Kazuko Miyazaki

Alveolar Bone Marrow as a Cell Source for Regenerative Medicine: Differences Between Alveolar and Iliac Bone Marrow Stromal Cells

We isolated and expanded BMSCs from human alveolar/jaw bone at a high success rate (70%). These cells had potent osteogenic potential in vitro and in vivo, although their chondrogenic and adipogenic potential was less than that of iliac cells.

Global changes in the nuclear positioning of genes and intra- and interdomain genomic interactions that orchestrate B cell fate

The genome is folded into domains located in compartments that are either transcriptionally inert or transcriptionally permissive. Here we used genome-wide strategies to characterize domains during B cell development. Structured interaction matrix analysis showed that occupancy by the architectural protein CTCF was associated mainly with intradomain interactions, whereas sites bound by the histone acetyltransferase p300 or the transcription factors E2A or PU.1 were associated with intra- and interdomain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitor cells, the transcriptionally inactive locus encoding early B cell factor (Ebf1) was sequestered at the nuclear lamina, which thereby preserved their multipotency. After development into the pro-B cell stage, Ebf1 and other genes switched compartments to establish new intra- and interdomain interactions associated with a B lineage–specific transcription signature.

The opposing roles of the transcription factor E2A and its antagonist Id3 that orchestrate and enforce the naive fate of T cells

It is established that the transcription factor E2A and its antagonist Id3 modulate the checkpoints consisting of the precursor to the T cell antigen receptor (pre-TCR) and the TCR. Here we demonstrate that Id3 expression was higher beyond the pre-TCR checkpoint, remained high in naive T cells and showed a bimodal pattern in the effector-memory population. We show how E2A promoted T lineage specification and how pre-TCR-mediated signaling affected E2A genome-wide occupancy. Thymi in Id3-deficient mice had aberrant development of effector-memory cells, higher expression of the chemokine receptor CXCR5 and the transcriptional repressor Bcl-6 and, unexpectedly, T cell–B cell conjugates and B cell follicles. Collectively, our data show how E2A acted globally to orchestrate development into the T lineage and that Id3 antagonized E2A activity beyond the pre-TCR checkpoint to enforce the naive fate of T cells.It is established that E2A and its antagonist, Id3, modulate developmental progression at the pre-TCR receptor (pre-TCR) and TCR checkpoints. Here we demonstrate that Id3 expression is elevated beyond the pre-TCR checkpoint, remains high in naive T cells and shows a bimodal pattern in the effector/memory population. We show how E2A promotes T-lineage specification and how pre-TCR mediated signaling affects E2A genome-wide occupancy. Thymi in Id3-deficient mice exhibited aberrant development of effector/memory cells, increased CXCR5 and Bcl6 expression, T-B cell conjugates and remarkably B cell follicles. Collectively, these data show how E2A acts globally to orchestrate T-lineage development and that Id3 antagonizes E2A activity beyond the pre-TCR checkpoint to enforce the naïve T cell fate.

Thymocyte proliferation induced by pre-T cell receptor signaling is maintained through polycomb gene product Bmi-1-mediated Cdkn2a repression.

Thymocytes undergo massive proliferation before T cell receptor (TCR) gene rearrangement, ensuring the diversification of the TCR repertoire. Because activated cells are more susceptible to damage, cell-death restraint as well as promotion of cell-cycle progression is considered important for adequate cell growth. Although the molecular mechanism of pre-TCR-induced proliferation has been examined, the mechanisms of protection against cell death during the proliferation phase remain unknown. Here we show that the survival of activated pre-T cells induced by pre-TCR signaling required the Polycomb group (PcG) gene product Bmi-1-mediated repression of Cdkn2A, and that p19Arf expression resulted in thymocyte cell death and inhibited the transition from CD4(-)CD8(-) (DN) to CD4(+)CD8(+) (DP) stage upstream of the transcriptional factor p53 pathway. The expression of Cdkn2A (the gene encoding p19Arf) in immature thymocytes was directly regulated by PcG complex containing Bmi-1 and M33 through the maintenance of local trimethylated histone H3K27. Our results indicate that this epigenetic regulation critically contributes to the survival of the activated pre-T cells, thereby supporting their proliferation during the DN-DP transition.

Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease

Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell–specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.

Polycomb Group Gene mel-18 Regulates Early T Progenitor Expansion by Maintaining the Expression of Hes-1, a Target of the Notch Pathway

Polycomb group (PcG) proteins play a role in the maintenance of cellular identity throughout many rounds of cell division through the regulation of gene expression. In this report we demonstrate that the loss of the PcG gene mel-18 impairs the expansion of the most immature T progenitor cells at a stage before the rearrangement of the TCR β-chain gene in vivo and in vitro. This impairment of these T progenitors appears to be associated with increased susceptibility to cell death. We also show that the expression of Hes-1, one of the target genes of the Notch signaling pathway, is drastically down-regulated in early T progenitors isolated from mel-18−/− mice. In addition, mel-18−/− T precursors could not maintain the Hes-1 expression induced by Delta-like-1 in monolayer culture. Collectively, these data indicate that mel-18 contributes to the maintenance of the active state of the Hes-1 gene as a cellular memory system, thereby supporting the expansion of early T progenitors.

Identification of Zfp521 / ZNF521 as a cooperative gene for E2A-HLF to develop acute B-lineage leukemia

E2A-hepatic leukemia factor (HLF) is a chimeric protein found in B-lineage acute lymphoblastic leukemia (ALL) with t(17;19). To analyze the leukemogenic process and to create model mice for t(17;19)-positive leukemia, we generated inducible knock-in (iKI) mice for E2A-HLF. Despite the induced expression of E2A-HLF in the hematopoietic tissues, no disease was developed during the long observation period, indicating that additional gene alterations are required to develop leukemia. To elucidate this process, E2A-HLF iKI and control littermates were subjected to retroviral insertional mutagenesis. Virus infection induced acute leukemias in E2A-HLF iKI mice with higher morbidity and mortality than in control mice. Inverse PCR detected three common integration sites specific for E2A-HLF iKI leukemic mice, which induced overexpression of zinc-finger transcription factors: growth factor independent 1 (Gfi1), zinc-finger protein subfamily 1A1 isoform a (Zfp1a1, also known as Ikaros) and zinc-finger protein 521 (Zfp521). Interestingly, tumors with Zfp521 integration exclusively showed B-lineage ALL, which corresponds to the phenotype of human t(17;19)-positive leukemia. In addition, ZNF521 (human counterpart of Zfp521) was found to be overexpressed in human leukemic cell lines harboring t(17;19). Moreover, both iKI for E2A-HLF and transgenic for Zfp521 mice frequently developed B-lineage ALL. These results indicate that a set of transcription factors promote leukemic transformation of E2A-HLF-expressing hematopoietic progenitors and suggest that aberrant expression of Zfp521/ZNF521 may be clinically relevant to t(17;19)-positive B-lineage ALL.

The establishment of B versus T cell identity

In B cell progenitors, E-proteins E2A and HEB (HeLa E-box binding protein) are crucial for the induction of a B lineage-specific program of gene expression and for orchestrating the assembly of the immunoglobulin loci. In the thymus E2A and HEB act differently, activating the expression of genes closely associated with the establishment of T cell identity and promoting the rearrangement of T cell receptor (TCR) loci. These findings have raised the question as to how E-proteins exert these different activities. We review here the distinct regulatory networks that establish B versus T cell identity, and how genomic architecture and location of genes is modulated in these lineage decisions. We conclude by proposing a model wherein stochasticity in the nuclear location of the early B cell factor 1 (Ebf1) locus in multipotent progenitors determines this lineage choice.

The Role of the Basic Helix-Loop-Helix Transcription Factor Dec1 in the Regulatory T Cells

Naturally occurring regulatory T (Treg) cells play a central role in the maintenance of immune homeostasis and in restraining the development of spontaneous inflammatory responses. However, the underlying mechanisms of Treg homeostasis remain incompletely understood. Of particular note, the IL-2Rα (CD25) is crucial for the homeostasis of Treg cells and the prevention of lymphoproliferative autoimmune disease. In this paper, we report that the basic helix-loop-helix transcription factor Dec1 is involved in the homeostasis of Treg cells and plays a role in their survival or expansion after adoptive transfer to lymphopenic recipients. Hence, it is crucial for the suppression of effector T cell-mediated inflammatory responses. Enforced expression of Dec1 upregulates CD25 expression during thymocyte development and increases the number of Treg cells in the periphery. Dec1 binds the transcription factor Runx1 and colocalizes with Runx1 in Treg cells. Specifically, we demonstrate that in Treg cells the Dec1/Runx1 complex binds to regulatory elements present in the Il-2rα locus. Collectively, these data show how Dec1 mechanistically acts in Treg cells.

Overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induced acute leukemia in p210BCR/ABL transgenic mice

Chronic myelogenous leukemia (CML) is a hematopoietic disorder, which begins as indolent chronic phase but inevitably progresses to fatal blast crisis. p210BCR/ABL, a constitutively active tyrosine kinase, is responsible for disease initiation but molecular mechanism(s) underlying disease evolution remains largely unknown. To explore this process, we employed retroviral insertional mutagenesis to CML-exhibiting p210BCR/ABL transgenic mice (Tg). Virus infection induced acute lymphoblastic leukemia (ALL) in p210BCR/ABL Tg with a higher frequency and in a shorter latency than wild-type littermates, and inverse PCR detected two retrovirus common integration sites (CISs) in p210BCR/ABL Tg tumors. Interestingly, one CIS was the transgene itself, where retrovirus integrations induced upregulation of p210BCR/ABL and production of truncated BCR/ABL with an enhanced kinase activity. Another CIS was Notch1 gene, where retrovirus integrations resulted in overexpression of Notch1 and generation of Notch1 lacking the C-terminal region (Notch1ΔC) associated with stable expression of its activated product, C-terminal-truncated Notch intracellular domain (NICDΔC). In addition, generation of Tg for both p210BCR/ABL and Notch1ΔC developed ALL in a shortened period with Stat5 activation, demonstrating the cooperative oncogenicity of Notch1ΔC/NICDΔC with p210BCR/ABL involving Stat5-mediated pathway. These results demonstrated that overexpression/enhanced kinase activity of BCR/ABL and altered expression of Notch1 induces acute leukemia in a transgenic model for CML.