Research Summary

■Research on Pluripotent Stem Cells

Mechanism of Self-Renewal in Pluripotent Stem Cells ① - Analysis of Dax1, Esrrb, and Oct3/4

Previous studies have revealed the protein-protein interaction between Dax1 and Oct3/4. In this research, Esrrb, a nuclear receptor, was identified as a factor interacting with Dax1. Esrrb is already known to interact with Oct3/4, enhancing its transcriptional activation. Our analysis of the correlation among Oct3/4, Dax1, and Esrrb revealed the following: (1) Esrrb interacts with Dax1, (2) Esrrb promotes the expression of Dax1 mRNA, and this transcriptional activation by Esrrb is inhibited by Dax1, (3) Oct3/4, Dax1, and Esrrb do not form a complex simultaneously, with Dax1 inhibiting and Esrrb promoting the transcriptional activation ability of Oct3/4. These findings suggest that a regulatory loop formed by these three transcription factors might control the self-renewal ability of embryonic stem (ES) cells (Uranishi et al. Mol Cell Biol. 2013).

Mechanism of Self-Renewal in Pluripotent Stem Cells ② - Analysis of ETV4/5

Recent studies have pointed out the similarities between ES cells and cancer cells. In this context, we discovered that the transcription factors ETV4 and ETV5, which are highly expressed in cancer cells, are also strongly expressed in ES cells. Using ETV4/5 double knockout (dKO) ES cells, we analyzed the function of ETV4/5 in ES cells. The results showed that ETV4/5 dKO ES cells: (1) remain positive for undifferentiated markers indicating an undifferentiated state, (2) exhibit reduced proliferation capacity with overexpression of CDK inhibitors, and (3) fail to adequately induce the expression of ectoderm markers upon differentiation induction. From these findings, we concluded that while ETV4/5 do not play a role in maintaining the undifferentiated state, they are involved in regulating proliferation and multi-lineage differentiation, thereby controlling stem cell properties (Akagi et al. J Biol Chem. 2015).

Mechanism of Self-Renewal in Pluripotent Stem Cells ③ - Analysis of Baf53a

The BAF complex is one of the chromatin remodeling complexes involved in various cellular activities. We analyzed the function of Baf53a, a component of the BAF complex, and found that: (1) disruption of the Baf53a gene leads to cell death, (2) cessation of Baf53a expression results in the accumulation of the tumor suppressor gene p53 and activation of Caspase3, a marker of apoptosis, and (3) these phenotypes can be rescued by Baf53a or its homolog Baf53b. These results indicate that Baf53a actively maintains the survival of ES cells (Zhu et al. Sci Rep. 2017).

In addition to the above studies, we have conducted functional analyses of various transcription factors and epigenetic modifiers. Our findings suggest that these factors form a transcription factor network in ES cells, potentially regulating the self-renewal mechanism of ES cells (Ueda et al. Stem Cells. 2017; Fujii et al. BBRC 2013; Ura et al. EMBO J. 2011; Ura et al. J Biol Chem. 2008, among others).

■Research on Hematopoiesis and Leukemia/Solid Tumors

Analysis of Genetic Abnormalities in Leukemia and Cancer Cells

Acute myeloid leukemia (AML) is associated with various chromosomal translocations in about half of the cases. The other half, known as normal karyotype AML, does not involve such translocations but exhibits mutations at the genetic level. We performed a comprehensive analysis of genetic abnormalities in two types of AML with chromosomal translocations (t(8;21) AML and t(15;17) AML) and normal karyotype AML. Our findings revealed that: (1) In t(8;21) AML, approximately 30% of samples had additional genetic abnormalities such as KIT mutations, and this subgroup had a poor prognosis. (2) In t(15;17) AML, we identified three subgroups based on additional abnormalities in FLT3, c-Myc, and other genes, suggesting multiple pathways to leukemogenesis. (3) In normal karyotype AML, nearly half of the samples exhibited uniparental disomy with genetic abnormalities such as JAK2 mutations. Additionally, we have also conducted studies on thyroid and esophageal cancers to elucidate genetic abnormalities and analyze the functions of mutant genes (Akagi et al. Haematologica. 2009a; Akagi et al. Blood. 2009; Akagi et al. Haematologica. 2009b; Akagi et al. Br J Cancer. 2008; Akagi et al. Int J Cancer. 2009).

Hematopoiesis Mediated by Transcription Factors ① - Analysis of C/EBPβ

Macrophages in the hematopoietic system play a role in innate immunity by phagocytosing foreign particles. The transcription factor C/EBPβ is known to regulate the differentiation and function of macrophages. To explore new functions of C/EBPβ in hematopoietic cells, we analyzed neutrophils from C/EBPβ knockout (KO) mice. We found that KO neutrophils (1) exhibited reduced responsiveness to LPS, with insufficient induction of IL-6 and IL-10, and (2) had increased cell death. These findings suggest that C/EBPβ is also involved in neutrophil function and survival (Akagi et al. Blood. 2008).

Hematopoiesis Mediated by Transcription Factors ② - Analysis of C/EBPβ and C/EBPε

The C/EBP family of transcription factors shares high structural homology and functional redundancy. To analyze the functions of C/EBP proteins in hematopoietic cells, we generated double knockout (dKO) mice for C/EBPβ and C/EBPε and analyzed their phenotypes. We found that dKO mice (1) died from severe infections within a few months after birth, (2) had neutrophils that exhibited morphological differentiation arrest, and (3) showed accumulation of undifferentiated hematopoietic stem and progenitor cells in the bone marrow. These phenotypes were not observed in single KO mice, indicating that C/EBPβ and C/EBPε have mutually compensatory roles and cooperatively regulate hematopoiesis (Akagi et al. PLoS ONE. 2010).

Study on Specific Granule Deficiency (SGD) in Neutrophils

The transcription factor C/EBPε is essential for neutrophil differentiation. Mutations in the C/EBPε gene in humans cause neutrophil differentiation arrest, leading to specific granule deficiency (SGD), a severe autosomal recessive disorder resulting in increased susceptibility to infections. We reported the second case of SGD in Japan and analyzed the novel mutant C/EBPε found in this patient. Our analysis revealed that the mutant C/EBPε (1) had significantly reduced transcriptional activation ability, (2) retained normal nuclear localization and DNA binding abilities, and (3) failed to interact with other transcription factors like PU.1 and Gata1. Based on these findings, we proposed a new model in which the novel mutant C/EBPε disrupts protein interactions, leading to functional loss and impaired neutrophil differentiation, resulting in SGD (Wada and Akagi et al. J Immunol. 2015; Muraoka et al. BBRC 2019).