Enhancer complexes located downstream of both human immunoglobulin Calpha genes

Abstract

To investigate regulation of human immunoglobulin heavy chain expression, we have cloned DNA downstream from the two human Calpha genes, corresponding to the position in the mouse IgH cluster of a locus control region (LCR) that includes an enhancer which regulates isotype switching. Within 25 kb downstream of both the human immunoglobulin Calpha1 and Calpha2 genes we identified several segments of DNA which display B lymphoid-specific DNase I hypersensitivity as well as enhancer activity in transient transfections. The corresponding sequences downstream from each of the two human Calpha genes are nearly identical to each other. These enhancers are also homologous to three regions which lie in similar positions downstream from the murine Calpha gene and form the murine LCR. The strongest enhancers in both mouse and human have been designated HS12. Within a 135-bp core homology region, the human HS12 enhancers are approximately 90% identical to the murine homolog and include several motifs previously demonstrated to be important for function of the murine enhancer; additional segments of high sequence conservation suggest the possibility of previously unrecognized functional motifs. On the other hand, certain functional elements in the murine enhancer, including a B cell-specific activator protein site, do not appear to be conserved in human HS12. The human homologs of the murine enhancers designated HS3 and HS4 show lower overall sequence conservation, but for at least two of the functional motifs in the murine HS4 (a kappaB site and an octamer motif ) the human HS4 homologs are exactly conserved. An additional hypersensitivity site between human HS3 and HS12 in each human locus displays no enhancer activity on its own, but includes a region of high sequence conservation with mouse, suggesting the possibility of another novel functional element.

Figure 1

Comparison of IgsH loci of mouse and human. Line A shows a map of the murine IgH locus, from which the region downstream from Cα is expanded in line B. The murine enhancers designated Cα3′E (11) and 3′αE (9) are shown as vertical ovals, along with the DNase I hypersensitivity site designations (12). We have distinguished the two copies of HS3 sequence as HS3A and HS3B; these are included in a large palindrome (arrows) that flanks HS12, according to the sequence analysis of Chauveau and Cogné (13). Line C shows the human IgH locus, illustrating the γ-γ-ε-α duplication units (brackets) and the possibility of two regions homologous to the murine LCR.

Figure 2

Regulatory loci downstream of human Cα1 and Cα2. Lines A and C, based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion (dashed box). Vertical ovals mark DNase I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown (oval). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C). The thick black lines under the maps of lines A and C (single lower case letters) represent hybridization probes used in this study.

Figure 3

Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. (A) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon (αm). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C, and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. (B) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. (C) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands <12 kb must result from DNase I cutting in the 3′ α2 region, with the size of these bands corresponding to their distance from the 3′ end of the 12-kb α2 HindIII fragment. This analysis allows assignment of three DNase I sites to the α2 locus, thus making it possible to assign the other hypersensitive sites seen in the BglII analysis of Fig. 2 A to the α1 locus.

Figure 3

Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. (A) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon (αm). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C, and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. (B) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. (C) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands <12 kb must result from DNase I cutting in the 3′ α2 region, with the size of these bands corresponding to their distance from the 3′ end of the 12-kb α2 HindIII fragment. This analysis allows assignment of three DNase I sites to the α2 locus, thus making it possible to assign the other hypersensitive sites seen in the BglII analysis of Fig. 2 A to the α1 locus.

Figure 3

Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. (A) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon (αm). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C, and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. (B) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. (C) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands <12 kb must result from DNase I cutting in the 3′ α2 region, with the size of these bands corresponding to their distance from the 3′ end of the 12-kb α2 HindIII fragment. This analysis allows assignment of three DNase I sites to the α2 locus, thus making it possible to assign the other hypersensitive sites seen in the BglII analysis of Fig. 2 A to the α1 locus.

Figure 4

Enhancer activity of selected regions downstream of human Cα1 and Cα2 genes. (A) Analysis of the locus downstream of α2, which was studied in detail. The map shows the position of DNase I sites; below this are diagrammed the restriction sites defining the boundaries of each fragment tested for enhancer activity by insertion into pGL3-Vκ, transfection into the human myeloma HS Sultan, and assay of resulting luciferase activity, as described in the text. The enhancer activities are given for constructs in the A orientation (the same orientation with respect to transcribed strands of immunoglobulin and luciferase) or the opposite B orientation, where examined. The luciferase activities were normalized to β-galactosidase activity encoded by a cotransfected plasmid, and expressed as fold-increase over the activity of an enhancerless control plasmid. For fragments showing enhancer activity, assays were performed at least in triplicate, and standard deviations are given. (B) Comparable analysis of selected fragments amplified from the homologous locus downstream from Cα1.

Figure 5

Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference (EMBL/GenBank/DDBJ accession numbers X96607 and X96608), while numbering for mouse HS4 is according to reference (EMBL/DDBJ/GenBank accession number S74166). (A) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. (B) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. (C) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. (D) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE (41, 42). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

Figure 5

Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference (EMBL/GenBank/DDBJ accession numbers X96607 and X96608), while numbering for mouse HS4 is according to reference (EMBL/DDBJ/GenBank accession number S74166). (A) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. (B) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. (C) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. (D) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE (41, 42). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

Figure 5

Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference (EMBL/GenBank/DDBJ accession numbers X96607 and X96608), while numbering for mouse HS4 is according to reference (EMBL/DDBJ/GenBank accession number S74166). (A) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. (B) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. (C) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. (D) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE (41, 42). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

Figure 5

Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference (EMBL/GenBank/DDBJ accession numbers X96607 and X96608), while numbering for mouse HS4 is according to reference (EMBL/DDBJ/GenBank accession number S74166). (A) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. (B) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. (C) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. (D) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE (41, 42). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).