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. 2015 Apr;29(4):1505-15.
doi: 10.1096/fj.14-263061. Epub 2014 Dec 30.

Setd1a regulates progenitor B-cell-to-precursor B-cell development through histone H3 lysine 4 trimethylation and Ig heavy-chain rearrangement

Betsabeh Khoramian Tusi  1 , Changwang Deng  1 , Tal Salz  1 , Leilani Zeumer  1 , Yangqiu Li  1 , Chi Wai Eric So  1 , Laurence M Morel  1 , Yi Qiu  2 , Suming Huang  2
Affiliations

Setd1a regulates progenitor B-cell-to-precursor B-cell development through histone H3 lysine 4 trimethylation and Ig heavy-chain rearrangement

Betsabeh Khoramian Tusi et al. FASEB J. 2015 Apr.

Abstract

SETD1A is a member of trithorax-related histone methyltransferases that methylate lysine 4 at histone H3 (H3K4). We showed previously that Setd1a is required for mesoderm specification and hematopoietic lineage differentiation in vitro. However, it remains unknown whether or not Setd1a controls specific hematopoietic lineage commitment and differentiation during animal development. Here, we reported that homozygous Setd1a knockout (KO) mice are embryonic lethal. Loss of the Setd1a gene in the hematopoietic compartment resulted in a blockage of the progenitor B-cell-to-precursor B-cell development in bone marrow (BM) and B-cell maturation in spleen. The Setd1a-cKO (conditional knockout) mice exhibited an enlarged spleen with disrupted spleen architecture and leukocytopenia. Mechanistically, Setd1a deficiency in BM reduced the levels of H3K4me3 at critical B-cell gene loci, including Pax5 and Rag1/2, which are critical for the IgH (Ig heavy-chain) locus contractions and rearrangement. Subsequently, the differential long-range looped interactions of the enhancer Eμ with proximal 5' DH region and 3' regulatory regions as well as with Pax5-activated intergenic repeat elements and 5' distal VH genes were compromised by the Setd1a-cKO. Together, our findings revealed a critical role of Setd1a and its mediated epigenetic modifications in regulating the IgH rearrangement and B-cell development.

Keywords: B-cell differentiation; Setd1a KO mice; long-range chromatin loops; promoter H3K4me3; transcriptional regulation.

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Figures

Figure 1.
Figure 1.
Strategies for conditional inactivation of Setd1a alleles and mutant phenotypes. A) Schematic representation of a KO strategy and the construct used for generating cKO of Setd1a. E, exon; P, primer; SA-IRES, splice acceptor-internal ribosome entry site. The Setd1a-targeted deletion allele was converted to cKO allele upon Flp recombination and Cre-induced excision of exon 4. B) PCR-based genotyping of WT (+/+), heterozygous floxed (fl/+), and homozygous floxed (fl/fl) littermates (top). PCR-based genotyping of the polydI/dC-induced allele deletion in the hematopoietic compartment (bottom). MW, molecular weight. C) RT-qPCR assays of Setd1a expression in the BM cells derived from 3 Setd1a-cKO (Setd1afl/fl:Mx1-cre+) and 3 WT control mice (Setd1a+/+:Mx1-cre+). D) RT-qPCR assays of Setd1a expression in BM B220+ B cells and spleen (SP) IgM+/B220+ B cells.
Figure 2.
Figure 2.
Loss of Setd1a in the hematopoietic compartment resulted in a blockage of B-cell development. A) Loss of Setd1a in the hematopoietic compartment resulted in splenomegaly. Whole spleens (top) and their weights (bottom; n = 10) are shown for Setd1a-cKO and WT control mice. Data are shown as the mean ± sd. *P ≤ 0.05. B) H&E staining (top) and immunohistochemical staining with anti- B220 antibody (bottom) for spleen sections in Setd1a mutant (Setd1afl/fl:Mx1-cre+; n = 3) revealed constricted and disrupted B-cell follicles compared with control (Setd1a+/+:Mx1-cre+; n = 3). The lymphoid follicles (F) are indicated. C) Representative FACS analysis of transitional B cells from T1 stage (AA4.1+/Cd23−/IgMhigh) to the more-matured T3 stage (AA4.1+/Cd23+/IgMlow) in the spleen. The Setd1a+/Δ mice (n = 2) exhibited a block in transition from T1 (AA4.1+/Cd23−/IgMhigh) to T2 (AA4.1+/Cd23+/IgMhigh) stages compared to the Setd1a+/+ control mice (n = 2).
Figure 3.
Figure 3.
Setd1a conditional deletion blocks pro-B to pre-B development. A) Representative FACS analysis of BM pro-B population (IgM− B220+ CD43+) and pre-B population (IgM− B220+ CD43−) comparing between the control (n = 4) and Setd1a-cKO (n = 5) mice induced with polydI/dC. Numbers indicate the percentage of gated IgM− leukocytes (left) and IgM− B220+ total BM cells (right). B) Representative FACS analysis of mature B-lymphocytes in spleen comparing between the control (n = 5) and Setd1a-cKO (n = 6) mice induced with polydI/dC. Numbers indicate the percentage of gated total spleen leukocytes. C) CFC assay analysis of formation of definitive pre-B colonies comparing between the control (n = 3) and Setd1a-cKO (n = 3) mice induced with polydI/dC. Data are shown as the mean ± sd. ***P ≤ 0.001.
Figure 4.
Figure 4.
Loss of Setd1a inhibits transcription of key regulators required for B-cell identity and development. A) qRT-PCR analysis of Pax5 transcripts in BM hematopoietic cells comparing the controls (Setd1a +/+:Mx1-cre+; n = 3) and Setd1a-cKO mutants (Setd1a fl/fl:Mx1-cre+; n = 3). Lin, lineage. B) Schematic representation of Pax5 locus and ChIP data from the Encyclopedia of DNA Elements (ENCODE) project showing H3K4me3 enrichment peaks at Pax5 promoter/enhancer regions. ChIP primers are indicated by black lines (top). ChIP analyses of H3K4me3 levels at the Pax5 promoter regions in BM-derived pro-B cells cultured in IL-7 (10ng/ml) comparing the Setd1a control and Setd1a-cKO mice (bottom). C) qRT-PCR analysis of Rag1 and Rag2 transcripts in BM hematopoietic cells comparing the controls (Setd1a +/+:Mx1-cre+; n = 2) and Setd1a-cKO mutants (Setd1a fl/fl:Mx1-cre+; n = 2). D) Schematic representation of Rag1 and Rag2 loci and ChIP data from the ENCODE project showing H3K4me3 enrichment peaks at their promoter regions. ChIP primers are indicated by gray lines (top). ChIP analyses of H3K4me3 levels at the Rag1/2 promoter regions in BM-derived pro-B cells cultured in IL-7 (10ng/ml) from Setd1a control and Setd1a-cKO mice (bottom). Data are shown as mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.
Figure 5.
Figure 5.
Setd1a deficiency impairs the binding of Pax5 and H3K4me3 enrichment in the rearranging IgH locus. A) Schematic representation of PAIR elements at the IgH locus (top). ChIP analyses of Pax5 recruitment at the PAIR element sites at IgH locus compared the control and Setd1a-cKO mice. B) Expression of PAIR4 and PAIR6 antisense transcripts is measured using RT-qPCR comparing the control and Setd1a-cKO mice. C) ChIP analysis in BM-derived pro-Bs cultured in IL-7 (10 ng/ml) revealed that loss of Setd1a disrupts H3K4me3 enrichment at the IgH locus. Data are shown as the mean ± sd. *P < 0.05; **P < 0.01.
Figure 6.
Figure 6.
3C analyses of long-range chromatin interactions between Eμ-interacting elements in the rearranging IgH locus. A) Schematic representation of the interacting regions at the IgH locus. Dashed vertical lines indicate HindIII cleavage sites. B) The 3C analyses of long-range interactions of the enhancer Eμ with the distal 3′ VHJ558, PAIR elements, proximal 5′ DFL, 3′RR regions, and the negative control regions across the IgH locus comparing the control and Setd1a-cKO mice. Dark-gray oval regions designated as B, C, and D were used as negative control regions. C and D) A total of 3 independent 3C experiments were quantitated by densitometry. Data are shown as the mean ± sd. *P < 0.05; **P < 0.01.
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