Functional replacement of the mouse E2A gene with a human HEB cDNA - PubMed (original) (raw)

Functional replacement of the mouse E2A gene with a human HEB cDNA

Y Zhuang et al. Mol Cell Biol. 1998 Jun.

Abstract

The mammalian E2A, HEB, and E2-2 genes encode a unique class of basic helix-loop-helix (bHLH) transcription factors that are evolutionarily conserved and essential for embryonic and postnatal development. While the structural and functional similarities among the gene products are well demonstrated, it is not clear why deletion of E2A, but not HEB or E2-2, leads to a complete arrest in B-lymphocyte development. To understand the molecular basis of the functional specificity between E2A and HEB/E2-2 in mammalian development, we generated and tested a panel of E2A knockin mutations including subtle mutations in the E12 and E47 exons and substitution of both E12 and E47 exons with a human HEB cDNA. We find that the alternatively spliced E12 and E47 bHLH proteins of the E2A gene play similar and additive roles in supporting B lymphopoiesis. Further, we find that HEB driven by the endogenous E2A promoter can functionally replace E2A in supporting B-cell commitment and differentiation toward completion. Finally, the postnatal lethality associated with E2A disruption is fully rescued by the addition of HEB. This study suggests that the functional divergence among E12, E47, and HEB in different cell types is partially defined by the context of gene expression.

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Figures

FIG. 1

(A) The 3′ portion of the mouse E2A genomic DNA (from _Kpn_I to _Bam_HI) was used in the gene targeting experiment. The exons contained within this piece of genomic DNA are shown as shaded boxes. The direction of the E2A gene, positions of E12 and E47 exons, and the 3′ polyadenylation site of the E2A gene are shown. The structures of E12ko and E47bm alleles are shown according to the scale of the wild type (wt). The E12ko allele was generated by insertion of a promoterless neo gene into the E12 exon (34). The E47bm allele was generated by cointegration of a point mutation and a selection marker into the E2A gene. “B” above the E47 exon of the E47bm allele indicates the position of the basic-region mutation and the addition of a _Bam_HI site. A PGK-Neo cassette running in the same direction as the E2A gene is inserted into the unique _Xba_I site downstream of the E2A gene. The names, positions, and directions of PCR primers used in the experiment are shown below the constructs. RV, _Eco_RV. (B) Detailed structure of the E47-specific exon. Sequences of the basic region of E47 wild-type and E47bm alleles are shown. Underlines indicate amino acid residues that are conserved in the bHLH gene family, and boldface indicates the changes made in E47bm. (C) RT-PCR analysis of thymocyte RNA prepared from E47bm homozygous, E47bm heterozygous, and wild-type animals. For each reverse-transcribed RNA sample, four sets of PCRs were performed with primers specific to (i) EF1α gene, (ii) E2A exons common to both E12 and E47, (iii) the E12-specific exon, and (iv) the E47-specific exon. To differentiate E47 wild-type and the E47bm alleles, PCR product generated with E47 primers was digested with _Bam_HI, which is present in the E47bm but not the wild-type allele (in lanes E47bm +/− and E47bm −/−). Results are representative of three repeated experiments with multiple individuals from each genotype. (D) Western analysis of E2A proteins in the thymus isolated from the wild-type, E2Ako, E47bm, and E12ko mice. Genotypes of the samples are indicated at the top. Size markers in kilodaltons are shown at the left, and positions of the full-length E2A proteins are indicated at the right.

FIG. 2

(A) Three-channel FACS analysis of live bone marrow (top two panels) and spleen B cells (bottom panel) prepared from 10- to 20-day-old pups carrying various E2A mutations. The genotype of each animal is indicated above each vertical panel, which is composed of three FACS analyses of the same animal. B220-FITC and 7AAD were included in all the stainings. In addition, CD19-PE or CD43-PE was used in the bone marrow samples, and IgM-PE was used in the spleen samples. The variation in the levels of CD19-PE staining is due to batch difference in the antibody used. To enrich lymphocytes and to exclude dead cells in the analysis, size gate and 7AAD gate were used for all the dot plots displayed. The relative percentages of pro-B, pre-B, and mature B cells in total live cells are given in each plot. FACS analysis of B cells in adult mice. Wild-type control, E47bm homozygous and E47bm/E12ko transheterozygous mice were analyzed with B220 and 7AAD plus CD19 for bone marrow, IgM for spleen, and CD5 for peritoneum samples. Presentation of data is as described for panel A. (C) Average B-cell numbers as a percentage of total nucleated bone marrow or spleen populations. Both sample size and standard deviation are shown with each data bar. Four genotype groups of animals, wild-type (wt), E47bm homozygous, E12ko homozygous, and E47bm/E12ko transheterozygous, are included. Pup is equivalent to 10 to 20 days old; adult is more than 2 months old.

FIG. 3

PCR analysis of Ig heavy-chain gene D-J rearrangements. Genomic DNA was purified from bone marrow of either 10- to 20-day-old neonates or 2-month-old adults. PCR was carried out with a J4-specific primer (24) and a D-segment-specific primer that hybridizes to the upstream sequence of most D segments (8). PCR products representing specific D-J rearrangements were revealed by a radiolabeled oligonucleotide that hybridizes to the internal side of the J4 primer. The running positions of DNA size markers and J segments are indicated on the left and right side, respectively. WT, wild type.

FIG. 4

(A) FACS analysis of spleen samples from stem cell transfusion experiments. Sources of donor stem cells are indicated at the top (BM, bone marrow; wt, wild type; FL, fetal liver). Each vertical panel represents separate FACS analysis for B cells (IgM), T cells (CD5), and myeloid cells (Mac1) from the same splenic sample. CD45.2 (Pharmingen) is an allotype marker that is present on the donor cells and absent on host cells. To eliminate nonspecific staining and dead cells, 7AAD was also included in all the staining reactions. Percentages of cells given in the quadrants are calculated based on total live cells rather than the size-gated population. Data are representative of two to five independent transfusion tests for each donor type. (B) FACS analysis of an E47bm mouse (representative of three mice) reconstituted with the wild-type HSC. Cells from bone marrow, spleen, and blood were analyzed with a B-cell marker (B220) and a myeloid cell marker (Mac1). Donor cells were derived from wild-type C57BL/6Ly5A mice and were negative for CD45.2, whereas host cells were positive for the CD45.2 marker. Gating and data presentation are as in panel A.

FIG. 4

FIG. 5

(A) Gene targeting construct for generating the E2Aheb allele. The Neo marker was introduced in the E2A gene as a fusion protein. IRES-driven human HEB cDNA was inserted downstream of the Neo cassette but in front of the E2A translation termination site. Both E12 and E47 exons are completely deleted from the targeting construct. Primers used for PCR genotyping the mutant and wild-type (wt) alleles are indicated (see Materials and Methods). Designations are as in Fig. 1A. (B) Western blot analysis of thymus (lanes 1 to 4) and spleen (lanes 5 to 8) nuclear extracts from 10-day-old neonatal mice of indicated genotypes. Wild-type, E2Ako heterozygote, E2Aheb heterozygote, and E2Aheb homozygote samples are included in the assay as indicated. The anti-HEB polyclonal sera used in this assay (Santa Cruz) cross-react with both human and mouse HEB proteins. Size markers in kilodaltons and the HEB band are indicated on the left and right, respectively. (C) Gel shift analysis of E-protein complex formation, using μE5 radiolabeled probe. μE5 probe (25) was incubated with nuclear extracts from splenocytes (lanes 1 to 6) or thymocytes (lanes 7 to 11) derived from wild-type (lanes 1, 2, 3, 7, 8, and 9) or E2Aheb (lanes 4, 5, 6, 10, and 11) animals. Anti-E2A antibody (Yae) was added in lanes 2, 5, and 8, and anti-HEB antibody (Santa Cruz) was added in lanes 3, 6, 9, and 11. The E2A- and HEB-dependent supershifts are indicated by arrows on the left and right sides, respectively. Assay conditions are as described by Sawada and Littman (22).

FIG. 6

(A) FACS analysis of mice carrying one or two copies of E2Aheb. Bone marrow (top two panels) and spleens (bottom panel) from 10-day-old wild-type (WT), E2Ako/E2Aheb transheterozygous, and E2Aheb homozygous mice were analyzed with B-cell markers. Plots were gated on live lymphocytes based on cell size and 7AAD staining. Percentages of cells shown in the quadrants represent the fractions of total live cells analyzed. (B) B-cell contents as a percentage of total live suspension cells for the five genotypes. Percentages of B cells in bone marrow and spleen are calculated based on the analysis of multiple litters with ages ranging from 1 to 3 weeks. Sample size and standard deviation are shown with each data bar. Relatively large standard deviations for most spleen samples are due to a sharp increase of B-cell content in the first 3 weeks of postnatal life.

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Functional replacement of the mouse E2A gene with a human HEB cDNA - PubMed (original) (raw)