We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin-and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.

The present study suggests that the educational program targeting oral function improvement is effective among the independent older population.

The Runx family of transcription factors is thought to regulate the differentiation of thymocytes. Runx3 protein is detected mainly in the CD4−8+ subset of T lymphocytes. In the thymus of Runx3-deficient mice, CD4 expression is de-repressed and CD4−8+ thymocytes do not develop. This clearly implicates Runx3 in CD4 silencing, but does not necessarily prove its role in the differentiation of CD4−8+ thymocytes per se. In the present study, we created transgenic mice that overexpress Runx3 and analyzed the development of thymocytes in these animals. In the Runx3-transgenic thymus, the number of CD4−8+ cells was greatly increased, whereas the numbers of CD4+8+ and CD4+8− cells were reduced. The CD4−8+ transgenic thymocytes contained mature cells with a TCRhighHSAlow phenotype. These cells were released from the thymus and contributed to the elevated level of CD4−8+ cells relative to CD4+8− cells in the spleen. Runx3 overexpression also increased the number of mature CD4−8+ thymocytes in mice with class II-restricted, transgenic TCR and in mice with a class I-deficient background, both of which are favorable for CD4+8− lineage selection. Thus, Runx3 can drive thymocytes to select the CD4−8+ lineage. This activity is likely to be due to more than a simple silencing of CD4 gene expression.

During thymic T cell development, immature CD4+/CD8+ thymocytes develop into either CD4+/CD8− helper or CD4−/CD8+ CTLs. The molecular mechanisms governing the complex selection and differentiation steps during thymic T cell development are not well understood. Here we developed a novel approach to investigate gene function during thymocyte development. We transfected ex vivo isolated immature thymocytes with gene-specific morpholino antisense oligonucleotides and induced differentiation in cell or organ cultures. A morpholino oligonucleotide specific for CD8α strongly reduces CD8 expression. To our knowledge, this is the first demonstrated gene knockdown by morpholino oligonucleotides in primary lymphocytes. Using this approach, we show here that the transcription factor Runx3 is involved in silencing of CD4 expression during CD8 T cell differentiation. Runx3 protein expression appears late in thymocyte differentiation and is confined to mature CD8 single-positive thymocytes, whereas Runx3 mRNA is transcribed in mature CD4 and CD8 thymocytes. Therefore, Runx3 protein expression is regulated at a post-transcriptional level. The knockdown of Runx3 protein expression through morpholino oligonucleotides inhibited the development of CD4−/CD8+ T cells. Instead, mature cells with a CD4+/CD8+ phenotype accumulated. Potential Runx binding sites were identified in the CD4 gene silencer element, which are bound by Runx protein in EMSAs. Mutagenesis of potential Runx binding sites in the CD4 gene silencer abolished silencing activity in a reporter gene assay, indicating that Runx3 is involved in CD4 gene silencing. The experimental approach developed here should be valuable for the functional analysis of other candidate genes in T cell differentiation.

In acute myeloid leukemia (AML), fusion genes often form as the result of specific chromosomal translocations.(1,2) Many of these fused genes, including MOZ, are transcription-related factors involved in the development or self-renewal of hematopoietic stem cells (HSC) and/or in hematopoietic cell differentiation. (3,4) Monocytic leukemia zinc finger protein (MOZ) is a Myst (MOZ, Ybf2 (Sas3), Sas2, Tip60)-type histone acetyltranseferase (HAT) that functions as a transcriptional coactivator for hematopoietic transcription factors such as AML1.(5,6) Recently, we and others used gene-targeted mice to reveal critical roles for MOZ in hematopoiesis, particularly in the self-renewal of HSC. (7,8) MOZ fusion genes can also transform HSC and myeloid progenitor cells into leukemia cells and confer unto them self-renewal activity. (9,10) Researching the functions of MOZ and MOZ fusion genes in normal and malignant hematopoiesis can aid in understanding the mechanisms of AML development and leukemic cell-renewal activity. Such discoveries may allow the development of improved AML therapies. Involvement of the MOZ gene in chromosomal translocationsChromosomal translocations, which are frequently detected in patients with AML, include t(8;21), inv(16), t(15;17) and t(11; v) (v: variable) result in the gene fusions AML1-ETO, CBFβ-MYH11, PML-RARα and MLL-fusions, respectively. MOZ was first identified as a gene involved in the translocation t(8;16)(p11;p13) resulting in the MOZ-CBP fusion gene. The MOZ-p300, MOZ-TIF2 and MOZ-NcoA3 fusion genes were later identified in AML from t(8;22)(p11;q13), (12,13) inv(8)(p11;q13) (14,15) and t(8; 20)(p11;q13), (16) respectively (Fig. 1). MOZ is also involved in a patient of pediatric therapy-related myelodysplastic syndrome with a novel chromosomal translocation t(2;8)(p23;p11).(17) MOZ-related factor (MORF; Myst4, Querkopf), a HAT that is highly homologous to MOZ, (18,19) also generates fusion genes with CBP and probably also with Gcn5 in various disorders, including AML (20,21) therapy-related myelodysplastic syndrome (22) and uterine leiomyomata.All MOZ fusion partner genes are involved in histone modification and transcriptional regulation. CBP and p300 are major HAT (24) that function as coactivators for various transcription factors. TIF2 (NcoA2/GRIP1) and NcoA3 (TRAM-1/RAC3/ pCIP/AIB-1) are adaptor proteins that combine nuclear receptors with CBP.(25) AML that express MOZ fusion genes are typically monocytic leukemias classified as M4/M5 among FrenchAmerican-British (FAB) subtypes;(18) MOZ-related translocation is found in about 6.5% of such AML subtypes. (16) In all MOZ fusion genes, breakpoints of MOZ are located in or around its acidic domain (Fig. 1). As a result, the N-terminal region of MOZ is retained and the C-terminal region is replaced with the fusion partners, such as CBP or p300. The N-terminal region of MOZ contains a H15 (histone H1/H5) domain related to nuclear localization, (5) a PHD (plant homeobox-like domain) zinc finger involved in binding to methylated histone,...