Immunologic Research 2006;36/1–3:27–32

From B Cell to Plasma Cell

Regulation of V(D)J Recombination
and Antibody Secretion

Abstract
B cell development culminates in the formation of plasma cells,
potent secretors of the immunoglobulins (Ig), proteins crucial for
the health of the organism. Two distinctive and crucial steps are
required during B cell differentiation. First, variable gene segments
encoding the antigen-binding region of Ig undergo directed
rearrangement through a process known as V(D)J recombination.
Second, alternative processing of the Ig heavy chain mRNA tran-
script enables plasma cells to secrete high levels of Ig protein. This
review focuses on the molecular mechanisms that control V(D)J
recombination in B cell progenitors and alternative RNA process-
ing in plasma cells.

Lisa Borghesi
Key Words Christine Milcarek
V(D)J recombination
Department of Immunology
E47
University of Pittsburgh School of
Hematopoiesis Medicine
hnRNP F
CstF-64
Plasma cell

V(D)J Recombinase Activity Is

First Upregulated in Uncommitted
Lymphoid Precursors
Because V(D)J recombination of immuno-
globulin genes is a required step in lympho-
cyte development, understanding the molecular
signals that initiate this process during
hematopoiesis is of great interest. V(D)J
recombination occurs in an ordered sequence
in which the immunoglobulin heavy chain
(IgH) rearranges before the Ig light chain, and
within IgH loci, D–JH joining precedes V–DJH

Lisa Borghesi or Christine Milcarek © 2006 27

Department of Immunology, Humana Press Inc.
University of Pittsburgh School of Medicine, 0257–277X/
200 Lothrop Street, Pittsburgh, PA 15261 (Online)1559-0755/06/
E-mail: borghesi@pitt.edu or milcarek@pitt.edu 36/1–3:27–32/$30.00
joining. Our work is focused on the very first
step in this process—initiation of D–JH
recombination—during progression of
uncommitted hematopoietic progenitors to
the B lymphocyte lineage.
The rag1 and rag2 genes are the compo-
nents of the V(D)J recombinase enzyme com-
plex that catalyzes double-strand DNA breaks.
Within the early B cell compartment, rag tran-
scripts were shown to be readily detectable in
pro-B cells, a stage of development in which
approx 60% of cells possess D–JH joins (1,2).
However, it was initially unclear whether rag
expression and, hence, V(D)J recombination,
occurred at earlier stages of hematopoietic
development. For example, rag2 transcription
in upstream pre-pro-B cells was detectable
using a rag2–GFP construct (3) but not by PCR
analysis of endogenous rag transcripts (2). To
address this issue, we began to characterize rag
expression and V(D)J recombinase activity in
early hematopoietic precursors in vivo.
To study regulation of V(D)J recombinase
activity in rare hematopoietic progenitors, we
exploited H2-SVEX transgenic mice that
express a fluorescent V(D)J recombination
reporter substrate. Consequent to V(D)J
recombination, cells express VEX, a modified
green fluorescent protein that is readily
detectable by flow cytometry. We found that
not only is VEX expression present in 90% of
pro-B cells, a population actively undergoing
recombination, but 20% of pre-pro-B cells are
also VEX+ (4). Moreover, VEX is expressed in
30–50% of common lymphoid progenitors
(CLPs), precursors that have not yet commit-
ted to a specific lymphoid lineage fate. These
data suggest that rather than being restricted to
committed B (or T) cells, V(D)J recombinase
activity is first upregulated in multipotent
hematopoietic progenitors (5). Examination of
double transgenic rag2–GFP × H2-SVEX
mice confirmed the simultaneous expression
of rag transcription and V(D)J recombinase
28
activity at the CLP stage of development (5).
In combination with experiments demonstrat-
ing that rag expression is first detectable in a
rare subset of upstream LSK (lin–scahikithi)
progenitors (6), this work defines a key devel-
opmental point at which V(D)J recombinase
activity is strongly upregulated in vivo.
Transcriptional Regulation of V(D)J
Recombination Initiation
Multiple cis-acting elements that regulate
both rag1 and rag2 have been identified
(7–9). The Erag enhancer of rag expression
was of particular interest because it has been
shown to regulate rag expression in pro-B but
not pro-T cells (10). To characterize a role for
Erag activity at the CLP stage of develop-
ment, we crossed Erag-deficient mice to the
H2-SVEX background. We found that mice
lacking Erag had a fivefold reduction in VEX
expression in both CLPs and pre-pro-B cells
(5,11). By contrast, VEX expression was
unaffected in pro-T cells from Erag null mice.
The decrease in recombinase activity in CLPs
and B cells but not T cells from Erag-deficient
animals suggests that B cell–specific regula-
tion already exists at this early stage of pre-
cursor development.
The transcription factor E47, a splice prod-
uct of the E2A gene, is a strong candidate for
regulating rag expression and recombinase
activity in CLPs. E2A is known to bind to a
rag2 enhancer (12), upregulate rag1 tran-
scripts (13), and trans-activate Erag in a B
cell line (10). However, it has been difficult to
determine whether E2A directly affects rag
transcription in hematopoietic progenitors in
vivo. E47 deficiency leads to an early block in
B cell development (14,15) rendering it
unclear whether the absence of detectable rag
transcripts in total bone marrow cells (16) is
due to the specific absence of rag expression
or is due to the absence of the progenitor cells
that are capable of expressing rag (i.e., CLPs
Borghesi and Milcarek
and pre-pro-B cells). To address this issue, we
first characterized the precise stage at which
B lineage development is blocked in E47-
deficient mice. Within the intact, upstream
subsets, we then examined each rag1 tran-
scription, V(D)J recombinase activity, and
D–JH joining.
We found that CLPs and pre-pro-B cells
were present in E47-null mice albeit at
reduced frequency but that pro-B/pre-B cells
were undetectable (11). Thus, these mice have
a complete block at the pro-B stage of devel-
opment. Within the intact CLP population, we
found severe recombination defects. rag1
transcripts were below the level of detection
as assessed by quantitative RT-PCR, and VEX
expression was reduced from 36% in E47-het-
erozygous animals to 0% in E47-null mice.
Consistent with the absence of recombination
machinery, the frequency of D–JH+ CLPs was
reduced from 6 out of 26 cells (23%) in E47
heterozygous mice to only 1 out of 52 (1.9%)
in E47-null mice. In contrast to the require-
ment of E47 for V(D)J recombinase activity
during B lineage development, loss of E47 has
no discernable effect on recombinase activity
in T lineage precursors. Together these data
indicate that E47 is absolutely required for
initiation of V(D)J recombination at the CLP
stage of development.
One of the unexpected implications of this
work is that CLPs may have a B lineage bias.
This idea is supported by the observations that
both Erag and E47 are required for recombi-
nase activity in CLPs and pre-pro-B cells, but
are dispensable for recombinase activity in T
lineage progenitors. Consistent with this
interpretation, T cells have been shown to
arise efficiently from early thymic progenitors
(ETPs) rather than CLPs (17). This concept
gains further credence by the observation that
ikaros-null mice generate near normal num-
bers of T cells despite the absence of the CLP
population (18). The factors that control rag
Regulation of Antibody Production
expression and V(D)J recombinase activity in
ETPs and the T lineage remain unclear.
Control of Ig Secretory mRNA Production
B-cell development and differentiation cul-
minate in the dual production of plasma cells
producing secreted Ig that is crucial for the
health of the organism and in memory cells
that continue to survive and provide the
anamnestic response. Blimp-1 is normally
repressed by BCL-6 in mature B cells; when
BCL-6 expression is turned down by lym-
phokine stimulation, blimp increases, thereby
further shutting BCL-6 off (19). Blimp
induces Ig secretion (20). Studies to date
indicate that this is an indirect effect, through
the induction of other proteins, because blimp
does not interact directly with the Ig promoter
or the polyadenylation machinery.
RNA processing events play the major role
in determining the ratios of the two forms of
Ig heavy chain mRNA. The crucial role for
polyadenylation was substantiated by our lab
and by others, as reviewed in ref. 21.
Polyadenylation at the weak secretory-specific
poly(A) site and splicing to the membrane-
specific exons at the suboptimal splice site, in
the last secretory-specific exon, are mutually
exclusive events. The changes in the cleavage
and polyadenylation of the precursor RNA tip
the balance in plasma cells to the use of the
first, weak poly(A) site. The CH4 (µ) or CH3
(γ) terminal-secretory Ig heavy chain exons
are composites of splicing and polyadenyla-
tion signals. This type of composite exon is
seen in numerous other genes, many of which
show differential poly(A) site utilization, so
understanding Ig heavy chain gene regulation
has much wider implications for the control of
gene expression in a number of systems (21).
Previously, we found that there was an
increase in the binding activity of CstF-64 and
the 100 kDa subunit of CPSF to poly(A) sites
29
in plasma cell/myeloma nuclear extracts as
compared with memory B cell/lymphomas
(22). In that study and subsequent studies we
could find no difference in the amount or form
of either protein. Subsequent microarray stud-
ies have detected no changes in the mRNAs
for these factors in a variety of B cell to
plasma cell comparisons. We hypothesized
that the change in the binding activity of
CstF-64 and CPSF-100 between B cell stages
might be brought about through the action of
“auxiliary polyadenylation factors” that could
augment the binding of the basal/constitutive
factors. CstF-64 activity, as judged by its
ability to bind to an RNA substrate, has also
been shown to vary following adenovirus (23)
and herpesvirus infection (24) with no change
in the level of the protein.
Auxiliary RNA Processing
Factors in Plasma Cells
While searching for factors that are differ-
entially expressed in plasma versus B cells,
we demonstrated that a change in the level of
hnRNP F is an important determinant in the
regulated use of alternative polyadenylation
sites (25). Those results and recent studies
(26) demonstrate that mammalian hnRNP F
can act as a negative regulator of CstF-64 in
the pre-mRNA cleavage reaction and that the
increased expression of hnRNP F in memory
B cells contributes to the suppression of the Ig
heavy chain secretory poly(A) site. The
hnRNP F protein is one member of a family
of closely related regulators including hnRNP
H, H′, H29, and F that will bind to poly(G).
There is significant variation in the ratio of
hnRNP H to F in different tissues suggesting
this ratio may serve a regulatory function
(27). Therefore, the relative amount of each
member of the pair may have many important
implications for RNA processing, much like
hnRNP A versus ASF/SF2 levels are impor-
tant for tissue-specific splicing patterns.
30
The U1A protein can be found both in a
small-ribonucleoprotein-particle (snRNP) that
contains U1 RNA, or in a distinctive fraction,
free of the snRNP, the SF-A complex. Both
components have been shown to influence post-
or co-transcriptional RNA processing reactions
in HeLa cells. Because U1A may influence the
processing of the immunoglobulin heavy chain
pre-mRNA in B cells, we wanted to see if the
levels of U1A in either of its two forms changed
following IL-6 stimulation to IgM secretion.
Using antibodies that specifically recognize
the two forms of U1A, snRNP-associated and
snRNP-free, we found that approx 16% of
U1A is in the SF-A form in B-cells. We mea-
sured the levels of U1A protein in its two states
in human B cell lines by both flow cytometry
and exhaustive immunoprecipitations. We
found a significant decrease in the amount of
snRNP-associated U1A following cytokine
stimulation that correlates with the changeover
to the secretory-specific poly(A) site use in the
SKW 6.4 cell line. Meanwhile, the number of
U1A molecules in the SF-A fraction of the pool
remains nearly constant following induction to
secretion. Our results (28) suggest that the
changing level of U1A in the snRNP fraction
may be important for influencing Ig heavy
chain mRNA processing.
Role of RNA Polymerase II
Association in mRNA Processing
The linkage between RNA processing
events and transcription has become increas-
ingly evident over the last several years (29).
Different promoters can direct either the
inclusion or exclusion of alternatively spliced
exons (30). The recruitment of different fac-
tors to control splice site selection occurs on
the carboxyl-terminal domain of RNA pol II
(CTD) at the start of transcription. Purified
CTD stimulates both polyadenylation and
splicing in vitro (31). The polyadenylation
factor CPSF loads onto the CTD of RNA
Borghesi and Milcarek
polymerase II in a directed fashion after first
interacting with the proteins at the promoter,
specifically certain TFIID-associated TAFII
proteins (32). Stimulation of 3′ processing in
vivo by CTD requires contact with the 50-kDa
subunit of the cleavage stimulation factor,
CstF. Different regions of the CTD with their
potential for unique patterns of phosphoryla-
tion may serve distinct functions in pre-
mRNA processing (33). Elongation by the
polymerase is also controlled by a number of
factors, including ELL2 (34). The expression
of ELL2 mRNA has been shown to be regu-
lated in plasma cell differentiation in several
studies including our own (unpublished) stud-
ies. The kinetics of polymerase elongation can
also influence the loading of splicing factors
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