Reading in B cells

B-cell hyperactivity as a result of enhanced germinal center reactions with defects in selection

Autoimmune diseases are most characterized by B cell’s escape from selection in the thymus. In lupus,
populations such as transitional B cells (CD24++CD38++), prena¨ıve and na¨ıve B cells are expanded in the
peripheral blood of patients . the central tolerance mechanisms are crucial in preventing B cell mediated
autoimmune diseases. For instance, the strong BCR signal from binding with high affinity to an autoantigen will
lead to deletion or receptor editing of the high affinity.
Reduced receptor editing have been documented in autoimmune-prone strains of mice and in humans with
lupus and type 1 diabetes.5,6

Recent publications have highlighted the role of Ikaros in pro- and pre-B cell development. Inactivation of Ikzf1
leads to arrestment at an aberrant‘pro-B cell’ stage characterized by increased cell adhesion and loss of
signaling via pre-BCR 1 (
Unexpectedly, derepression of expression of the transcription factor Aiolos did not compensate for the
loss of Ikaros in pro-B cells.)
ChIP analysis of the binding of Ikaros to regulatory elements of the Rag locus

Ikaros appears to promote SLP-65 activation by as yet unknown mechanisms, and is required for cell cycle
arrest in response to pre-BCR signaling [46].

consistent with a role for Aiolos in limiting pre-B cell proliferation, there are increased numbers of pre-B cells
in Aiolos-deficient mice2

The relevance of Ikaros proteins for the termination of human pre-B cell proliferation is underscored by the
seminal finding that IKZF1 is mutated in most ALL cases [12] and [13].

Few factors have been identified as indispensable in B cell development. Ikzf1, the founding member of Ikaros
transcription factor family, has been shown to be crucial in lymphopogenesis. Ikaros attenuates IL-7 signal 3.
Ikaros has been associated with type 1 diabetes

IRF-4 and -8 induce the expression of Ikaros and Aiolos to downregulate pre-BCR and inhibit pre-B-cell
expansion (22).

Foxp3+ Helios+ regulatory T cells are expanded in SLE patients and correlated with disease score4. Nagpal et
al. (2013) showed that Ikaros represses protein phosphatase 2A (PP2A) 5

The lack of folliculin interacting proteins FNIP1 leads to pro-B to pre-B cell block6

In the absence of co-stimulatory signals, activation of nuclear factor of activated T cells (NFAT) induces the
initiation of a transcriptional programme that results in a state of unresponsiveness, including PTP1B and Ikaros
7

Irf8, mice deficient in this transcription factor, both germline and conditional knockout, produced anti-dsDNA
antibodies, and B cell anergy was breached in the Irf8-deficient mice ( Pathak et al., 2013). However, the
effects on pre-B cells were not determined. Since several observations showed that receptor editing and L-
chain gene rearrangements are defective in B cells of patients with autoimmune disease ( Radic and Zouali,
1996 and Zouali, 2008), it will be of interest to probe the con and
liver harvested at indicated time points. Liver mononuclear cells were harvested and counted.
1
Isolated MNCs were stained for specific surface markers of each cell type.
(B, C) Percentages (B) and absolute numbers (C) of different immune cell types in liver of vehicle-injected
(grey) and PA-injected (black) B6 mice … hours after injection. Liver MNCs were harvested and stained for …,
, , , contribution of IRF4 and 8 to receptor editing in autoimmunity.

In humans, the relative number of self-reactive B cells decreases from ∼40 to 20% as newly formed immature
B cells transition into the naive mature B cell compartment (4). This progressive decline in self-reactive cells
fails to occur in patients with systemic lupus erythematosus or rheumatoid arthritis, supporting the conclusion
that peripheral selection is essential for maintaining B cell self-tolerance (5, 6).

Transitional B cells B220+, IgM+, CD21/35−, CD43− . In mouse, transitional B cells are generally defined as
IgMhiCD24+ . Transitional type 1 (T1) B cells are IgMhi and negative for IgD (IgD−), L-selectin (L-selectin−),
CD23 (CD23−), and CD21 low (CD21lo), and transitional type 2 (T2) B cells are IgMhi, IgDhi, CD21hi, CD23+,
and L-selectin+ (11)

To delete Irf4 in pre-B cells, we generate Irf-4 flox and Cre controlled by distal germline promoter, which only
becomes highly active in pre-B cells (fractions C' and D) 8 The Cκ-iYFP insertion has the additional advantage
of serving as a reporter for both germline and rearranged transcripts since both include the Cκ exon.
The role of GCN4 in B cell central tolerance
The level of recombining sequence (RS) rearrangement is measured as in Panigrahi (2008) 9 showed that RS
rearrangement levels are highest in late pre–B cells.
RS rearrangement provides a measurement for repeated rearrangement attempts at κ (receptor editing). λ-
expressing B cells can form without undergoing RS rearrangement, indicating that RS is not required for the
production of λ (12).
the defect in secondary rearrangement is more severe at the immunoglobulin λ locus than at the κ locus,
indicating that IRF-4 is more critical for the λ rearrangement.10 In this study, Since receptor editing in the bone
marrow occurs at the pre-B and immature B stages, we isolated both pre-B and immature B cells from the bone
marrow of IRF-4+/− and IRF-4−/− mice. Therefore, targeting Irf4 is becoming a new focus in autoimmune disease
studies.
http://books.google.com/books?id=1FmPBl5C9kcC&pg=PA351&lpg=PA351&dq=%22histone+acetylation
%22+b+cell+autoimmune&source=bl&ots=4zfbqp4mMr&sig=lxwaRhf_s93imNfJTm7gKeVAha8&hl=en&sa
=X&ei=rFkjVIXiEIeuogTylIHgBw&ved=0CCUQ6AEwAQ#v=onepage&q=%22histone%20acetylation
%22%20b%20cell%20autoimmune&f=false
Several studies have indicated inefficient B cell receptor editing in lupus and other autoimmune diseases.
However, few studies have looked into epigenetic control of central tolerance checkpoints.
3.Background and Significance
This study aims to investigate the role of the histone acetyltransferase GCN5 in establishing B cell central tolerance
and in pathogenesis of lupus. In normal condition, committed pro-B cells in the bone marrow undergo extensive
V(D)J recombination to generate a diverse repertoire of receptors, 40 to 60% of which are autoreactive11. Two
central tolerance checkpoints help eliminate autoreactive B cell receptors (BCRs)12 26. First of all, in the bone
marrow, heavy chain undergoes additional rearrangement in pro B cells, and light chain undergoes κ genes
recombination and rearrangement of the recombining sequence (RS) (6, 7) Secondly, clonal deletion between
newly emigrated B cells in the peripheral blood and mature naive B cells in the spleen, is clearly defective
in SLE patients (14, 15). in pre-B cells, before cells emigrate to the periphery. In autoimmune diseases such as
lupus, these checkpoints appear to be defective, resulting in the emergence of increased numbers of B cells
expressing autoreactive BCRs. Several studies have indicated that compromised B cell central tolerance
mechanisms, especially receptor editing, lead to anti-nuclear antibodies production13 14(Liu JI 200715 Lupus
2
susceptibility genes may breach tolerance to DNA by impairing receptor editing of nuclear antigen-reactive B
cells.) Previously, the lupus mouse model MRL/lpr was shown to have reduced levels of RS rearrangement9
(paper: Chang The lupus susceptibility locus Sle1 facilitates). Evidence of defect in the second checkpoint has
also emerged, as in lupus patients, autoreactive antibodies fail to be removed at the transition between newly
emigrated (CD19+ CD10+IgM+CD27−) and mature naive B cell (CD19+ CD10−IgM+CD27−)16. Understanding the
mechanism of B cell central tolerance is crucial for understanding lupus pathogenesis.

This project aims to explore epigenetic control of B cell central tolerance.

Multiple epigenetic marks, including miRNA, histone modifications, and DNA methylation, control different
aspects of B cells17. increased histone acetylation and reduced lupus symptoms in lupus-prone mice treated with
HDAC inhibitors18. Post translational modification of histone tails alters the physical properties and
configuration structure of chromatin and modulate the accessibility of transcription factors to DNA-binding
sites.8 Histone modifications include acetylation, phosphorylation, methylation, ubiquitylation, and
sumoylation.49 and 50 Most histone modifications are dynamic and reflect the balance between competing
enzymatic activities that establish and remove these posttranslational epigenetic marks. Histone
acetyltransferases comprise of two classes: Type A histone acetyltransferases possess the properties normally
associated with chromatin-modifying enzymes as they are localized to the nucleus and contain bromodomain,
include GCN5, p300/CBP, among others;
Type B HATs are located in the cytoplasm and are responsible for acetylating newly synthesized histones prior
to their assembly into nucleosomes, include Hat-1, among others. Previously, another type A HAT, p300, was
shown to suppress SLE19, as defective p300 acetyltransferase (AT) activity exclusively in B lymphocytes results
in an autoimmune disease similar to SLE with full symptoms.
Epigenetic induction of Blimp1 drives plasma cell differentiation17. Defective pre-B cells in Blimp-1-/-20
In in vitro experiment with B cell line, GCN5 deficiency causes strong resistance against the apoptosis
induced by B-cell receptor–mediated stimulation through transcriptional regulation of various apoptosis-
related genes [Kikuchi 2008]. In addition, GCN5 activates the phosphatidylinositol 3-kinase/Akt survival
pathway in B cells exposed to oxidative stress, by controlling gene expression of Syk and Btk [23];
promotes the superoxide-generating system in B cells, by controlling gp91-phox gene expression [24
GCN5 deficiency significantly or slightly down-regulated gene expression of Blimp-1 (to undetectable
level), Ikaros (to ∼25%), IRF-4 (to ∼5%), OBF-1 (to ∼45%,OBF-1 is essential for the generation of
antibody-secreting cells and the development of autoimmunity in MRL-lpr mice. JA 2007), Pax-5 (to
∼45%, involved in various BCR signaling21), PU.1 (to ∼50%), and Xbp-1 (to ∼60%), but obviously up-
regulated those of Bach2 (to ∼200%, induces negative selection and opposes BCL6 function prior to the
pre-BCR checkpoint22), Bcl-6 (to ∼220%, positive selection of pre-B cells that have passed the
checkpoint22), E2A (to ∼160%), and EBF1 (to ∼170%). These genes fall within two groups: those
controlling apoptosis in response to BCR signal (…) and those regulate receptor editing (Irf-4, Ikaros).
Among several factors involved in establishing two central tolerance checkpoints, Irf4 appears to be the most
essential. Irf4 is a transcription factor that is present in all hematopoietic cells. In IRF-4-deficient mice, the
percentages of λ-expressing B cells were significantly decreased in the bone marrow and spleens of IRF-4−/−
mice (4% vs. 9%). The products of RS and λ1 rearrangement were significantly reduced in the IRF-4−/− mice,
indicating a defect in secondary rearrangement. Irf4 also interacts with multiple factors that play important role
in B cell development, such as Ikzf11, E2A, PU.1 and Spi-B, which regulate κ 3′ enhancer and λ enhancers.
IRF4, when expressed in IRF4,8−/− pre-B cells, induces κ germline transcription, enhances V(D)J rearrangement
activity at the κ locus, and promotes L chain rearrangement and transcription. Chromatin immunoprecipitation
assay further reveals that IRF4 expression leads to histone modifications and enhanced chromatin accessibility
at the κ locus. 23 Recently, the histone acetyltransferase (HAT) GCN5 was found to be essential for Irf4

3
expression in immature B cells24. Study conducted in chicken cell line DT40 showed that expression of Ikaros
(to ∼25%) and IRF-4 (to ∼5%) are suppressed in GCN5-deficient DT40 mutants (GCN5−/−). Also, drastic
resistance against apoptosis induced by PMA/ionomycin in the GCN5−/− cell line25.

However, there has been no study on GCN5 activity in the context of autoimmune diseases. Also, the above
studies was conducted in steady-state DT40 cell line and does not reflect the dynamic of Irf4 control by GCN5
during early B cell stages and in the context of autoimmune disease. Given the involvement of GCN5−/− in
both mechanisms of central tolerance (apoptosis and RS editing), we hypothesize that GCN5 is a master
regulator B cell central tolerance. Our project has two aims:

Studies into tolerance checkpoints have been limited due to difficulty of measuring tolerance efficiency at each
stage. Previous studies largely relied on measuring serum and mature B cell-derived autoantibodies. It is not
known which factors control tolerance mechanisms. Factors that have been identified as crucial in early B cell
development often play dynamic role throughout B cell development. This proposal takes advantage of recent
techniques to measure RS rearrangement and additional rearrangement, as well as stage-specific knock-out, to
study the role of Irf4 in each tolerance checkpoint.

In an in vitro system26, when receptor-editing signals are given to bone marrow immature B cells by
antiidiotype antibody or after in vivo exposure to membrane-bound self-antigen, MRL/lpr 3-83
transgenic immature B cells undergo less endogenous rearrangement and up-regulate recombination
activating gene messenger RNA to a lesser extent.
Receptor editing happens in pro-to-pre B cell stage for H chain and pre-to-naïve B cell stage for L chain. This
mechanism provides the cell with a new receptor through continued rearrangement at the light chain loci (or on
rare occasions, at the heavy chain locus) and replacement of the former light chain with a new light chain (8).
Receptor editing has been shown to be a highly efficient mechanism to replace these autoreactive receptors with
a nonself-reactive receptor (4, 9–11).

Aim 1: Functional interaction of GCN5 with Blimp1 and Irf4 in the context of lupus.
Hypothesis: We hypothesize that in lupus mice, GCN5 binding to Irf4 is reduced, leading to impaired receptor
editing, while GCN5 binding to Blimp-1 is…, leading to resistance to apoptosis.

We will use cell sorting to isolate pre- and pro- B cells from bone marrow Mrl-lpr and C3H control mice. We
will then isolate pre- and pro- B cells (CD43+CD24+, as in27), immature transitional T1 cells from peripheral
blood (IgM+CD21/35− CD43− ).

GCN5 interaction with other proteins will be studied as in28. Coprecipitated chromatin with anti-GCN5 antibody
(ChIP) will be performed as follow. Sorted cells (1×106) will be lysed in the presence of 1 mM PMSF and 100
μg/ml aprotinin. Lysates will be sonicated to shear DNA to lengths between 200 and 1000 bp and then
centrifuged at 12,000 g at 4°C for 10 min. Immunoprecipitation will be performed with 2 μg anti-GCN5
antibody (or 2 μg IgG as the negative control) or 2 μl anti-acetylated histone antisera (or 2 μl normal rabbit
serum as the negative control). Immunoprecipitated and input DNAs will be analyzed by PCR with the
appropriate primers corresponding to the sequences around the 5′-flanking region of the chicken IRF-4 gene
[27, 28]. PCR will be performed, and the level of gene detected in lupus mice will be compared to that in
control mice.
The levels of RAG 1 and RAG 2 expression will also be measured, as these genes are normally upregulated
early in receptor editing, as a response to BCR stimulation26.
4
We aim to determine whether altered expression of Irf4, Blimp1, and Ikaros is associated with under-
acetylation, and whether increasing histone acetylation by inhibiting histone deacetylase might correct central
tolerance.
Predicted outcome: If there is no difference in the level of binding of GCN5 and the TFs above,

If GCN5-/- and mutation result in no consequence: Previously, GCN5 was found to have overlapping role in
PCAF complex in mouse embryogenesis24
We’ll hypothesize that PCAF compensates for GCN5. To test this hypothesis, we will generate…

Aim 2: The effect of GCN5 B cell specific knock out, mutation, and overexpression on central tolerance.
2a) Generation of a conditional GCN5 knockout and GCN5 mutant mouse models
Mutation of the GCN5 gene will be confirmed by Southern (DNA) blot analysis of tail DNA

GCN5 is well characterized in yeast, located on chromosome 11 in mouse. Gcn5 in mice leads to embryonic
death 29, 30. GCN5-/- embryos complete gastrulation but do not form somites, a neural tube, or a notochord. Loss
of Gcn5 leads to increased apoptosis and mesodermal defects during fetal development16. The murine GCN5
gene consists of … exons (Figure 1A) and corresponds with KAT2A in human. A construct will be generated to
target the integration of loxP sites to flank GCN5 exons 12 and 13, which is important for…. In the presence of
Cre recombinase, exon 3 can be deleted with the subsequent introduction of a stop codon (Figure 1A). This will
create a truncation mutant of Hat1 that produces a product less 50 amino acids long. In the event that alternate
splicing occurs in the Hat1 gene that could skip exon 3, only exon 9 can be spliced to exon 2 and retain the
proper reading frame. In this case, the protein that would be produced would lack the entire Hat1 active site.
We’ll generate homozygous GCN5−/− mutant on B6 background by crossing GCN5fl/fl with CD19-Cre. We also
generate GCN5 transgenic (or ectopic expression c)… As a mouse line expressing Cre recombinase from the
gene encoding CD79a (Cd79a-Cre; also known as mb1-Cre)17 initiates deletion of loxP-flanked genes at the
transition from pre-pro-B cell to committed pro-B cell31
We then isolate pre- and pro- B cells

GCN5 bearing two point mutations (E568A and D609A) will be created as previously described32. Specifically,
Genomic sequences covering the entire Gcn5 coding region were isolated previously (39). The targeting vector
was constructed by placing a 6.9-kb XbaI/EcoRV genomic fragment containing exons 8 to 19 of Gcn5 into
Bluescript KS(+). The E568A mutation will be introduced into exon 12, and D609A into exon 13 of Gcn5 by
site-directed mutagenesis. A PGKneobpA neomycin resistance expression cassette was inserted into intron 12
accompanying an EcoRI site. A MC1-TK (thymidine kinase) cassette was added at the 3′ end for negative
selection. For genotyping, genomic DNA was digested with EcoRI/EcoRV and probed with a 0.5-kb
EcoRI/HindIII fragment (5′ probe) or a 0.6-kb XbaI/EcoRI fragment (3′ probe). Creation of Gcn5hat/+
mice.The Gcn5 targeting vector was linearized and electroporated into 129/SvEv-derived embryonic stem (ES)
cells (AB1). Doubly resistant cells [resistant to G418 and 1-(2-deoxy-2-β-d-arabinofuranosyl)-S- iodouracid
(FLAU)] will be genotyped by Southern blot analyses using probes described above. Correctly targeted ES
clones were microinjected into C57BL/6J blastocysts. Chimeras were mated with wild-type C57BL/6J mice,
and heterozygotes were identified among agouti progeny by Southern blot analysis of tail genomic DNA using
the probes described above. Heterozygous mice were intercrossed to generate mice homozygous for the Gcn5
point mutations.

We will measure Vκ-RS rearrangement levels in fraction D, fraction E (k+)

Example of HAT k/o: There is a single homolog of Hat1 in the murine genome that is highly similar to human
and yeast Hat1. The murine Hat1 gene consists of 11 exons (Figure 1A). A construct was generated to target
5
the integration of loxP sites to flank Hat1 exon 3. In the presence of cre recombinase, exon 3 can be deleted
with the subsequent introduction of a stop codon (Figure 1A). This will create a truncation mutant of Hat1
that produces a product less 50 amino acids long. In the event that alternate splicing occurs in the Hat1
gene that could skip exon 3, only exon 9 can be spliced to exon 2 and retain the proper reading frame. In this
case, the protein that would be produced would lack the entire Hat1 active site.
The targeting construct was transfected into mouse embryonic stem (ES) cells and cells grown with
antibiotic selection. Cell lines in which the targeting construct was properly integrated were identified (data
not shown). These cells were then injected into blastocysts to generate chimeric mice. The chimeras were
then mated with wild type mice (C57/Bl6) and the pups were screened by Southern blot to determine
whether germline transmission of the Hat1 flox allele had been achieved. Several animals with a Hat1 flox/Hat1+
genotype were identified
2b)
To investigate the effects of GCN5 deficiency on the gene expression of various B-cell differentiation–related
transcription factors including Bach2, Bcl-6, Blimp-1, E2A, EBF1, Ikaros, IRF-4, MITF, OBF-1, Pax-5, PU.1
and Xbp-1, we will semiquantitative RT-PCR on total RNAs prepared from pre- and pro- B cells
We will also examine the effect of GCN5 deficiency on the protein levels of Blimp-1 and IRF-4 by immunoblot
analysis with specific antibodies. Anti-Blimp-1 and anti-IRF-4 antibodies recognized single positive bands of
about 90 and 52 kDa, respectively.

**
Production of auto-antibodies is largely attributed to loss of tolerance. IRF4 deficiency completely protected
lupus mice from glomerulonephritis despite increased activation of antigen-presenting cells in C57BL/6-
(Fas)lpr mice, resulting in a massive increase in plasma levels of TNF and IL-12p40 (Maciej Lech 2011).
IRF4-deficient mice exhibited a profound reduction in serum immunoglobulin concentrations and did not mount
detectable antibody responses. 33 This same study (1997) showed that spleens from IRF4−/− mice display
increased membrane IgM (mIgM)high mIgDlow, and decreased mIgMlow mIgDhigh, B cell populations. The
frequency of CD23+ B220+ B cells was markedly reduced, and the CD23high B220+ B cell subpopulation was
absent (Fig. 2), indicating a block at a late stage of peripheral B cell maturation (8). Consistent with such a
block was the absence of germinal centers in B cell follicles of spleens and lymph nodes. Immunoglobulin
secretion in response to stimulation with LPS or monoclonal antibody (mAb) to CD40 was severely impaired in
IRF4−/− B cells

(46–48): In pro-pre B cell lines, blocking NF-κB induction reduces κ transcription and rearrangements.

On the other hand, overexpression of IRF-4 is linked to poor prognosis in chronic lymphocytic leukemia and to
the pathogenesis of adult T-cell leukemia and lymphoma.7,8 These studies indicate that IRF-4, when
overexpressed, functions as an oncoprotein. On the other hand, in contrast to its role in promoting tumor
progression in late stages of B lymphopoiesis, expression of IRF-4 is down-regulated in some myeloid and early
B-lymphoid malignancies.
Complete loss of IRF-4 facilitates BCR/ABL transformation of B-lymphoid progenitors, indicating that IRF-4
functions in inhibiting B-lymphoid transformation by BCR/ABL. Forced expression of IRF-4 inhibits
BCR/ABL transformation of B-lymphoid progenitors in vitro 34

We’ll isolate splenic B220+ IgM+ B cells of adult (3–4-mo-old) C57BL/6 mice (n = 5) and CD19+ 9G4−
peripheral B cells
6
ments in B cell populations.iRS-KDE rearrangements were amplified with 59-ATTGATGCT-GCCGTAGCC-
39and59-AGGCTTCCTAGGGAGGTCAG-39primers and were detected with the59-
TCTGCAGCTGCATTTTTGCCA-39FAM-labeled hydrolysis probe

Drugs that inhibit FKBP52, such as ascomycin, increase IRF4 function (86).

Markers for different stages of B cells from BM - Figure 1a from 35

There was a high co-occurrence of binding sites for IRF4 (73%) and PU.1 (54%) at all Ikaros peaks (9,878

like Pax-5-/- pro-B lines, Ik-/- pro-B lines can be differentiated into macrophages when cultured with macrophage
colony-stimulating factor[67]. Thus, in addition to contributing to B lineage specification through the activation
of IL-7R, EBf1 and Flt3 expression, Ikaros also locks in the B lineage by shutting off alternative cell fates.

the abnormal activation threshold in Ikaros-deficient B cells may have real consequences for tolerance.
Mutations that compromise B cell activation thresholds often lead to autoantibody production[79,80].

* In mature B cells: While it is clear that Ikaros regulates B cell responses to stimulation, the mechanism
remains a mystery. Data from T cells have suggested that Ikaros maintains activation thresholds by integrating
the inputs of multiple signaling cascades into a transcriptional response to stimulation[5,74]. The rationale for
this is two fold: (1) in comparison with WT cells, Ik+/- and Ik+/DN T cells are resistant to inhibitors of signaling
pathways that lie downstream of the TCR (MAPK, Ras, PI3K/Akt, LCK/FYN, PKC, calcineurin), indicating

7
that no one pathway is responsible for increased proliferation; and (2) Ikaros, colocalizes with heterochromatin
and replication foci, and thus its loss might result in widespread gene deregulation[5,74]. This latter point may
be especially relevant in cycling cells, which must synthesize their DNA and re-establish heterochromatin with
each cycle. To date however, it is unclear if Ikaros deficiency grossly changes the transcriptional response to
BCR, TCR or mitogen stimulation in lymphocytes, and thus, this model lacks strong experimental support.

* 3 ways that Ikaros may act:

2- Second, in vitro assays have shown that Ikaros can recruit the C terminal binding protein (CtBP) and CtBP
interacting protein (CtIP) co-repressors, which may in turn repress transcription by interacting directly with the
basal transcriptional machinery (TATA binding protein and transcription factor IIB)[32,34,35]
3- Ikaros competes with positive factors to repress transcription of genes such Igll1 [Lambda5; competes with
early B cell factor (EBF)][36], dntt (terminal deoxynucleotidyl transferase; competes with Ets)[37], and Hes1
(competes with the RBP-Jk/Notch complex)[38].

Besides, Ikaros may activate the transcription of certain target genes, possibly by recruiting SWI/SNF, or
promoting transcriptional elongation.

Depletion of B cells results in increase in survival and reduction in multiorgan inflammation in human with
IPEX (immune dysregulation, polyendocrinopathy, enteropathy,X-linked) syndrome36.

Ikaros up-regulates the expression of the genes encoding folliculin (Flcn) and folliculin-interacting protein 1
(Fnip1), important for energy homeostasis, which were recently identified as essential checkpoints in pre-B
cells (Baba et al., 2012; Park et al., 2012).

Two folliculin interacting proteins, FNIP1 and FNIP2. pro-B to pre-B cell block in Fnip1−/− mice, which
cannot be rescued by rapamycin treatment and is thus mTOR independent. Transcriptome analyses of Fnip1−/−
pro-B cells reveal compromised expression of key genes implicated in B-cell surface expression, including
VpreB1/VpreB2, λ5, and Rag1/Rag2. We also show that expression of prerecombined heavy and light-chain
genes in Fnip1−/− mice helps bypass the pro-B cell arrest to the transitional bone marrow (BM) IgM+
compartment. Strikingly, the reconstituted immature B cells fail to migrate to the periphery. Finally, in support
of an in vivo functional link between Flcn and Fnip1, we show that tamoxifen-inducible deletion of folliculin in
mice recapitulates the Fnip1−/− pro-B cell arrest 6

An important question is how Ikaros is regulated during pre-B cell differentiation. Ikaros transcripts and protein
are down-regulated in fraction C and up-regulated in fraction C′ cells, suggesting that Ikaros function may dip
when the pre-BCR is first expressed, and that pre-BCR signaling may up-regulate Ikaros expression.
Furthermore, FOXO1 may regulate Ikaros through correct mRNA splicing (Alkhatib et al., 2012), and SYK
may influence Ikaros localization and binding activity (Uckun et al., 2012). Additional research will be needed
to determine how Ikaros activity is modulated during differentiation. 3 mice carrying a germline hypomorphic
Ikaros mutation, which show a partial block in pro-B to pre-B cell development (Kirstetter et al., 2002).

The protein tyrosine phosphastase PTP1B has been shown to downregulate CD40, BAFF-R, TLR-4 signalling37.
Lower PTP1B level in B cells is associated with rheumatoid arthritis; however, it is not clear whether this is
cause or effect. While specific inhibitors for PTP1B have entered clinical trials We aim to study the role of
PTP1B in the development of antigen-specific B cells.

8
PTP1B 2014 paper: Ptpn1f/f-mb1cre mice show no impairment of B cell development; PTP1B-negative B cells
respond better to anti-CD40 and BAFF stimulus; PTP1B-deficient splenic B cells show increased p38 and Akt
activation; Ptpn1f/f-mb1cre mice have an elevated TD immune response.
The downstream signaling of activated B cells includes several tyrosine phosphorylation steps, which are under
the tight control of protein tyrosine phosphatases (PTPs; Pao et al., 2007a; Kurosaki and Hikida, 2009). Most
nonreceptor PTPs play an inhibitory role in the regulation of B cell activation; therefore, they are important to
maintain immunological tolerance. Indeed, loss of PTP function can lead to autoimmune disorders (Vang et al.,
2008).

Figure 2: Key steps in the development of antigen-specific B cells.

B Cell Maturation Antigen (BCMA) is an important receptor that binds two ligands: BAFF and APRIL.
Although BCMA was reported to be important in maintaining the long-lived PC compartment in immunized B6
mice, we previously demonstrated that the spontaneous development of long-lived PCs, autoantibody
production, and lethality in two autoimmune-prone mouse models were significantly worsened in the absence of
BCMA. These observations suggest that, in autoimmune-prone mice, signals through BCMA on B cells help
control GC B cell homeostasis and the stringent elimination of autoreactive GC B cells

Neutrophils in SLE BM produce increased levels of IFN-α and APRIL (paper: Neutrophil-Mediated IFN
Activation in the Bone Marrow Alters B Cell Development)
IFN-regulated chemokines = IP-10 (CXCL10), MCP-1 (CCL2), and MIP-3b (CCL19)

Flow cytometric analysis of CD19+ BM cells separates B cells into early (CD24+++, CD38+++ expression) versus
mature (excluding CD24+++, CD38+++) populations. Early B cells were further divided into pro/pre-, immature,
and early transitional (T1 and T2) B cells based on the expression of IgD and IgM. IFN-high lupus patients
exhibit reduced pro/pre-B cells and elevated early transitional T1 and T2 cells compared with NC and IFN-low
SLE.

lyn−/− mice manifest considerable reduction in the numbers of mature follicular and immature transitional (T1,
T2, and T3) B cells in lymphoid organs but equal numbers of newly formed immature B cells in the bone
marrow  the absence of Lyn inhibitory signaling in B cells alone is sufficient to drive autoimmunity.

Gotot et al demonstrated that Treg cells are essential to suppress autoreactive B cells in an antigen-specific
manner and to prevent them from producing autoantibodies. This suppression requires PD-1 expression on
autoreactive B cells and expression of the two PD-1 ligands on Treg cells. These findings demonstrate in vivo
that Treg cells use PD-1 ligands to directly suppress autoreactive B cells

9
lack of IRF4 prevents LN in B6lpr mice, wherein B6lpr/Irf4 −/− mice hardly developed any glomerular Ig and
complement deposits or glomerular abnormalities based on the morphometrical assessment of the activity and
chronicity index for LN [37]
On the other hand, IRF4+/+ mice developed severe EAE [48]. dense inflammatory infiltrates in the spinal
sections of IRF4+/+ mice but not in those of IRF4−/− mice
There’s no report on disregulation of Irf4 in B cells in the context of autoimmune disease 38

the absence of IRF-4 expression in c-rel−/− B cells coincided with a greater sensitivity of these cells to the
antiproliferative activity of IFNs.39
NF-kb activity may be regulated differently in autoimmune context. For example, during inflammatory
responses, p50 homo-dimers would be replaced by transcription activating p50/p65 or p50/Rel heterodimers to
enhance κ gene expression required to secrete large amounts of antibodies of defined specificity.40

significant reduction in the number and percentage of B lineage cells was apparent in IkBα transduced chimeric
mice. IkBα expression decreased the percentage of pre-B and immature B cell subsets in the bone marrow

1. Schwickert TA, Tagoh H, Gultekin S, Dakic A, Axelsson E, Minnich M, et al. Stage-specific control of
early B cell development by the transcription factor Ikaros. Nat Immunol 2014, 15(3): 283-293.

2. Wang JH, Avitahl N, Cariappa A, Friedrich C, Ikeda T, Renold A, et al. Aiolos regulates B cell
activation and maturation to effector state. Immunity 1998, 9(4): 543-553.

3. Heizmann B, Kastner P, Chan S. Ikaros is absolutely required for pre-B cell differentiation by
attenuating IL-7 signals. J Exp Med 2013, 210(13): 2823-2832.

4. Alexander T, Sattler A, Templin L, Kohler S, Gross C, Meisel A, et al. Foxp3+ Helios+ regulatory T
cells are expanded in active systemic lupus erythematosus. Ann Rheum Dis 2013, 72(9): 1549-
1558.

5. Nagpal K, Watanabe KS, Tsao BP, Tsokos GC. Ikaros represses protein phosphatase 2A (PP2A)
expression through an intronic binding site. J Biol Chem 2014.

6. Baba M, Keller JR, Sun HW, Resch W, Kuchen S, Suh HC, et al. The folliculin-FNIP1 pathway deleted
in human Birt-Hogg-Dube syndrome is required for murine B-cell development. Blood 2012,
120(6): 1254-1261.

7. Muller MR, Rao A. NFAT, immunity and cancer: a transcription factor comes of age. Nat Rev
Immunol 2010, 10(9): 645-656.

8. Amin RH, Cado D, Nolla H, Huang D, Shinton SA, Zhou Y, et al. Biallelic, ubiquitous transcription
from the distal germline Ig{kappa} locus promoter during B cell development. Proc Natl Acad Sci U
S A 2009, 106(2): 522-527.

9. Panigrahi AK, Goodman NG, Eisenberg RA, Rickels MR, Naji A, Luning Prak ET. RS rearrangement
frequency as a marker of receptor editing in lupus and type 1 diabetes. J Exp Med 2008, 205(13):
2985-2994.

10. Pathak S, Ma S, Trinh L, Lu R. A role for interferon regulatory factor 4 in receptor editing. Mol Cell
Biol 2008, 28(8): 2815-2824.

10
11. Nemazee D. Receptor editing in lymphocyte development and central tolerance. Nat Rev Immunol
2006, 6(10): 728-740.

12. Luning Prak ET, Monestier M, Eisenberg RA. B cell receptor editing in tolerance and autoimmunity.
Ann N Y Acad Sci 2011, 1217: 96-121.

13. Witsch EJ, Cao H, Fukuyama H, Weigert M. Light chain editing generates polyreactive antibodies in
chronic graft-versus-host reaction. J Exp Med 2006, 203(7): 1761-1772.

14. Mazari L, Ouarzane M, Zouali M. Subversion of B lymphocyte tolerance by hydralazine, a potential

mechanism for drug-induced lupus. Proc Natl Acad Sci U S A 2007, 104(15): 6317-6322.

15. Liu Y, Li L, Kumar KR, Xie C, Lightfoot S, Zhou XJ, et al. Lupus susceptibility genes may breach
tolerance to DNA by impairing receptor editing of nuclear antigen-reactive B cells. J Immunol
2007, 179(2): 1340-1352.

16. Yurasov S, Wardemann H, Hammersen J, Tsuiji M, Meffre E, Pascual V, et al. Defective B cell
tolerance checkpoints in systemic lupus erythematosus. J Exp Med 2005, 201(5): 703-711.

17. Li G, Zan H, Xu Z, Casali P. Epigenetics of the antibody response. Trends Immunol 2013, 34(9): 460-
470.

18. Reilly CM, Regna N, Mishra N. HDAC inhibition in lupus models. Mol Med 2011, 17(5-6): 417-425.

19. Forster N, Gallinat S, Jablonska J, Weiss S, Elsasser HP, Lutz W. p300 protein acetyltransferase
activity suppresses systemic lupus erythematosus-like autoimmune disease in mice. J Immunol
2007, 178(11): 6941-6948.

20. Shapiro-Shelef M, Lin KI, McHeyzer-Williams LJ, Liao J, McHeyzer-Williams MG, Calame K. Blimp-1
is required for the formation of immunoglobulin secreting plasma cells and pre-plasma memory B
cells. Immunity 2003, 19(4): 607-620.

21. Schebesta A, McManus S, Salvagiotto G, Delogu A, Busslinger GA, Busslinger M. Transcription

factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration,
and immune function. Immunity 2007, 27(1): 49-63.

22. Swaminathan S, Duy C, Muschen M. BACH2-BCL6 balance regulates selection at the pre-B cell
receptor checkpoint. Trends Immunol 2014, 35(3): 131-137.

23. Ma S, Turetsky A, Trinh L, Lu R. IFN regulatory factor 4 and 8 promote Ig light chain kappa locus
activation in pre-B cell development. J Immunol 2006, 177(11): 7898-7904.

24. Kikuchi H, Nakayama M, Kuribayashi F, Imajoh-Ohmi S, Nishitoh H, Takami Y, et al. GCN5 is

essential for IRF-4 gene expression followed by transcriptional activation of Blimp-1 in immature
B cells. J Leukoc Biol 2014, 95(3): 399-404.

25. Kikuchi H, Nakayama T. GCN5 and BCR signalling collaborate to induce pre-mature B cell
apoptosis through depletion of ICAD and IAP2 and activation of caspase activities. Gene 2008,
419(1-2): 48-55.

11
26. Lamoureux JL, Watson LC, Cherrier M, Skog P, Nemazee D, Feeney AJ. Reduced receptor editing in
lupus-prone MRL/lpr mice. J Exp Med 2007, 204(12): 2853-2864.

27. Mojica MP, Perry SS, Searles AE, Elenitoba-Johnson KS, Pierce LJ, Wiesmann A, et al. Phenotypic
distinction and functional characterization of pro-B cells in adult mouse bone marrow. J Immunol
2001, 166(5): 3042-3051.

28. Holmlund T, Lindberg MJ, Grander D, Wallberg AE. GCN5 acetylates and regulates the stability of
the oncoprotein E2A-PBX1 in acute lymphoblastic leukemia. Leukemia 2013, 27(3): 578-585.

29. Xu W, Edmondson DG, Evrard YA, Wakamiya M, Behringer RR, Roth SY. Loss of Gcn5l2 leads to
increased apoptosis and mesodermal defects during mouse development. Nat Genet 2000, 26(2):
229-232.

30. Yamauchi T, Yamauchi J, Kuwata T, Tamura T, Yamashita T, Bae N, et al. Distinct but overlapping
roles of histone acetylase PCAF and of the closely related PCAF-B/GCN5 in mouse embryogenesis.
Proc Natl Acad Sci U S A 2000, 97(21): 11303-11306.

31. Kwon K, Hutter C, Sun Q, Bilic I, Cobaleda C, Malin S, et al. Instructive role of the transcription
factor E2A in early B lymphopoiesis and germinal center B cell development. Immunity 2008,
28(6): 751-762.

32. Bu P, Evrard YA, Lozano G, Dent SY. Loss of Gcn5 acetyltransferase activity leads to neural tube
closure defects and exencephaly in mouse embryos. Mol Cell Biol 2007, 27(9): 3405-3416.

33. Mittrucker HW, Matsuyama T, Grossman A, Kundig TM, Potter J, Shahinian A, et al. Requirement
for the transcription factor LSIRF/IRF4 for mature B and T lymphocyte function. Science 1997,
275(5299): 540-543.

34. Acquaviva J, Chen X, Ren R. IRF-4 functions as a tumor suppressor in early B-cell development.
Blood 2008, 112(9): 3798-3806.

35. Kikuchi K, Lai AY, Hsu CL, Kondo M. IL-7 receptor signaling is necessary for stage transition in
adult B cell development through up-regulation of EBF. J Exp Med 2005, 201(8): 1197-1203.

36. Mihai CT, Rotinberg P, Brinza F, Vochita G. Extremely low-frequency electromagnetic fields cause
DNA strand breaks in normal cells. J Environ Health Sci Eng 2014, 12(1): 15.

37. Medgyesi D, Hobeika E, Biesen R, Kollert F, Taddeo A, Voll RE, et al. The protein tyrosine
phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell
autoimmunity. J Exp Med 2014.

38. Xu WD, Pan HF, Ye DQ, Xu Y. Targeting IRF4 in autoimmune diseases. Autoimmun Rev 2012,
11(12): 918-924.

39. Grumont RJ, Gerondakis S. Rel induces interferon regulatory factor 4 (IRF-4) expression in
lymphocytes: modulation of interferon-regulated gene expression by rel/nuclear factor kappaB. J
Exp Med 2000, 191(8): 1281-1292.

12
40. Zhong H, May MJ, Jimi E, Ghosh S. The phosphorylation status of nuclear NF-kappa B determines
its association with CBP/p300 or HDAC-1. Mol Cell 2002, 9(3): 625-636.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0054896  histone acetylation

measurement method

1.AbstractThe question to be addressed and the approaches to be taken should be succinctly described.
Theabstract should be ½ page or less.
2.Specific AimsProvide a short introductory paragraph that delineates the broad, long-term objectives and what
thespecific research is intended to accomplish. State the hypotheses to be defended. One page or less
isrecommended.
3.Background and SignificanceBriefly sketch the background leading to the present proposal, critically evaluate
existing knowledge inthe field, and identify gaps that the project is intended to fill. State concisely the
importance of the workto a basic biological or clinical issue. Remember that this section must provide a
rationale for the specificaims that have been formulated.Upto three pages is recommended.
4.Research DesignDescribe the research design and the procedures that will be used to address the specific
aims. Includehow the data will be collected, analyzed, and interpreted. Indicate possible outcomes of
experiments and how these will influence the direction of the studies. Discuss potential difficulties and
limitations. Theexperimental methods should not include too many details regarding buffers, concentrations
ofantibodies,etc., although the committee may ask the student about such details during the oralpresentation.
This section should form the major portion of the application and should be at least fourpages in length.

Sections1-4should be no more than 10 single-spaced pages in length. Font size should beat least
11or12pointwith½ inchmarginson all sides. Adherence to these formatting instructions ismandatory forthe
submissionof the proposal

DNMT1 mRNA expression isreduced in T cells from active lupuspatients. This finding suggest that T-cell DNA
hypomethylation in lupus might becaused by reduced DNMT1 expression. SLE T cellsexhibit increased
andprolonged expressionof cell-surface CD40L, spontaneouslyoverproduce IL-10, butoverproduce INF-γ

DNA methylation involves the covalent modification of the 5th carbon in cytosine residues of CG
dinucleotides. Approximately 70% to 80% of CG dinucleotides are methylated in normal mammalian cells.

treatment of MRL/lpr splenocytes with histone deacetylases inhibitors in vitro resulted in the reduced
expression of several cytokines, which include interleukin-12 (IL-12), interferon-γ (IFN-γ), IL-6, and IL-10.
These effects on cytokine gene expression were associated with increased histones H3 and H4 acetylation
following treatment with histone deacetylase inhibitors. 53

13