MicroRNA-126-mediated control of cell fate in B-cell myeloid progenitors as a potential alternative to transcriptional factors.

Lineage specification is thought to be largely regulated at the level of transcription, where lineage-specific transcription factors drive specific cell fates. MicroRNAs (miR), vital to many cell functions, act posttranscriptionally to decrease the expression of target mRNAs. MLL-AF4 acute lymphocytic leukemia exhibits both myeloid and B-cell surface markers, suggesting that the transformed cells are B-cell myeloid progenitor cells. Through gain- and loss-of-function experiments, we demonstrated that microRNA 126 (miR-126) drives B-cell myeloid biphenotypic leukemia differentiation toward B cells without changing expression of E2A immunoglobulin enhancer-binding factor E12/E47 (E2A), early B-cell factor 1 (EBF1), or paired box protein 5, which are critical transcription factors in B-lymphopoiesis. Similar induction of B-cell differentiation by miR-126 was observed in normal hematopoietic cells in vitro and in vivo in uncommitted murine c-Kit(+)Sca1(+)Lineage(-) cells, with insulin regulatory subunit-1 acting as a target of miR-126. Importantly, in EBF1-deficient hematopoietic progenitor cells, which fail to differentiate into B cells, miR-126 significantly up-regulated B220, and induced the expression of B-cell genes, including recombination activating genes-1/2 and CD79a/b. These data suggest that miR-126 can at least partly rescue B-cell development independently of EBF1. These experiments show that miR-126 regulates myeloid vs. B-cell fate through an alternative machinery, establishing the critical role of miRNAs in the lineage specification of multipotent mammalian cells.

Imatinib mesylate directly impairs class switch recombination through down-regulation of AID: its potential efficacy as an AID suppressor.

Activation-induced cytidine deaminase (AID) is essential for class switch recombination and somatic hypermutation. Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that class switch recombination on B-cell activation is apparently inhibited by IM through down-regulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor.

A versatile method to profile hepatitis B virus DNA integration.

BACKGROUND: HBV DNA integration into the host genome is frequently found in HBV-associated HCC tissues and is associated with hepatocarcinogenesis. Multiple detection methods, including hybrid capture-sequencing, have identified integration sites and provided clinical implications; however, each has advantages and disadvantages concerning sensitivity, cost, and throughput. Therefore, methods that can comprehensively and cost-effectively detect integration sites with high sensitivity are required. Here, we investigated the efficiency of RAISING (Rapid Amplification of Integration Site without Interference by Genomic DNA contamination) as a simple and inexpensive method to detect viral integration by amplifying HBV-integrated fragments using virus-specific primers covering the entire HBV genome. METHODS AND RESULTS: Illumina sequencing of RAISING products from HCC-derived cell lines (PLC/PRF/5 and Hep3B cells) identified HBV-human junction sequences as well as their frequencies. The HBV-human junction profiles identified using RAISING were consistent with those determined using hybrid capture-sequencing, and the representative junctions could be validated by junction-specific nested PCR. The comparison of these detection methods revealed that RAISING-sequencing outperforms hybrid capture-sequencing in concentrating junction sequences. RAISING-sequencing was also demonstrated to determine the sites of de novo integration in HBV-infected HepG2-NTCP cells, primary human hepatocytes, liver-humanized mice, and clinical specimens. Furthermore, we made use of xenograft mice subcutaneously engrafted with PLC/PRF/5 or Hep3B cells, and HBV-human junctions determined by RAISING-sequencing were detectable in the plasma cell-free DNA using droplet digital PCR. CONCLUSIONS: RAISING successfully profiles HBV-human junction sequences with smaller amounts of sequencing data and at a lower cost than hybrid capture-sequencing. This method is expected to aid basic HBV integration and clinical diagnosis research.

Carboxy-terminal domain of AID required for its mRNA complex formation in vivo.

Activation-induced cytidine deaminase (AID) is essential for the class switch recombination (CSR) and somatic hypermutation (SHM) of Ig genes. Originally, AID was postulated to be an RNA-editing enzyme, because of its structural homology with a known RNA-editing enzyme, APOBEC1. In support of this idea, AID shares many of the properties of RNA-editing enzymes, including nucleocytoplasmic shuttling and a dependency on de novo protein synthesis. However, it has not been shown whether AID recognizes a specific mRNA and edits it to generate an enzyme involved in CSR or SHM. Here, we examined the association between AID and polyadenylated [poly(A)(+)] RNA in vivo, using UV cross-linking coupled with a poly(A) capture method that relies on biotinylated oligo(dT) and streptavidin-conjugated beads. We found that both exogenous AID expressed in transfected CH12 cells and endogenous AID expressed in BL2 cells were associated with poly(A)(+) RNA. Similar protein-poly(A)(+) RNA complexes were formed by APOBEC1 and APOBEC3G. However, the interactions of all of these cytidine deaminase family members, including AID, with poly(A)(+) RNA were indirect. This was expected for APOBEC1, which is known to act through an RNA-interacting cofactor, APOBEC1 complementation factor (ACF). In addition, the carboxy-terminal region of AID, which is essential for class switching, was also required for its interaction with poly(A)(+) RNA. These results suggest that the CSR activity of AID requires an ACF-like cofactor that specifically interacts with the carboxy-terminal domain of AID.

Alteration of processing induced by a single nucleotide polymorphism in pri-miR-126.

MicroRNAs (miRNAs) are small non-coding RNAs that inhibit expression of specific target genes at the post-transcriptional level. Sequence variations in miRNA genes, including pri-miRNAs, pre-miRNAs and mature miRNAs, could potentially influence the processing and/or target selection of miRNAs. In this study, we have found the single nucleotide polymorphism (SNP) at the twenty-fourth nucleotide (+24) of the mature miR-126 in the genome of RS4;11 cells, derived from a MLL-AF4 ALL patient. Through a series of in vivo analyzes, we found that this miR-126 SNP significantly blocks the processing of pri-miRNA to mature miRNA, as well as reduces miRNA-mediated translational suppression. Moreover, its frequency is different among races. Thus, our study emphasizes the importance of identifying new miRNA SNP and its contribution to miRNA biogenesis which is possible link to human genetic disease.

Familial acanthosis nigricans with the FGFR3 mutation: Differences of pigmentation between male and female patients.

Familial acanthosis nigricans caused by the mutation of the fibroblast growth factor receptor 3 (FGFR3) gene is characterized by short stature, hypochondroplasia and acanthosis nigricans. We herein report a Japanese family that showed a missense mutation of c.1948A>C (p.K650Q) in FGFR3. The pigmentation of acanthosis nigricans was more prominent in male patients than in female patients in this family. We immunohistochemically analyzed the distribution of melanocytes. Although pigmentation in the basal layer was denser in the proband, there was no difference in the distribution and number of melanocytes.

Transgenic mouse model for imaging of ATF4 translational activation-related cellular stress responses in vivo.

Activating transcription factor 4 (ATF4) is a translationally activated protein that plays a role in cellular adaptation to several stresses. Because these stresses are associated with various diseases, the translational control of ATF4 needs to be evaluated from the physiological and pathological points of view. We have developed a transgenic mouse model to monitor the translational activation of ATF4 in response to cellular stress. By using this mouse model, we were able to detect nutrient starvation response, antivirus response, endoplasmic reticulum (ER) stress response, and oxidative stress in vitro and ex vivo, as well as in vivo. The reporter system introduced into our mouse model was also shown to work in a stress intensity-dependent manner and a stress duration-dependent manner. The mouse model is therefore a useful tool for imaging ATF4 translational activation at various levels, from cell cultures to whole bodies, and it has a range of useful applications in investigations on the physiological and pathological roles of ATF4-related stress and in the development of clinical drugs for treating ATF4-associated diseases.

Transgenic mouse model for imaging of interleukin-1ß-related inflammation in vivo.

Inflammation is a biological response associated with symptoms of various diseases, and its study is important in gaining an understanding of the pathological conditions of such diseases and in making strategic plans for promoting healing. It is therefore essential to develop technologies for the detection of inflammatory conditions. Interleukin-1ß (IL-1ß) is a proinflammatory cytokine produced and secreted mainly by monocytes and macrophages in response to inflammatory stimulation. The activation of IL-1ß is regulated through transcriptional induction by the promoter and post-translational processing by the inflammasome. Here we have developed a reporter gene to monitor the activation status of IL-1ß by using a dual regulation system and, by using the reporter gene, we have established a mouse model that permits low-invasive visualization of the inflammatory status. Previous reporter systems dependent on the transcription or processing of IL-1ß show problems in terms of background noise or signal specificity. Our reporter system overcomes these weaknesses by combining advantages from regulation by a promoter and processing of IL-1ß. Our mouse model detected specific physiological inflammation in the liver and pancreas caused by hepatitis or pancreatitis models, respectively. Our reporter gene and mouse model are therefore expected to become useful bioresources for future medical science.

miRNAs in normal and malignant B cells.

MicroRNAs (miRNA) are small noncoding RNAs that posttranscriptionally regulate expression of target mRNAs. In animals, miRNAs control many developmental and physiological processes. It has recently revealed that miRNAs critically work in B cell development and some B cell malignancy and that their expression profiles well correlate with its prognosis and phenotype. This review overviews these interesting findings and discuss a possibility that miRNA is a promising target for therapeutic and diagnostic tool for B cell malignancy.