LBP-1a is the predominant LSF/LBP-1 family member in B-cells
In order to distinguish LSF and LBP-1a, anti-peptide antibodies were produced against divergent regions (). The antibody specificities and sensitivities were tested against recombinant proteins () and mammalian cell extracts in which each protein was independently overexpressed (). In both instances, each antibody was highly specific for its cognate protein. The LBP-1a antibody recognizes multiple protein species from untransfected 293T extracts (); due to alternative splicing, the cellular LBP-1a gene can encode two major products: LBP-1a and the larger LBP-1b [12
]. LBP-1b is expressed in a tissue-specific manner [13
], and is not evident in primary B cells ().
Figure 1 LBP-1a is the predominant LSF family member in primary B cells. A) Sequences of LBP-1a (aa 274–336) and LSF (aa 277–332) are aligned. Antipeptide antibodies were produced against nonconserved epitopes (bold). B) Ten ng of His-LBP-1a or (more ...)
With these new reagents, we determined the relative abundance of LSF and LBP-1a in primary B-cells. By comparing the reactivity of each antibody against whole cell lysates from mouse splenic B-cells (, right panels) to its reactivity against a standard curve of purified recombinant His-LSF or His-LBP-1a (, left panels), we concluded that primary mouse B cells contain at least 5-fold more LBP-1a (on the order of 104 molecules/cell) than LSF.
In primary, resting B-lymphocytes LBP-1a binds genomic DNA around particular S regions
We previously demonstrated in vitro
that recombinant LSF, as well as related proteins in mouse splenic cellular extracts, could bind specific sequences within Sμ, Sα, and Sε [10
]. S regions can be clustered into two groups: Sμ, Sε, and Sα contain repeated pentameric sequences, whereas Sγ1, Sγ2a, Sγ2b, and Sγ3 contain 49–52 base-pair repeats [2
]. We computationally analyzed the likelihood of LSF/LBP-1a binding sites within each S region. In some (Sμ, Sε, Sα, and Sγ3), numerous LSF/LBP-1a recognition sites were predicted; in others (Sγ1, Sγ2a, and Sγ2b), exceedingly few ( and Fig. S1A in supporting material
). A similar pattern was evident when human S regions were analyzed (Fig. S2
Figure 2 LBP-1a binds to Sμ and Sα, but not Sγ1, regions in resting B cells and is released after LPS stimulation. A) Schematic of mouse immunoglobulin S region sequences with predicted LSF/LBP-1a binding sites. Long horizontal lines, to (more ...)
We tested these predictions by ChIP assays in primary murine splenic B-lymphocytes, focusing on Sμ, which is operative in all CSR, and Sα and Sγ1, which are target switch regions of the two classes of repeats. Binding of LBP-1a, the more abundant LSF family member in B-cells (), was monitored. Amplification of specific switch regions in the genomic DNA was analyzed by semi-quantitative PCR analysis (see Materials and Methods); the highly repetitive nature of these genomic sequences prevented design of real-time PCR primers. Binding in vivo
was robust at both Sμ and Sα (), where the amplicons were located at least 1.5 kb and 3.5 kb, respectively, downstream of transcriptional regulatory elements (the intronic promoters and the enhancer region), and either at the border of or well within S region sequences. However, as expected given the low density of predicted high affinity binding sites (roughly one per 3500 bp) LBP-1a binding was not detectable around Sγ1 in the region of the amplicon (). Similarly, LBP-1a occupied Sε and Sγ3, the respective amplicons being at least 2.5 kb and 3.2 kb downstream of the intronic promoters, with minimal or no occupancy of Sγ2a and Sγ2b in the regions probed (Fig. S1B in supporting material
). The experimental findings at all heavy chain gene loci are therefore consistent with predictions of LSF/LBP-1a binding sites within these S region sequences.
LPS stimulation of B-lymphocytes decreases binding of LBP-1a to S regions
Functionality of LBP-1a association with S regions in vivo
was tested initially by assaying binding upon stimulation of primary B-lymphocytes. In previous studies, inherent DNA-binding activity of LSF/LBP-1a to S sites in mouse splenic extracts, assayed by EMSA, decreased after stimulation to undergo CSR [10
]. Using the specific antibody (), levels of LBP-1a were measured in whole cell extracts of purified splenic B-lymphocytes stimulated by LPS for increasing amounts of time (). Surprisingly, LBP-1a levels (and LSF protein levels; data not shown) substantially increased by 24 h after stimulation. In parallel LPS-stimulated cultures, we directly measured binding of LBP-1a to genomic S regions by ChIP. Occupancy at both Sμ () and Sα () was readily apparent at 0 and 10 h, but markedly decreased at 24 and 48 h post-stimulation. Thus, although protein levels increased at late time points after stimulation, binding decreased.
The decrease in DNA-binding with LPS stimulation is likely due to direct modification of LBP-1a. The closely related paralog, LSF, is directly targeted by mitogenic signal transduction pathways in multiple cell types, altering LSF DNA-binding potential, in part through modification of S291 [14
]. LBP-1a contains an analogous serine; we hypothesize that LBP-1a is targeted downstream of cytokine signaling cascades in resting B cells, being modified to reduce binding to S regions.
The kinetics of release of LBP-1a from S region sequences following stimulation of B cells are consistent with LBP-1a inhibiting early events in CSR. Induction of AID mRNA and some germline transcripts occurs by 12 to 24 h post-stimulation [16
], with Iα sterile transcripts [18
], formation of R-loops [19
], and alterations in histone acetylation by 48 h post-stimulation [20
]. The inverse correlation between binding of LBP-1a and activation steps of CSR suggests that LBP-1a is a repressor.
LBP-1a-mediated repression of CSR to IgA, but not IgG1, in murine splenic B-cells
In a B cell line capable of undergoing CSR only from Sμ to Sα in vitro
, LSF/LBP-1a did indeed repress CSR [10
]. However, the question remained as to whether, in primary B cells, CSR in general would be subject to such inhibition, since all CSR in IgM-expressing B cells involves the Sμ switch region with which LSF/LBP-1a interacts, or if CSR only at certain heavy chain gene loci would be inhibited. To distinguish between these possibilities, we generated bone marrow chimeric mice in which a dominant negative form of LSF (LSFdn) was expressed in donor hematopoietic cells. LSF family members oligomerize with each other and bind DNA as obligate tetramers [11
]. Two aa substitutions in the DNA-binding region of LSFdn prevent DNA-binding [21
]; due to oligomerization this is a dominant phenotype that blocks all LSF family members [11
]. This decrease in DNA-binding of endogenous LSF family members by LSFdn expression has previously been demonstrated in multiple studies, including in a murine B cell line [10
Bone marrow cells from donor mice were transduced in vitro
with retrovirus capable of expressing both LSFdn and GFP. Transplantation of these cells into lethally irradiated mice repopulated the recipient hematopoietic system. The resulting transduced, GFP-expressing splenic B cells were isolated and analyzed for CSR upon stimulation in vitro
. Two control B cell populations were used. First, non-transduced cells in the donor bone marrow gave rise to GFP-negative splenic B cells in the same mouse, providing an ideal internal control for the GFP-positive, LSFdn-expressing cells (see Fig. S3 in supporting material
for demonstration of GFP-negative versus GFP-positive populations). Second, other recipient mice were transplanted with donor bone marrow transduced with parental retrovirus, capable of expressing only GFP, in order to control for the possibility that transduction with any retrovirus would alter the efficiency of CSR.
Expression of LSFdn was first validated in the LSFdn group of mice. Isolated GFP-positive and GFP-negative populations of primary splenic B-cells were harvested and immunoblotted with antibody specific to LSF. Endogenous murine LSF was detected in GFP- cells, whereas at least 50-fold higher levels of exogenous human LSFdn were detected in GFP+ cells from the same mouse (). Considering relative expression levels of LBP-1a and LSF (), LSFdn expression was also at least 10-fold higher than that of LBP-1a. Even equimolar concentrations, both in vitro
and in vivo
, are sufficient to dramatically suppress wild type DNA-binding of LSF family members [21
Figure 3 LSFdn expression in B-cells increases switching to IgA, but not to IgG1, upon induction of CSR. A) Isolated B-cells (5 × 106) from LSFdn chimeric mice were sorted into transduced (GFP+) and non-transduced (GFP−) populations based on GFP (more ...)
To determine whether this expression of LSFdn affects the frequency of CSR, purified splenic B-cells from both chimeric LSFdn mice and chimeric control mice were stimulated to undergo CSR in vitro. Unlike results in the I.29μ B cell line, in the absence of stimulation, there was no detectable IgA or IgG1 expression in B cells isolated from LSFdn mice (data not shown). That alleviation of repression by LBP-1a is not sufficient to induce CSR in primary resting B cells in the absence of signaling is consistent with extensive analyses indicating that specific activation signals are absolutely required.
Due to the differences in density of LBP-1a occupancy at switch regions Sα vs. Sγ1 (), and to the relatively high efficiency of switching that can be induced in vitro
to these two regions, we compared switching to IgA with that to IgG1 [2
]. Cells were gated on the basis of GFP expression and surface levels of IgG1 and IgA expression after stimulation were monitored by flow cytometry (see Fig. S3 in supporting material
for example). In initial experiments, comparison of the degree of CSR in BALB/cByJ B cells to that in the chimeric B cells indicated that CSR in the chimeric cells was less robust (approximately one fourth the levels of CSR), suggesting that bone marrow transplantation compromises the degree of CSR achievable in primary B cells in culture. We note that the depression in frequency of CSR in chimeric mouse B cells unfortunately precluded investigation of the inherently less efficient switching in vitro
to isotypes other than IgA and IgG1.
By normalizing the percentage of transduced cells undergoing CSR to that of nontransduced cells in the same B cell population from the same mouse, we could most accurately determine the effect of LSFdn expression on CSR. The ratios for each individual mouse are plotted for expression of IgA and IgG1 (, respectively), compiled from multiple independent experiments. These experiments included activation of B cells using multiple protocols; the effect of LSFdn on IgA CSR induced either by LPS, IL-5 and TGF-β1 (left panel) or by LPS and IL- 4 (right panel) are shown in . Importantly, for chimeric control mice, splenic B cells switched expression from IgM to either IgG1 or IgA at the same frequencies, irrespective of whether or not they expressed GFP, resulting in a ratio for each isotype of 1.0. For the chimeric LSFdn-expressing mice, expression of IgG1 was also unaltered in the transduced cells (ratio of 0.94, ). In stark contrast, the percentage of LSFdn-expressing cells presenting surface IgA was significantly elevated (1.8-fold, P<0.005) compared to their non-transduced counterparts (, left panel). A distinct cytokine cocktail appeared to result in a similar derepression of IgA expression by LSFdn (1.6-fold, P=0.067; , right panel). These data demonstrate that LSF family members do not downregulate CSR in general, but only to certain isotypes (e.g. IgA).
How LBP-1a reduces CSR requires further investigation. Effects of LBP-1a, or other LSF family members, on CSR could potentially be either indirect or direct. Indirect effects might result, for instance, from LBP-1a affecting the degree of cell cycling. However, we view this possibility as unlikely for the following reasons. First, in a B cell line capable of undergoing CSR, expression of LSFdn, although similarly affecting CSR [10
], did not detectably alter cell growth properties (data not shown). Second, since induction of IgG1 and of IgA in primary B cells both require multiple cell divisions [23
], it is difficult to conceive of how indirect effects of LBP-1a would impact switching to one, but not to the other. Our combined findings that LBP-1a binds Sμ and Sα, but apparently not Sγ1, and that inhibition of LSF/LBP-1a increases levels of immunoglobulin class switching to IgA, but not to IgG1, instead suggest that LBP-1a inhibits CSR directly, as a consequence of LBP-1a binding S region sequences upstream of the respective heavy chain coding region.
Direct regulation of CSR by LBP-1a could result from either transcriptional or non-transcriptional mechanisms. Regulation of germline transcription by LBP-1a is unlikely, given that LSFdn did not alter the levels of germline transcripts in a B cell line in which it regulated CSR [10
], and that the demonstrated binding of LBP-1a at the heavy chain loci is distant from the transcriptional regulatory regions. Alternative mechanisms for regulation of CSR by LBP-1a include, although are not limited to, inhibition of S-S synapsis, counteracting AID deamination or strand breakage, or reduction in chromatin accessibility. With regards to the latter, activation of CSR correlates with histone acetylation in S regions [5
]. We note that LBP-1a may have the capability of inhibiting chromatin accessibility, in that the highly similar LSF interacts with multiple inhibitory chromatin modifying factors including histone deacetylases, Sin3A corepressor and the polycomb protein RING [11