| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Biological Sciences, Hunter College of the City University of New York, New York, NY 10021; and
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, NY 10021
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Eµ was the first transcriptional enhancer discovered in the IgH
locus, lying in the intron separating the JH gene
segments and Cµ (1, 2, 3) (see Fig. 1
).
Originally identified by virtue of its effects on the transcription of
cloned genes introduced into B-lineage cells, Eµ has since been shown
to play an important role in VH gene assembly as well (4, 5). Its importance later in B cell development was brought into
question, however, by the discovery that a number of Ig-secreting cell
lines lacking Eµ continued to produce IgH chains at high levels
(6, 7, 8, 9, 10).
|
E (11, 12). 3'E, recently renamed hs1,2, maps
approximately 16 kb downstream of murine BALB/c C exons, and lies
approximately 200 kb 3' of the JH gene segments where
VH gene assembly takes place. We found that it lay 
70 kb
downstream of the highly transcribed IgH gene of one of the
Eµ-deficient cell lines. We subsequently deleted hs1,2 from the IgH
chromosome of this cell line (9921), replacing it with a
neor marker gene (13). The result was loss of
heavy chain gene expression. In a similar neo replacement
experiment performed in mice, it was found that loss of hs1,2 resulted
in aberrant heavy chain class switch recombination on the affected IgH
chromosome (14). These two studies suggested that, like Eµ, hs1,2
served a role both in gene transcription and in DNA recombination.
Because both experiments involved insertion of a foreign transcription
unit (neor) at the site of hs1,2
deletion, however, it was not possible to conclusively attribute both
these activities to hs1,2 alone. It was possible that the
neor transcription unit itself contributed to a
more generalized disruption of a larger control region. Reporter genes
inserted into the locus control region of the ß-like globin genes
have been shown to have this effect (15, 16). Significantly, it was
soon discovered that hs1,2 was surrounded by additional enhancers
within the 3' region of the IgH locus, suggesting that it might not
function alone.
Hs3A (formerly C3'E) maps immediately 3' of C and upstream of
hs1,2 (17) (see map in Fig. 1
). Hs3B, closely related in sequence to
hs3A, lies 13 kb downstream of hs1,2, and hs4 lies another 4 kb farther
3' (18, 19, 20). It has been noted that the 25-kb region from hs3A to
hs3B might be considered a large, inverted repeat (18, 21). Each of the
3' IgH enhancers has been tested for its effect on the transcription of
reporter genes transiently transfected into one or more cell lines. Hs4
was the sole enhancer with detectable activity in pre-B cell lines (19, 20). Hs1,2 and hs4 were tested for function in surface Ig+
cell lines where hs4 proved moderately active. There was some
controversy with regard to hs1,2 function in surface Ig+
cell lines (20, 22, 23). All of the 3' IgH enhancers (hs1,2, hs3A,
hs3B, and hs4) proved functional in Ig-secreting plasmacytomas (11, 12, 17, 18, 19, 23, 24). Hs1,2 was also assayed in cis to a reporter
gene in transgenic mice, where it appeared to function only after B
cell stimulation (25, 26, 27).
A combination of some of the IgH 3' enhancers was analyzed a few years
ago in the context of both a c-myc and an Ig
(light
chain) promoter (19). Many of the IgH/c-myc chromosome
translocations characteristic of human Burkitt’s lymphoma and of
murine and rat plasmacytoma result in juxtaposition of the 3' end of
the IgH locus with the 5' end of the c-myc locus. It has
been postulated, therefore, that the 3' IgH enhancers serve to
deregulate expression of the translocated copy of c-myc.
Consistent with this hypothesis, Madisen and Groudine (19) found that
the combination of hs1,2,3B,4 yielded a unit that could drive
position-independent, copy number-dependent expression of a reporter
gene in a stably transfected plasmacytoma (Ig-secreting) cell line.
Moreover, this combination of enhancers led to a shift in the
transcription start site within the reporter gene’s c-myc
promoter to that commonly used in a deregulated, translocated
c-myc gene.
In the present study we have broadened these analyses to ask how all of the presently identified 3' enhancers and the intronic enhancer Eµ function in combination over the course of B cell development and in the context of an IgH promoter. We have found that the 3' region enhancers synergize to yield a high level of activity at the surface Ig-positive stage of B cell development, a time when individual enhancers have little or no activity on their own. This synergy is much less pronounced at the terminally differentiated plasma cell stage, when individual enhancers take on greater activity. Our studies also show that while Eµ is active through all stages of B cells analyzed, its relative contribution toward the expression of a transfected reporter gene is low in surface Ig+ cells.
These data document clear changes in the transcriptional control of IgH gene expression as B cells mature. It can be expected, therefore, that the array of transcription factors essential to Ig gene expression in early stage B cells will be distinct from that required for Ig gene expression in late stage or Ig-secreting cells. Several transcription factors that are essential to early Ig gene expression and that are required, therefore, for the generation of B cells have recently been identified through gene knockout studies (28, 29, 30, 31). Our data suggest developmental stages at which we might expect additional sets of transcription factors to take on essential functions at the IgH locus.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ig-secreting cell lines, representative of plasmacytes. S194 plasmacytoma (IgA-secreting plasmacytoma derived from BALB/c) was a gift from Dr. B. K. Birshtein, Albert Einstein College of Medicine (Bronx, NY). P3X63Ag8 (IgG1-secreting plasmacytoma derived from BALB/c; TIB 9) was obtained from American Type Culture Collection (Rockville, MD).
Abelson virus-transformed pre-B cells.
18-8 (32) is an Abelson virus-transformed mouse pre-B cell line that is
capable of switching from µ to 
2b production in culture; it was a
gift from Dr. H. Radomska, Beth Israel Hospital, Harvard Medical School
(Boston, MA). 18-81 (32) is a clonal derivative of the cell line 18-8;
it was a gift from Dr. B. K. Birshtein.
Murine surface Ig+ B cells. A20 (33) is an IgG+ cell line derived from BALB/c, TIB-208, and was obtained from American Type Culture Collection. M12.4.1 (34) is a BALB/c-derived, IgG2a+, hgprt- variant of M12.4 and was a gift from Dr. B. K. Birshtein.
Human surface Ig+ B cells. Namalwa (35) (CRL-1432) and Raji (36) (CCL-86) cells were obtained from American Type Culture Collection.
Rat-1 fibroblasts. Rat-1 fibroblasts (37) were obtained from American Type Culture Collection (CRL-1764).
Most of the cell lines were maintained in RPMI 1640 medium (Life Technologies, Gaithersburg, MD; catalogue no. 31800-089), 20% bovine calf serum (BCS) (HyClone Laboratories, Logan, UT; catalogue no. SH30072.03), 1% penicillin-streptomycin (Life Technologies; catalogue no. 15140-122), 2 mM L-glutamine (Life Technologies; catalogue no. 21051-016), and 50 µM ß-ME (Sigma, St. Louis, MO; catalogue no. M-7522). The exceptions were P3X63Ag8, Rat-1, Namalwa, and Raji cells, which were maintained in DMEM (Life Technologies; catalogue no. 12100-061) and 15% BCS. All cells were maintained at about 37°C in an atmosphere of 7 to 8% CO2.
Vector constructions
A schematic representation of each construct is shown in Figure 3. The VH promoter fragment (140 bp) used in all the
constructs was obtained as a PCR product from a cloned plasmacytoma
2b gene (from the MPC11 plasmacytoma) (38). It extends from -129 bp
to +6 bp with respect to the cap site of the heavy chain transcript
(nucleotides 21–162, GenBank accession no. M17056; two cap sites at
nucleotides 153 and 156). This promoter includes the octamer sequence
shared by all Ig promoters (nucleotides 99–106, GenBank accession no.
M17056) as well as an upstream heptamer (nucleotides 91–97). The
140-bp KpnI (an authentic site)/BglII (introduced
site) fragment was subsequently ligated with
KpnI/BglII-digested vector (luciferase expression
vector, pGL2 Basic, Promega, Madison, WI), generating the
"backbone" construct pGL2BV.
|
1.3 kb downstream of
luciferase-encoding sequences). The following enhancer regions were
inserted into pGL2BV to generate reporter constructs 2
to 7. Eµ. A 1-kb XbaI fragment (38) spanning Eµ was engineered to have BamHI/SalI ends and was ligated to BamHI/SalI-digested pGL2BV.
Hs1,2.
A 4-kb XbaI fragment containing hs1,2 was engineered to have
BamHI/SalI ends and was ligated to
BamHI/SalI-digested pGL2BV. The 4-kb
XbaI fragment was isolated from phage clone M2, which
contains 18 kb BALB/c genomic DNA mapping from within C and
downstream (11). Hs1,2 covers an 1.3-kb region beginning 700 bp
from the 5' end (with respect to IgH transcription) of the 4-kb
XbaI fragment. The nucleotide sequence of an homologous
region in the 129 mouse strain can be found in GenBank (accession no.
X96607;21). Deletion of an overlapping region (5.1 kb) in the
Eµ-deficient cell line 9921 resulted in loss of 2a gene
transcription (13).
Hs3A.
A 1-kb Xba fragment containing hs3A was subcloned from M2 (11)
(see description above) and engineered to have
BamHI/SalI ends (the DNA sequence of this
fragment can be found in GenBank, accession no. U65625) (18). The
resulting fragment was cloned into
BamHI/SalI-digested pGL2BV.
Hs4,3B. A 2.5-kb BssHII fragment containing a fusion of hs3B and hs4 was isolated from the plasmid HS3.4 (19) (HS3.4 was provided by Dr. M. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA; sequence of hs3B, GenBank accession no. S74164). The pGL2BV was digested with SalI; the ends were made flush with Klenow and then ligated with the 2.5-kb hs3B,4 fragment.
Hs3B. The pGL2BV(hs3B) was directly derived from pGL2BV(hs4,3B). The latter plasmid was digested with SpeI and XhoI to discard a 1.4-kb hs4-containing fragment. The ends of the resulting vector were made flush with Klenow, and the vector was self-ligated to yield pGL2BVhs3B. The SpeI and XhoI sites lay within the hs4,3B fragment, so that hs3B remains within the SalI site of pGL2BV.
Hs4. pGL2BV(hs4,3B) was digested with SacI to remove a 1.2-kb hs3B-containing fragment. Again, SacI sites mapped within the hs4,3B fragment. The resulting vector was gel-isolated and then self-ligated to form pGL2BVhs4.
Constructs 8 to 10 were generated as follows.
PGL2BV(hs4,3B,3A,1,2,Eµ): construct 10 (Fig. 3).
The Xba fragments containing hs1,2 and hs3A were first joined by
ligation into XbaI-digested pBS SK+ (catalogue
no. 21120, Stratagene, La Jolla, CA) and then removed by digestion of
the resulting plasmid, pBS(hs3A,1,2), with
BamHI/SalI. The latter fragment was ligated with
BamHI/SalI-digested pGL2BV, resulting
in vector pGL2BV(hs3A,1,2). The 1-kb Eµ fragment was
engineered to carry SalI ends and was then inserted into
SalI-digested pGL2BV(hs3A,1,2). The result was
pGL2BV(hs3A,1,2,Eµ). The ends of the 2.5-kb hs4,3B
fragment were first made flush with Klenow and then inserted into a
BamHI site (also made flush with Klenow) within
pGL2BV(hs3A,1,2,Eµ). The result is an enhancer construct
in which all IgH enhancers lie between the
BamHI/SalI sites of pGL2BV in the
order indicated in Figure 3.
PGL2BV(hs1,2,4,3B): construct 8 (Fig. 3).
The ends of the 4-kb (hs1,2) fragment were made flush with Klenow and
then ligated to XhoI-digested pGL2BV(hs4,3B)
(XhoI ends also made flush with Klenow). The
XhoI site lies at one end of the hs4,3B fragment in
pGL2BV(hs4,3B). The enhancers in
pGL2BV(hs1,2,4,3B), therefore, are positioned in the order
indicated and within the SalI site of the pGL2BV
vector.
pGL2BV(hs3A,1,2,3B,4): construct 9 (Fig. 3).
An intermediate construct, pGL2BV(hs3A,1,2,3A), was
made first (see above). It was then cut with SalI, the ends
were made flush with Klenow, and it was ligated with a flush-ended
2.5-kb BssHII fragment containing hs3B and hs4. All
enhancers in this construct lie between
BamHI/SalI sites of pGL2BV.
Transient transfections
All cells were grown to log phase and then transfected with DNA either in the presence of DEAE-dextran as described in the product information from Promega (catalogue no. E1210; 18-81, 18-8, M12.4.1, A20, Namalwa, and Raji cells), by a DEAE-dextran supplemented electroporation method (39) (S194 and P3X63Ag8 cells), or by a calcium phosphate method (40) (Rat-1 cells). In electroporation experiments, 4 x 106 cells were used per transfection, and 125 ng of ß-galatosidase expression vector was added to each transfection tube as a control for transfection efficiency. The molar ratio of the control plasmid to test plasmid (both supercoiled) was, in all cases, 1:7, and the total amount of DNA transfected per cell line was also kept constant (3 µg/transfection) through addition of carrier DNA when needed (BamHI-linearized pBS, Stratagene). DNAs were added first to a 0.4-cm (width) electroporation cuvette (Bio-Rad, Hercules, CA), followed by 10 µg of DEAE-dextran (Promega, catalogue no. E1210) and then by 4 x 106 cells. An electric pulse was delivered at 960 µF and 300 V (S194) or 280 V (P3x63Ag8) by a Bio-Rad Gene Pulser electroporator and Capacitance Extender (Bio-Rad). The cells were then placed in a 37°C incubator with 7 to 8% CO2. Transfections were harvested after 18 to 24 h.
For the DEAE-dextran transfections, the DNAs were first diluted to a final volume of 326 µl with PBS. The molar ratio of control plasmid (ß-galactosidase) to test plasmid was again 1:7, but 1 µg of ß-galactosidase plasmid was included in each transfection, and the total amount of DNA used per transfection was kept constant at 6 µg. Seventeen microliters of 10 mg/ml DEAE-dextran were added to the DNA, and the samples were immediately vortexed. Cells (4 x 106) were pelleted, resuspended in the DEAE-dextran/DNA mixture, and then placed in an incubator (37°C) for 30 min. After the 30-min incubation, the cells were removed and placed in 3.5 ml chloroquine-supplemented (0.08 mM) medium and incubated for an additional 2 h at 37°C. The cells were then pelleted, resuspended in 5 ml of serum-containing medium, and returned to the incubator for 48 h before harvesting.
Luciferase assay
At harvest, all cells were collected, washed twice with PBS, and transferred to 1.5-ml microfuge tubes. Cell pellets were then lysed using 200 µl of the 1x reporter lysis reagent (luciferase assay kit, catalogue no. E1501, Promega). Luciferase assay reagent (50 µl; Promega) were added to 10 µl of the lysate, and luminescence was measured for 1 min in a scintillation counter (LS6000, Beckman, Palo Alto, CA) or for 30 s (B cells) or 10 s (non-B cells) in a luminometer (1LA-911-optocomp1, Tropix, Bedford, MA). Duplicate samples were counted, and the average was taken for statistical analyses.
Luciferase values were normalized for transfection efficiency by
dividing each by the amount of activity obtained from the
ß-galactosidase reporter gene control. The fold enhancement (±SD)
for each enhancer or group of enhancers was determined by dividing the
normalized luciferase values for each test construct by the luciferase
activity generated by the enhancerless (IgH promoter only) construct.
At least six independent transfections, using at least two different
plasmid preparations, were performed with each construct. In some
cases, a p value is given for the significance of
differences in activity among enhancer constructs in the same cell line
or among cell lines with the same enhancer construct. The p
value was obtained by Student’s t test. p
0.05 or less was regarded as evidence that the datasets had
significantly different means.
ß-Galactosidase assay
ß-Galatosidase assays were performed using the ß-galatosidase assay kit from Promega (catalogue no. E2000). Briefly, 150 µl of the ß-galatosidase reagent was added to 150 µl of the cell lysates, and the mixtures were incubated at 37°C for 30 min. Five hundred microliters of 1 M Na2CO3 were added to stop the reaction, and the mixture was then transferred to a disposable cuvette (Bio-Rad, catalogue no. 223-9955). Absorbance at 420 nm was measured with a spectrophotometer (DU series 600, Beckman).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
.
PGL2BV consists of a luciferase reporter gene driven by a
VH promoter. While many studies of the IgH and IgL
enhancers have used heterologous promoters (17, 19, 22, 24), it is
evident from our work and that of others that there are significant
differences in the way that particular promoters respond to the same
enhancers (19, 41, 42). To more accurately assess enhancer activity in
the relevant context, therefore, we used a bona fide VH
promoter in our reporter constructs. This promoter includes the
consensus heptamer and octamer sequences characteristic of Ig promoters
and is the promoter driving 2b expression in the murine myeloma
MPC11. It is also the promoter driving 2a expression in the 9921
derivative of MPC11 that has lost Eµ as a consequence of a heavy
chain class switch (7, 10).
|
, all constructs
carried enhancers at a 3' position relative to luciferase-coding
sequences. While the actual spacing involved in vivo was not recreated,
positioning the enhancers 3' of the promoter again more closely
mimicked the situation in the endogenous IgH locus. The importance of
positioning has been directly demonstrated in experiments measuring the
activity of the consensus octamer (found in all Ig promoters and in all
of the IgH enhancers identified to date). Transcription factor
requirements for octamer activity vary considerably, depending upon the
position of the octamer with respect to the promoter (43, 44, 45, 46). In
practice, octamer-containing enhancers such as Eµ lead to
considerably greater reporter gene activity when placed adjacent to a
promoter than when placed at a distance downstream (12). Activity of individual IgH enhancer elements at different stages of B cell development
Eµ and the individual 3' IgH enhancers were tested in cell lines
representative of pre-B cells, surface Ig+ B cells,
and Ig-secreting plasmacytes, respectively (constructs 1–6 in Fig. 3).
Transfections were performed in at least two distinct cell lines
representative of a given stage of development to control for cell
line-specific variations. The activity of each element is expressed as
enhancement above the enhancerless construct and the data are
summarized in Figures 4 to 7.
|
and
6, that
there was no appreciable difference in the relative activities of
individual enhancers in the murine and human lines, supporting the idea
that the 3' IgH enhancer elements are also functionally conserved. All
the enhancer constructs were also introduced into a non-B-lineage cell
line to demonstrate the tissue specificity of their activities. As
shown in Figure 8, the test constructs
showed little activity over background when assayed in the fibroblastic
cell line, Rat-1.
|
|
). The 3' enhancers, when assayed individually,
however, showed very weak activity throughout. Hs4 and hs1,2 were
somewhat more active in the Ig-secreting cells than in earlier stage B
cells (hs4, p < 0.001; hs1,2, p <
0.001; by Student’s t test; see Materials and
Methods). While hs4 had detectable activity over background
(enhancerless control) in the pre-B and surface Ig+ cell
lines, the fold enhancement was not statistically different from that
in a non-B-lineage cell line (pre-B vs rat, p = 0.34;
Figs. 4 and 8).
|
|
rearrangements, and some even undergo heavy chain class switching so
that the same cell line in different laboratories may be phenotypically
distinct (32, 50). The differences among the three studies could also
be due, however, to differences in reporter gene construction. As
described earlier, only in our studies was hs4 placed downstream of the
reporter gene. Synergistic interactions reveal 3' enhancer element activity in surface Ig+ B cells
In one of the pre-B cell lines tested (18-8), the 3' enhancers
were moderately active when assayed in combination with one another or
with Eµ (compare Eµ alone with hs4,3B,3A,1,2,Eµ construct; Fig. 4; p < 0.001). The overall activity of the 3'
enhancers in this cell line as measured in these experiments was
slight, however, compared with that in cells representing other stages
of B cell development (
Figs. 5–7).
In surface Ig+ cell lines, the effect of the combined 3'
IgH enhancers on reporter gene activity was dramatic (Figs. 5 and 6;
note the change in scale compared with Fig. 4). At this stage, there
was pronounced synergy among the 3' elements. While each of the 3'
elements was weak when assayed individually, the combination of even
two elements (hs3B and hs4) gave an activity much greater than the sum
of their individual activities. The functional synergy was even more
marked when all four 3' elements were combined in a single construct
(hs3A,1,2,3B,4; Figs. 5 and 6). The transcriptional enhancement
achieved was as high as 80-fold (Fig. 6) and in most cases surpassed
that achieved by Eµ (the exception was M12.4.1, combination of 3'
enhancers 
Eµ; p = 0.32). The contribution of
individual 3' enhancers to the overall synergy appeared to vary
somewhat from cell line to cell line (e.g., compare the effect of
adding hs3A to the rest of the 3' enhancers in A20 vs M12.4.1 cells)
and probably reflects some differences in the transcription factor
profile in these established cell lines. However, the overall pattern
of substantial and synergistic 3' enhancer activity was shared among
the cell lines.
At the plasma cell stage, synergy among the 3' enhancer elements was less evident. The combination of all 3' elements constituted a more effective enhancer than any of the 3' elements alone, but the effect was close to additive. In addition, Eµ was no longer surpassed in activity by a combination of the 3' elements. Rather, Eµ and the 3' elements appeared to contribute equally to reporter gene expression in the Ig-secreting cells. Again, there were cell line variations. The 3' enhancers were, overall, more powerful in S194 than in P3X63Ag8. The particularly low activity of hs4 in P3X63Ag8 may explain this difference and may reflect the loss of a transcription factor in this cell line that contributes substantially to hs4 function.
We assigned a numerical value to the level of functional synergy among
3' enhancer elements by comparing the activities of all 3' enhancers
combined with the sum of their individual activities (see Table I). A value of 1 means that the whole is
equal to the sum of the parts and signifies neither interference nor
synergy. Values above 1, therefore, signify varying degrees of synergy,
and values below 1 indicate varying degrees of interference. As shown
in Table I, synergy among the 3' elements was pronounced at the surface
Ig+ stage of B cell development (average = 10 ±
4), but not at earlier or later stages (surface Ig+ vs
pre-B or Ig secreting, p < 0.001). When Eµ was added
to the 3' enhancer elements, no substantial synergy between this
intronic enhancer and the 3' enhancers was revealed at any of the
developmental stages (Table I). This is consistent with an earlier
study of Eµ combined with hs1,2 in which no synergy was detected
(22). In Ig-secreting cells, there was even some evidence of
interference between Eµ and the 3' enhancers (functional synergy
values were <1.0 for S194 and P3X63Ag8; Table I), something also
reported by others when Eµ and hs1,2 were assayed together in cells
at this developmental stage (22, 51).
|
Since the IgH locus is subject to the effects of both Eµ and the
3' enhancers, it was of interest to examine how their relative
contributions to gene expression changed as a function of B cell
developmental stage. This can be expressed as the percent activity of
each (Eµ and the combined 3' enhancers; constructs 2 and 9,
respectively, in Fig. 3) compared with the activities of all enhancers
combined (construct 10). Table II
summarizes the data expressed in this fashion.
|
100%. The one
exception was P3X63Ag8, where Eµ alone had greater activity than all
enhancers combined so that the calculated contribution by Eµ was
180%.
As summarized in Table II, Eµ’s contribution was very high in pre-B
cells, low in surface Ig+ cells, and then became high
again in Ig-secreting cells. The contribution made by the 3' enhancers
was reciprocal to that of Eµ in pre-B and Ig+ cells: low
in pre-B cells and high in surface Ig+ cells. Like Eµ,
however, the 3' enhancers made a substantial contribution to reporter
gene activity at the Ig-secreting cell stage.
A careful analysis of the data presented in Figures 4 through 7 reveals
that the change in relative contributions by Eµ and the 3' elements
reflects alterations in 3' enhancer function and not in Eµ activity.
Among the cell lines examined, Eµ activity did not vary more than 2-
to 6-fold, and the variation showed no relationship to developmental
stage. Conversely, enhancement by the combination of 3' enhancers
showed dramatic changes, beginning near zero in the pre-B cells, rising
to 20- to 80-fold in surface Ig+ cells, and then dropping
to 3- to 8-fold at the plasma cell stage (significance of differences
among developmental stages, p < 0.001). While the
absolute level of 3' enhancer activity declined in the latter
Ig-secreting cells, the relative contributions made by these elements
to reporter gene activity remained high (50 and 60%; Table II).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
For these studies, we assayed the IgH locus enhancers in a downstream position from their usual promoter target, a VH promoter. We analyzed enhancer function in eight different B-lineage cell lines, representing three distinct stages in B cell development. Significantly, we found that the 3' enhancers, in combination, were active in cells where individual 3' enhancers showed no activity. In particular, there was marked synergy among the 3' enhancers at the surface Ig+ stage of B cell development, which led to an overall enhancement of transcription far beyond that achieved by any of the individual enhancers alone, including Eµ. When measured individually, only one of the 3' region enhancers, hs4, had measurable, but modest, activity at this stage.
The overall pattern of IgH enhancer activity can be summarized as follows. Eµ is the most powerful of the enhancers in pre-B cells. As gene knockout studies in mice have shown, it functions as a facilitator of gene rearrangement (V to DJ joining) as well as of transcription in these early B-lineage cells (4, 5).
In surface Ig+ cells, Eµ declines in importance, and the 3' elements begin to function, but only as an interdependent complex. Interestingly, in preliminary experiments, we have found that this synergy is not evident when a non-VH promoter is used, suggesting that protein binding motifs within the promoter and within each of the interacting enhancers contribute to this effect (unpublished results). Work is in progress to identify the relevant DNA sequences and transcription factors. Given the purported importance of the 3' region to the process of heavy chain class switching (14), it will also be of interest to determine whether this functional synergy is operational in the context of one or more of the intronic promoters driving germ-line transcription of CH genes before class switching.
Finally, in Ig-secreting cells, functional synergy among the 3'
enhancers has declined, so that their contribution, as a whole, is
slightly less than or equal to that of Eµ. Another group has
reported, in contrast, that the 3' enhancers are synergistic in
Ig-secreting cells. As cited earlier, this is the only other study in
which some of the 3' IgH enhancers were assayed in combination (19).
Interestingly, the Ig-secreting cell line used in that study, TEPC
1165, was an IL-6-dependent plasmacytoma. Transfections were performed,
therefore, in the presence of IL-6, and IL-6 has been shown to both
induce and up-regulate transcription factors that can bind Ig enhancer
elements (53, 54). It is possible that IL-6 induces one or more of the
transcription factors that contribute to enhancer synergy, mimicking
the synergy naturally evident in surface Ig+ cell lines
(Table I).
When Eµ and the 3' enhancers were combined in the Ig-secreting cells
(as in construct 10; Fig. 3), the resulting activity was equal to or
less than that of Eµ alone. This suggests a functional redundancy for
Eµ and the 3' enhancers, respectively. Only one enhancer region (Eµ
or the 3' enhancers) has to be active to maintain high level gene
activity, and in transient assays, one has a slightly repressive effect
on the other. This could be due to their dependence on a common
transcription factor(s) that is in limited supply. In any case, these
data are consistent with the isolation of several Ig-secreting
plasmacytomas that lack Eµ and yet continue to transcribe IgH genes
at high levels (6, 7, 8, 9, 10).
In one such plasmacytoma, 9921, we replaced hs1,2 with a
neor marker gene (by homologous recombination)
and found that all IgH transcription ceased (13). The data in Figure 7
would not predict such a dominant role for hs1,2 in the transcription
of an Eµ-deficient IgH gene. The combination of hs3B and hs4 is
almost equal to that of all the 3' enhancers together. The present data
are more consistent, therefore, with the idea that the hs1,2
deletion/neor insertion has disrupted the normal
interactions among hs3A, hs3B, and hs4 with the VH
promoter. This single enhancer deletion, therefore, has led to the
"technical" knockout of all the 3' region enhancers. This might
also be true of the hs1,2 deletion/neor
insertion experiment in mice (14). Experiments to test this model
within the 9921 cell line are in progress.
In summary, transcriptional control of the IgH locus changes over the course of B cell development. Not only do previously inactive enhancer regions become active, but, as shown here, active enhancers take on modified modes of action. A specific implication is that the multiple protein binding motifs of these enhancer regions are differentially occupied at different times. A few years ago, the first tissue-specific transcription factor shown to bind Ig promoters and IgH enhancers was identified (55). This factor, Oct-2, was long assumed to be critical to the expression of Ig genes, so that the finding that Ig-expressing B cells could develop in its absence was generally met with great surprise (56). We and others have suggested, however, that Oct-2 plays an essential role in some later aspects of B cell development and Ig expression (56, 57, 58). Other transcription factors with the ability to bind IgH enhancer sequences have proven essential to early stages of B cell development and have yet to be tested for their importance in later stage B cells (e.g., PU.1 and E2A) (28, 29, 31). Certainly, stage-specific dependence on particular transcription factors should no longer be surprising in the context of the newly emerging view of the IgH locus as being under shifting modes of control.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Laurel A. Eckhardt, Department of Biological Sciences, Hunter College of The City University of New York, 695 Park Ave., New York, NY 10021. E-mail address:
Received for publication November 14, 1997. Accepted for publication January 9, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
constant region. Mol. Cell. Biol. 13:1547.3'E and hs3). J. Immunol. 158:4780.[Abstract]
-HS4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation. Nucleic Acids Res. 23:975. light chain enhancers, but not heavy chain enhancers. Nucleic Acids Res. 22:4216.E (hs1, 2). Proc. Natl. Acad. Sci. USA 93:4392. enhancer at early stages of B cell differentiation. Mol. Cell. Biol. 13:3611. immunoglobulin promoters and enhancers display developmentally controlled interactions. Nucleic Acids Res. 21:4941. genes. J. Exp. Med. 186:845.1 and C2 genes. J. Immunol. 159:1310.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  P. J. ter Brugge, V. B. T. Ta, M. J. W. de Bruijn, G. Keijzers, A. Maas, D. C. van Gent, and R. W. Hendriks A mouse model for chronic lymphocytic leukemia based on expression of the SV40 large T antigen Blood, July 2, 2009; 114(1): 119 - 127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Zhang, A. Alaie-Petrillo, M. Kon, F. Li, and L. A. Eckhardt Transcription of a Productively Rearranged Ig VDJC{alpha} Does Not Require the Presence of HS4 in the Igh 3' Regulatory Region J. Immunol., May 15, 2007; 178(10): 6297 - 6306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Laurencikiene, V. Tamosiunas, and E. Severinson Regulation of {epsilon} germline transcription and switch region mutations by IgH locus 3' enhancers in transgenic mice Blood, January 1, 2007; 109(1): 159 - 167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Pawlitzky, C. V. Angeles, A. M. Siegel, M. L. Stanton, R. Riblet, and P. H. Brodeur Identification of a Candidate Regulatory Element within the 5' Flanking Region of the Mouse Igh Locus Defined by Pro-B Cell-Specific Hypersensitivity Associated with Binding of PU.1, Pax5, and E2A. J. Immunol., June 1, 2006; 176(11): 6839 - 6851. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. A. Dunnick, J. Shi, K. A. Graves, and J. T. Collins The 3' end of the heavy chain constant region locus enhances germline transcription and switch recombination of the four {gamma} genes J. Exp. Med., May 2, 2005; 201(9): 1459 - 1466. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. E. Garrett, A. V. Emelyanov, M. A. Sepulveda, P. Flanagan, S. Volpi, F. Li, D. Loukinov, L. A. Eckhardt, V. V. Lobanenkov, and B. K. Birshtein Chromatin Architecture near a Potential 3' End of the Igh Locus Involves Modular Regulation of Histone Modifications during B-Cell Development and In Vivo Occupancy at CTCF Sites Mol. Cell. Biol., February 15, 2005; 25(4): 1511 - 1525. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. S. Park, J. S. Kim, L. Tessarollo, J. D. Owens, L. Peng, S. S. Han, S. Tae Chung, T. A. Torrey, W. C. Cheung, R. D. Polakiewicz, et al. Insertion of c-Myc into Igh Induces B-Cell and Plasma-Cell Neoplasms in Mice Cancer Res., February 15, 2005; 65(4): 1306 - 1315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. C. Kim, C. R. Edmonston, X. Wu, A. Schaffer, and P. Casali 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 J. Biol. Chem., October 1, 2004; 279(40): 42258 - 42269. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. E. W. Sulentic, W. Zhang, Y. J. Na, and N. E. Kaminski 2,3,7,8-Tetrachlorodibenzo-p-dioxin, an Exogenous Modulator of the 3'{alpha} Immunoglobulin Heavy Chain Enhancer in the CH12.LX Mouse Cell Line J. Pharmacol. Exp. Ther., April 1, 2004; 309(1): 71 - 78. [Abstract] [Full Text]
|
|
|
|
|
|
M. A. Sepulveda, A. V. Emelyanov, and B. K. Birshtein NF-{kappa}B and Oct-2 Synergize to Activate the Human 3' Igh hs4 Enhancer in B Cells J. Immunol., January 15, 2004; 172(2): 1054 - 1064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Heckman, T. Cao, L. Somsouk, H. Duan, J. W. Mehew, C.-y. Zhang, and L. M. Boxer Critical Elements of the Immunoglobulin Heavy Chain Gene Enhancers for Deregulated Expression of Bcl-2 Cancer Res., October 15, 2003; 63(20): 6666 - 6673. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Gordon, S. Saleque, and B. K. Birshtein Yin Yang 1 Is a Lipopolysaccharide-Inducible Activator of the Murine 3' Igh Enhancer, hs3 J. Immunol., June 1, 2003; 170(11): 5549 - 5557. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Podojil and V. M. Sanders Selective Regulation of Mature IgG1 Transcription by CD86 and {beta}2-Adrenergic Receptor Stimulation J. Immunol., May 15, 2003; 170(10): 5143 - 5151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Sharif, H. S. Radomska, D. M. Miller, and L. A. Eckhardt Unique Function for Carboxyl-Terminal Domain of Oct-2 in Ig-Secreting Cells J. Immunol., October 15, 2001; 167(8): 4421 - 4429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Laurencikiene, V. Deveikaite, and E. Severinson HS1,2 Enhancer Regulation of Germline {epsilon} and {gamma}2b Promoters in Murine B Lymphocytes: Evidence for Specific Promoter-Enhancer Interactions J. Immunol., September 15, 2001; 167(6): 3257 - 3265. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Shi and L. A. Eckhardt Deletional analyses reveal an essential role for the hs3b/hs4 IgH 3' enhancer pair in an Ig-secreting but not an earlier-stage B cell line Int. Immunol., August 1, 2001; 13(8): 1003 - 1012. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Mundt, I. C. Nicholson, X. Zou, A. V. Popov, C. Ayling, and M. Bruggemann Novel Control Motif Cluster in the IgH {{delta}}-{{gamma}}3 Interval Exhibits B Cell-Specific Enhancer Function in Early Development J. Immunol., March 1, 2001; 166(5): 3315 - 3323. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Adams, H. Ackerly, K. Cunningham, and W. Dunnick A DNase I hypersensitive site near the murine {gamma}1 switch region contributes to insertion site independence of transgenes and modulates the amount of transcripts induced by CD40 ligation Int. Immunol., December 1, 2000; 12(12): 1705 - 1713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zelazowski, Y. Shen, and C. M. Snapper NF-{kappa}B/p50 and NF-{kappa}B/c-Rel differentially regulate the activity of the 3'{alpha}E-hsl,2 enhancer in normal murine B cells in an activation-dependent manner Int. Immunol., August 1, 2000; 12(8): 1167 - 1172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Stevens, L. Wang, and R. G. Roeder Functional Analysis of the OCA-B Promoter J. Immunol., June 15, 2000; 164(12): 6372 - 6379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Hu, Q. Pan, E. Pardali, F. C. Mills, R. M. Bernstein, E. E. Max, P. Sideras, and L. Hammarstrom Regulation of Germline Promoters by the Two Human Ig Heavy Chain 3' {alpha} Enhancers J. Immunol., June 15, 2000; 164(12): 6380 - 6386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Stevens, J. Ong, U. Kim, L. A. Eckhardt, and R. G. Roeder Role of OCA-B in 3'-IgH Enhancer Function J. Immunol., May 15, 2000; 164(10): 5306 - 5312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Collins and W. A. Dunnick Cutting Edge: IFN-{gamma} Regulated Germline Transcripts Are Expressed from {gamma}2a Transgenes Independently of the Heavy Chain 3' Enhancers J. Immunol., December 1, 1999; 163(11): 5758 - 5762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Seidl, J. P. Manis, A. Bottaro, J. Zhang, L. Davidson, A. Kisselgof, H. Oettgen, and F. W. Alt Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus PNAS, March 16, 1999; 96(6): 3000 - 3005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Saleque, M. Singh, and B. K. Birshtein Ig Heavy Chain Expression and Class Switching In Vitro from an Allele Lacking the 3' Enhancers DNase I-Hypersensitive hs3A and hs1,2 J. Immunol., March 1, 1999; 162(5): 2791 - 2803. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. LUO and R.G. ROEDER B-cell-specific Coactivator OCA-B: Biochemical Aspects, Role in B-Cell Development and Beyond Cold Spring Harb Symp Quant Biol, January 1, 1999; 64(0): 119 - 132. [Abstract] [PDF]
|
|
|
|
|
|
R.G. ROEDER Role of General and Gene-specific Cofactors in the Regulation of Eukaryotic Transcription Cold Spring Harb Symp Quant Biol, January 1, 1998; 63(0): 201 - 218. [Abstract] [PDF]
|
|
|
|
|
|
K. Kanda, H.-M. Hu, L. Zhang, J. Grandchamps, and L. M. Boxer NF-kappa B Activity Is Required for the Deregulation of c-myc Expression by the Immunoglobulin Heavy Chain Enhancer J. Biol. Chem., October 6, 2000; 275(41): 32338 - 32346. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
