Yukio Mishima

Bcl11b is required for differentiation and survival of αβ T lymphocytes

The gene Bcl11b, which encodes zinc finger proteins, and its paralog, Bcl11a, are associated with immune-system malignancies. We have generated Bcl11b-deficient mice that show a block at the CD4−CD8− double-negative stage of thymocyte development without any impairment in cells of B- or γδ T cell lineages. The Bcl11b−/− thymocytes showed unsuccessful recombination of Vβ to Dβ and lacked the pre–T cell receptor (TCR) complex on the cell surface, owing to the absence of Tcrb mRNA expression. In addition, we saw profound apoptosis in the thymus of neonatal Bcl11b−/− mice. These results suggest that Bcl11b is a key regulator of both differentiation and survival during thymocyte development.

Homozygous deletions and point mutations of the Rit1/Bcl11b gene in γ-ray induced mouse thymic lymphomas

Allelic loss (LOH) mapping and sequence analysis were conducted for gamma-ray induced mouse thymic lymphomas and a novel tumor suppressor gene, Rit1/Bcl11b, on chromosome 12 was isolated. Bi-allelic changes were found in 17 of the 66 p53-proficient lymphomas with Rit1 LOH but in only 2 of the 54 p53-deficient lymphomas. This suggests an association between the presence of functional p53 and inactivation of the Rit1 gene in the lymphoma development. Introduction of Rit1 into HeLa cells lacking Rit1 expression suppressed cell growth. These results indicate that loss-of-function mutations of Rit1 contribute to mouse lymphomagenesis and possibly to human cancer development.

Expression of TCRαβ Partly Rescues Developmental Arrest and Apoptosis of αβ T cells in Bcl11b−/− Mice

Bcl11b−/− mice show developmental arrest at the CD44−CD25+ double-negative 3 (DN3) or immature CD8+single-positive stage of αβ T cell. We have performed detailed analysis of sorted subsets of Bcl11b−/− thymocytes, DN3 and CD44−CD25− double-negative 4 (DN4) cells. Surface expression of TCRβ proteins was not detected in DN3 thymocytes and markedly reduced in DN4 thymocytes, whereas expression within the cell was detected in both, suggesting some impairment in processing of TCRβ proteins from the cytoplasm to the cell surface. This lack of expression, resulting in the absence of pre-TCR signaling, could be responsible for the arrest, but the transgenic TCRβ or TCRαβ expression on the cell surface failed to promote transition from the DN3 to CD4+CD8+ double-positive stage of development. This suggests that the pre-TCR signal cannot compensate the deficiency of Bcl11b for development. Bcl11b−/− DN3 thymocytes showed normal DNA rearrangements between Dβ and Jβ segments but limited DNA rearrangements between Vβ and DJβ without effect of distal or proximal positions. Because this impairment may be due to chromatin accessibility, we have examined histone H3 acetylation in Bcl11b−/− DN3 cells using chromatin immunoprecipitation assay. No change was observed in acetylation at the Vβ and Dβ gene locus. Analysis of Bcl11b−/− DN4 thymocytes showed apoptosis, accompanied with lower expression of anti-apoptotic proteins, Bcl-xL and Bcl-2, than wild-type DN4 thymocytes. Interestingly, the transgenic TCRαβ in those cells reduced apoptosis and raised their protein expression without increased cellularity. These results suggest that Bcl11b deficiency affects many different signaling pathways leading to development arrests.

Lack of Bcl11b tumor suppressor results in vulnerability to DNA replication stress and damages

Bcl11b/Rit1 is involved in T-cell development and undergoes chromosomal rearrangements in human T-cell leukemias. Thymocytes of Bcl11b−/− newborn mice exhibit apoptosis at a certain developmental stage when thymocytes re-enter into the cell-cycle. Here, we show that Bcl11b-knockdown T-cell lines, when exposed to growth stimuli, exhibited apoptosis at the S phase with concomitant decreases in a cell-cycle inhibitor, p27 and an antiapoptotic protein, Bcl-xL, owing to transcriptional repression. This repression was a likely consequence of the impairment of Sirt1, a nicotinamide adenine dinucleotide-dependent deacetylase associating with Bcl11b. Activation of the apoptotic process cleaved the mediator protein, Claspin, and inhibited phosphorylation of cell-cycle checkpoint kinase 1 (Chk1) that plays a central role in sensing and responding to incomplete replication. Bcl11b−/− thymocytes also failed to phosphorylate Chk1 when UV irradiated. These results implicate Bcl11b in the remedy for DNA replication stress and maintenance of genomic integrity.

Predisposition to mouse thymic lymphomas in response to ionizing radiation depends on variant alleles encoding metal-responsive transcription factor-1 (Mtf-1 )

Genetic predisposition to cancers is significant to public health because a high proportion of cancers probably arise in a susceptible human subpopulation. Using a mouse model of γ-ray-induced thymic lymphomas, we performed linkage analysis and haplotype mapping that suggested Mtf-1, metal-responsive transcription factor-1 (Mtf-1), as a candidate lymphoma susceptibility gene. Sequence analysis revealed a polymorphism of Mtf-1 that alters the corresponding amino acid at position 424 in the proline-rich domain from a serine in susceptibility strains to proline in resistant strains. The transcriptional activity of Mtf-1 encoding serine and proline was compared by transfecting the DNA to Mtf-1-null cells, and the change to proline conferred a higher metal responsiveness in transfections. Furthermore, the resistant congenic strains possessing the Mtf-1 allele of proline type exhibited higher radiation inducibility of target genes than susceptible background strains having the Mtf-1 allele of serine type. Since products of the targets such as metallothionein are able to suppress cellular stresses generated by irradiation, these results suggest that highly inducible strains having Mtf-1 of proline type are refractory to radiation effects and hence are resistant to lymphoma development.

Multi-step lymphomagenesis deduced from DNA changes in thymic lymphomas and atrophic thymuses at various times after γ -irradiation

Whole-body γ-irradiation to mice causes thymic atrophy where a population of precancerous cells with mutation can be found. Thus, clonal growth and DNA changes at Bcl11b, Ikaros, Pten, Notch1 and Myc were examined in not only thymic lymphomas but also in atrophic thymuses at various times after irradiation. Clonal expansion was detected from the distinct patterns of rearrangements at the TCRβ receptor locus in a fraction of atrophic thymuses at as early as 30 days after irradiation. This expansion may be in part owing to the rearranged TCRβ signaling because the transfer of bone marrow cells with the rearrangement and the wild-type locus into severe-combined immunodeficiency mice showed preferential growth of the rearranged thymocytes in atrophic thymus. Loss of heterozygosity (LOH) at Bcl11b and trisomy of Myc were found at high frequencies in both lymphomas and atrophic thymuses, and in contrast, LOH at Ikaros and Pten were rare in atrophic thymuses but prevalent in lymphomas. Notch1 activation was detected in lymphomas and in atrophic thymuses only at a late stage. Similar patterns of DNA changes were found in atrophic thymuses induced in Bcl11b+/− mice. These results suggest the order of genetic changes during lymphomagenesis, Bcl11b and Myc being at the early stage; whereas Ikaros, Pten and Notch1 at the late stage.

Novel repetitive sequence families showing size and frequency polymorphism in the genomes of mice

A middle repetitive sequence, PR1, originally found in mouse rDNA appeared as satellite-like bands when EcoRI and BglII digests of genomic DNA were subjected to Southern blot hybridization using PR1 as probe. The copy number and sizes of PR1-related satellite-like bands, designated as PR1 families, differed remarkably among the subspecies and laboratory strains of mice when the EcoRI digests of genomic DNAs were compared. These bands were not detected in rat and human DNAs. A unit of PR1 sequence was determined by examining cloned EcoRI 3.5 kb (kb, 10(3) bases) fragment and 6.6 kb rDNA by cross-hybridization and sequence analysis: 3.5 kb and 6.6 kb DNAs are composed of homologous PR1 regions and the flanking non-homologous sequences. The results indicate that amplification of different sequences containing PR1 has occurred in different subspecies and strains of mice, and that the segments of satellite-like bands are likely to have been created by recombination of the PR1 sequence with other DNA segments before amplification. The chromosomal distribution of the 3.5 kb PR1 family was studied by back-crossing the female F1 between BALB/c and DDD/1 to male DDD/1. The segregation data strongly suggest that most, if not all, of this family are located on a single chromosome. The stability of these PR1 families in the genomes of cultured cells of a given strain was also examined. An extra band homologous to PR1 appeared in their genomes, but was not detected in other tissues, indicating that some PR1 families may change even during cell propagation.

PAIRING OF DNA FRAGMENTS CONTAINING (GGA:TCC)N REPEATS AND PROMOTION BY HIGH MOBILITY GROUP PROTEIN 1 AND HISTONE H1

Tandemly repeated DNA sequences of (GGA:TCC) n are found in tracts up to 50 base pairs long, dispersed at thousands of sites throughout the genomes of eukaryotes. Here we demonstrate the formation of complexes paired between two DNAs containing such repeats in vitro and show enhancement of the pairing by glutathione S-transferase fusion proteins of high mobility group protein 1 and histone H1. This assembly depends on incubation time at 37 °C and concentrations of the proteins and DNA, and the enhancement is inhibited by distamycin and actinomycin D interacting DNA through the minor groove. Structure of the DNA-DNA complex is deduced by comparison of its mobility in gel electrophoresis with those of synthetic markers of heterotetramers. Three synthetic and genomic DNA fragments containing repeats that have different arrangements exhibit different efficiencies of DNA pairing, implying that the pairing is affected by the number of repeat units and the arrangement of repeats in a sequence. Intriguingly, pairing occurs between homologous fragments but not between heterologous DNAs among the three. These results suggest that the repeat-mediated DNA pairing plays a role in organization of higher order architecture of chromatin and possibly chromosome segregation requiring sequence-specific association events of DNA molecules.

Bcl11b transcription factor plays a role in the maintenance of the ameloblast-progenitors in mouse adult maxillary incisors

Rodent incisors maintain the ability to grow continuously and their labial dentin is covered with enamel. Bcl11b zinc-finger transcription factor is expressed in ameloblast progenitors in mouse incisors and its absence in Bcl11b(KO/KO) mice results in a defect in embryonic tooth development. However, the role of Bcl11b in incisor maintenance in adult tissue was not studied because of death at birth in Bcl11b(KO/KO) mice. Here, we examined compound heterozygous Bcl11b(S826G/KO) mice, one allele of which has an amino acid substitution of serine at position 826 for glycine, that exhibited hypoplastic maxillary incisors with lower concentrations of minerals at the enamel and the dentin, accompanying the maxillary bone hypoplasia. Histological examinations revealed hypoplasia of the labial cervical loop in incisors, shortening of the ameloblast progenitor region, and impairment in differentiation and proliferation of ameloblast-lineage cells. Interestingly, however, juvenile mice at 5days after birth did not show marked change in these phenotypes. These results suggest that attenuated Bcl11b activity impairs ameloblast progenitors and incisor maintenance. The number of BrdU label-retaining cells, putative stem cells, was lower in Bcl11b(S826G/KO) incisors, which suggests the incisor hypoplasia may be in part a result of the decreased number of stem cells. Interestingly, the level of Shh and FGF3 expressions, which are assumed to play key roles in the development and maintenance of ameloblasts and odontoblasts, was not decreased, though the expressed areas were more restricted in ameloblast progenitor and mesenchyme regions of Bcl11b(S826G/KO) incisors, respectively. Those data suggest that the incisor maintenance by Bcl11b is not directly related to the FGF epithelial-mesenchymal signaling loop including Shh but is intrinsic to ameloblast progenitors and possibly stem cells.

Bcl11b heterozygosity promotes clonal expansion and differentiation arrest of thymocytes in γ‐irradiated mice

Bcl11b encodes a zinc‐finger transcription factor and functions as a haploinsufficient tumor suppressor gene. Bcl11bKO/KO mice exhibit differentiation arrest of thymocytes during β‐selection as has been observed with other mouse models involving knockouts of genes in the Wnt/β‐catenin signaling pathway. Recurrent chromosomal rearrangement at the BCL11B locus occurs in human T‐cell leukemias, but it is not clear how such rearrangement would contribute to lymphomagenesis. To address this issue, we studied clonal cell growth, cell number, and differentiation of thymocytes in Bcl11bKO/+ mice at different time points following γ‐irradiation. Analysis of D‐J rearrangement at the T cell receptor β‐chain (TCRβ) locus and cell surface markers by flow cytometry revealed two distinct populations of clonally growing thymocytes. In one population, thymocytes share a common D‐J rearrangement but retain the capacity to differentiate. In contrast, thymocytes in the second population have lost their ability to differentiate. Since the capacity to self renew and differentiate into multiple cell lineages are fundamental properties of adult stem cells, the differentiation competent population of thymocytes that we have isolated could potentially function as cancer stem cells. We also demonstrate increased expression of β‐catenin, a well‐known oncogenic protein, in Bcl11bKO/+ thymocytes. Collectively, the Bcl11bKO/+ genotype contributes to clonal expansion and differentiation arrest in part through an increase in the level of β‐catenin. (Cancer Sci 2010)