human HS1 gene product. The HS1 gene is expressed specifically in hematopoietic cells and encodes p75HS1, which carries both helix-turn-helix and Src homology 3 motifs. p75HS1 showed rapid tyrosine phosphorylation and association with a Src-like kinase, Lyn, after crosslinking of membrane-bound IgM. Thus, p75HS1 may be an important substrate of Lyn and possibly other protein-tyrosine kinases upon B-cell antigen receptor-mediated signaling.