Severe T cell lymphopenia
While surveying the descendants of chemically mutagenized mice for lymphocyte deficiencies (Siggs et al., 2011
), we identified a single male mouse devoid of T cells. This phenotype, nicknamed scanT
because of the low (or scant) frequency of T cells, was transmitted as a recessive trait (). scanT
mutant mice were born at the expected Mendelian ratio, were typically fertile, and were outwardly normal in behavior.
Figure 1. T cell aplasia in scanT mice. (A) Initial generations of the scanT pedigree. Black symbols, T-deficient phenotype; gray symbols, wild-type phenotype; open symbols, not tested. (B) Percentages of B (CD19+) and T (CD3ε+) cells in the peripheral (more ...)
scanT thymi were severely hypoplastic (), containing around 2% of the number of wild-type cells, and lacked corticomedullary definition (). Thymocyte development was impaired from the early thymic precursor (ETP) stage and beyond (). Of the lymphocytes that were present in the thymus, almost all were B cells, representing a 20-fold increase over wild-type numbers ().
Mutation of a previously uncharacterized zinc finger protein
We mapped the scanT
genetic lesion first by genome-wide linkage to chromosome 12 () and then by fine mapping to a 5-Mbp interval between markers D12Mit33 and D12Mit4 (). Because none of the 35 annotated protein-encoding genes in the interval (Table S1
) had previously been implicated in T cell development, we sequenced the coding exons and flanking splice junctions of three (Zbtb1
, and Ppp2r5e
) based on their predominant expression in lymphocytes (http://biogps.gnf.org/
). 93.4% of all target nucleotides (6,081/6,511) were covered on both strands of wild-type and scanT
samples with a Phred quality score of >30, and a single missense transition was identified in Zbtb1
(C74R; ). PolyPhen-2 (Adzhubei et al., 2010
) assigned a score of 0.954 to this mutation, predicting a deleterious effect with 93% confidence. 43.2% of the total coding critical region (59,447/137,702 nt) was also covered at least three times by SOLiD 3 sequencing, with no additional mutations found.
Figure 2. Identification of a missense mutation in ZBTB1. (A and B) Chromosomal mapping (A) and fine mapping (B) of the scanT phenotype. LOD, logarithm of odds score. (C) DNA sequence chromatograms of the mutated nucleotide in Zbtb1 (thymine to cytosine), resulting (more ...) Zbtb1
encodes a 713-aa member of the POK (POZ [Poxviruses and zinc finger] and Krüppel) or BTB-ZF family of transcriptional regulators and had no previously described function. Mouse ZBTB1 is 94% identical to its counterpart in man. Two characteristic domains are shared by BTB-ZF family members: an N-terminal BTB/POZ domain and a series of C-terminal C2
Krüppel-type zinc finger motifs. ZBTB1 itself contains an N-terminal BTB domain, eight zinc finger motifs, and two nuclear localization sequences (Matic et al., 2010
). The residue mutated in the scanT
pedigree (C74) is predicted to lie within the A3 helix of the BTB domain ( and Fig. S1
; Stogios et al., 2005
) and is highly conserved across the vertebrate lineage (). Two unique transcripts are predicted to arise from the Zbtb1
locus, both of which harbor the scanT
mutation, yet only one of which (Ensembl release 65 accession no. ENST00000394712) is predominantly expressed in T cells and other leukocytes in mouse and man (Fig. S2
). The other (Ensembl release 65 accession no. ENST00000358738) is known to be transcribed in HeLa cells by the nonconventional single-polypeptide nuclear RNA polymerase IV (Kravchenko et al., 2005
To confirm that the mutation in Zbtb1 was responsible for the scanT phenotype, wild-type Zbtb1 was retrovirally transduced into scanT hematopoietic progenitors (). GFP+ T cells were recovered from recipients of Zbtb1-transduced scanT progenitors (), presumably as a consequence of restored thymic development. These data indicated that the mutation in Zbtb1 could account for T cell deficiency in scanT mice.
In a minority of cases (3/16 in a 3-mo-old cohort of Zbtb1scanT
homozygotes), T cells were detected at diminished frequencies in the blood of germline mutant mice. We hypothesized that these cells arose from a somatic mutation that suppressed the scanT
phenotype, considering the strong selective advantage that such a mutation would confer and given that a similar phenomenon is known to occur in human T cell–deficient SCID (Hirschhorn et al., 1996
). To test for somatic suppressor mutations, DNA was isolated from sorted T and B cells. Sequencing of the mutated Zbtb1
locus revealed a heterozygous mutation within the T cell compartment of one phenotypic revertant (but not the other two; ), indicating that most, if not all, T cells in this mouse were clonally derived. This mutation either occurred after the CLP stage or did not confer a selective advantage in the B cell lineage because it was not observed in DNA from CD19+
cells (). The mutation in question occurred at the same nucleotide as the scanT
mutation (the first base of codon 74), yet rather than a reversion to the wild-type codon (TGC, encoding cysteine) changed the codon to serine (AGC). Given the physical similarities of serine and cysteine, we propose that Zbtb1C74S
is permissive to T cell development and T cell clonal expansion, whereas Zbtb1C74R
Defects in lymphoid but not myeloid development
The effects of Zbtb1 mutation were not restricted to the T cell lineage. Numbers of all lymphocyte lineages (T, B, and NK) were reduced in the spleens of mutant mice, whereas numbers of myeloid cells were not (). An examination of B cell development in mutant bone marrow revealed an accumulation of B cell progenitors at the Hardy Fraction C stage, followed by a reduction of progenitors at each subsequent stage (). In the mutant spleen, numbers of transitional and follicular B cells were significantly reduced by an average of 72% and 44%, respectively (). The relatively minor reduction in follicular as compared with transitional B cell numbers may indicate mild compensatory expansion in the follicular compartment. Mutant B cells nevertheless responded to T-independent (but not T dependent) immunization (), implying a developmental rather than a functional B cell defect.
Figure 3. scanT mice have generalized deficiencies of lymphoid but not myeloid cells. (A) Total numbers of major lymphoid and myeloid cell subsets in the spleen of 8-wk-old sex-matched littermates, gated as follows: T (CD3ε+), B (CD19+), NK (NK1.1+CD3ε (more ...)
A cell-intrinsic defect
To distinguish between potential hematopoietic and nonhematopoietic origins of the Zbtb1 mutant phenotype, we created radiation chimeras. Rag1 mutants reconstituted with Zbtb1 mutant bone marrow developed B cells but not T cells (). This defect was intrinsic to Zbtb1 mutant T cell progenitors, rather than other radiosensitive hematopoietic cells, because T cells in mixed bone marrow chimeras were exclusively derived from wild-type donors (). In fact, all blood lymphocytes (but not CD11b+ myeloid cells) were wild-type donor derived, implying a general competitive failure of Zbtb1 mutant lymphoid progenitors (). Finer phenotyping of the spleen, bone marrow, and thymus of mixed bone marrow chimeric mice confirmed this to be the case. B, T, and NK cells in the spleen were exclusively wild-type derived (CD45.1+), whereas little or no competitive disadvantage was seen in the myeloid compartments (). Cells of wild-type origin dominated the B cell compartment from the Fraction A stage onward and similarly outcompeted Zbtb1 mutant NK progenitors in bone marrow and T cell progenitors in the thymus ().
Figure 4. A cell-intrinsic T cell deficiency and competitive failure of lymphoid reconstitution. (A and B) Lethally irradiated Rag1 mutant mice (CD45.2+) were reconstituted with unmixed scanT or wild-type bone marrow (CD45.2+; A) or a mixture of scanT or wild-type (more ...)
Uncompromised hematopoietic progenitor function
However, the scanT
mutation did not appear to affect hematopoietic development before lymphoid specification. Percentages and numbers of early hematopoietic progenitors, including CLPs, were comparable in heterozygous and homozygous mutant bone marrow () and were reconstituted at equivalent percentages in mixed bone marrow chimeras (). Hematopoietic stem cell (HSC) turnover, as measured by incorporation of the thymidine analogue 5-ethynyl-2′deoxyuridine (EdU), occurred at similar rates in wild-type and mutant mice (). Wild-type and Zbtb1
mutant bone marrow also generated equivalent numbers of hematopoietic colonies in the spleens of lethally irradiated recipients, both at days 8 (indicative of megakaryocyte/erythroid progenitor function) and 12 (reflective of multipotent progenitor function; ; Na Nakorn et al., 2002
). Myeloid engraftment did not occur in unconditioned Zbtb1
mutant recipients of wild-type bone marrow, which is consistent with the interpretation of intact HSC function ().
Figure 5. Normal development and function of hematopoietic progenitors. (A and B) Frequencies (A) and numbers (B) or hematopoietic progenitors in heterozygous and homozygous Zbtb1 mutant bone marrow. LT-HSC (long-term HSC; CD135−CD34− or CD150+ (more ...)
In certain aspects, Zbtb1
mutants reflect the phenotype of mice lacking Notch1. Both Zbtb1
mutants are profoundly T cell deficient and show an accumulation of B cells in the thymic rudiment (Radtke et al., 1999
). Yet although Zbtb1
mutants have a competitive disadvantage in the B and NK lineages, Notch1
mutants do not (Radtke et al., 1999
). Germline mutation of Zbtb1
appears permissive to embryonic development, whereas mutation of Notch1
is not (Swiatek et al., 1994
), indicating a more specialized role for ZBTB1 in determining lymphoid fate. This is consistent with the conservation of Zbtb1
only among vertebrate genomes and might implicate the human ZBTB1
locus in genetically obscure cases of T cell–deficient SCID (Fischer, 2007
Although there are no previous studies of a physiological role for ZBTB1, one describes some of its biochemical characteristics (Matic et al., 2010
). ZBTB1, like many of its BTB-ZF counterparts, acts as a potent transcriptional repressor, as determined by the repressive activity of a Gal4-ZBTB1 fusion protein. This repressive activity, as well as the colocalization of ZBTB1 with the SMRT transcriptional repressor, is also regulated by SUMOylation (Matic et al., 2010
Our data raise several questions about the function of ZBTB1 in T cell and lymphoid specification. In particular, why is ZBTB1 critical for T cell development but only essential for B and NK development under competition? Can the reduction in ETP numbers be explained by their failure to migrate to the thymus, by a failure to proliferate within it, or a combination of both? Programmed death of T cell progenitors is unlikely to account for this because T cells also fail to develop in the absence of the proapoptotic protein BIM (which can rescue erythropoiesis in mice deficient for the BTB-ZF relative LRF; Fig. S3
; Maeda et al., 2009
In summary, our work defines ZBTB1 as a regulator of lymphoid development, joining the ranks of several other lymphoid-promoting BTB-ZF proteins. As is the case with other BTB-ZF family members, ZBTB1 presumably acts as a transcriptional suppressor, and the determination of precisely which genes it regulates will be of central importance to our understanding of lymphopoiesis.