Asxl1 ablation in mouse embryonic stem cells impairs neural differentiation without affecting self-renewal.

Additional sex comb-like1 (Asxl1) is known as a chromatin modulator that plays dual functions in transcriptional regulation depending on the cell type. Recent studies using Asxl1 knockout mice revealed that Asxl1 is important for the proliferation and differentiation of hematopoietic progenitor cells, and the development of organs. Although we previously reported Asxl1 as a Sox2 target gene, its function in embryonic stem cells (ESCs) remains largely unknown. For this purpose, we isolated ESCs from the blastocyst inner cell mass of Asxl1-/- mice. Asxl1 deficiency in ESCs exhibited no effect on cell proliferation, expression of core pluripotent transcription factors, or alkaline phosphatase activity, suggesting dispensability of Asxl1 for self-renewal of ESCs. By contrast, the differentiation of Asxl1-/- ESCs was significantly affected as shown by size reductions of embryoid bodies accompanied with apoptosis, aberrant expression of differentiation genes, downregulation of bivalent neurogenesis genes, and abnormal axon formation in neurons. Overall, our findings indicated that Asxl1 played a critical role in regulating genes associated with neural differentiation without affecting self-renewal of mouse ESCs.

The systemic effects of sclerostin overexpression using ΦC31 integrase in mice.

Sclerostin, encoded by the Sost gene, is mainly produced by osteocytes in bone and antagonizes the Wnt/ß-catenin signaling pathway, which is a requisite for bone formation. Currently, human anti-sclerostin antibodies are being tested in phase III clinical trials. In addition, serum sclerostin levels are reported to be associated with bone mineral density and fracture risk in normal individuals; however, the correlation between serum sclerostin and bone mass remains controversial. To study the effects of the continuous exposure of exogenous sclerostin on bone, a ΦC31 integrase system, which has the characteristics of site-specificity and efficiency, was applied for the delivery of the Sost gene in this study. We injected Sost-attB plasmid with or without ΦC31 integrase plasmid into the mouse tail vein using a hydrodynamic-based method. The site-specific integration of the Sost gene into the mouse genome was confirmed by examining a pseudo-attP site on the hepatic genomic DNA. Sclerostin was expressed in the hepatocytes, secreted into the blood flow, and maintained at high concentrations in the mice with both Sost-attB plasmid and ΦC31 integrase plasmid injections, which was observed by serial measurement. Moreover, the mice with long-term high levels of serum sclerostin showed trabecular bone loss on micro-CT analysis. Peripheral B cell populations were not affected. Our results suggested that sclerostin could be expressed in the liver and sustained successfully at high levels in the blood by using the ΦC31 integrase system, leading to trabecular bone loss. These findings may help to further ascertain the effects of sclerostin introduced exogenously on the skeleton.

Generation of conditional alleles for Foxc1 and Foxc2 in mice.

The Forkhead box transcription factors, Foxc1 and Foxc2, are crucial for development of the eye, cardiovascular network, and other physiological systems, but their cell-type specific and postdevelopmental functions are unknown, in part because conventional (i.e., whole-organism) homozygous-null mutations of either factor result in perinatal death. Here, we describe the generation of mice with conditional-null Foxc1(flox) and Foxc2(flox) mutations that are induced via Cre-mediated recombination. Mice homozygous for the unrecombined alleles are viable and fertile, indicating that the conditional alleles retain their wild-type function. The embryos of Foxc1(flox) or Foxc2(flox) mice crossed with Cre-deleter mice that are homozygous for the recombined allele (i.e., Foxc1(Δ/Δ) or Foxc2(Δ/Δ) embryos) lack expression of the corresponding gene and show the same developmental defects observed in conventional homozygous mutant embryos. We expect these conditional mutations to enable characterization of the cell-type specific functions of Foxc1 and Foxc2 in development, disease, and adult animals.

Positive regulation of additional sex comb-like 1 gene expression by the pluripotency factor SOX2.

Additional sex comb-like 1 (ASXL1) has been suggested to be an enhancer of trithorax and polycomb proteins, and functions as a dual co-regulator of retinoid acid (RA) signaling. However, the mechanism by which ASXL1 gene expression is regulated remains unresolved. Concomitant downregulation of both SOX2 and ASXL1 during the RA-induced differentiation of P19 cells prompted us to investigate the role of SOX2 in the regulation of ASXL1. Knockdown of SOX2 in SOX2-rich NT2 cells resulted in the reduction of ASXL1 expression, whereas SOX2 overexpression in SOX2-deficient H1299 cells increased ASXL1 expression. Using a cloned ASXL1-luciferase reporter, we demonstrated that SOX2 directly transactivates the ASXL1 promoter. Serial deletion and mutation studies mapped the SOX2 response element region in the ASXL1 promoter to -1600 to -1400 bp. We showed by chromatin immunoprecipitation assay that SOX2 directly binds to the ASXL1 promoter region. Finally, formation of embryonic bodies by ASXL1-depleted murine E14TG2a embryonic stem cells was significantly impaired, similar to SOX2-knockdown cells. From these results, we suggest that ASXL1 may be a direct target of SOX2 and may play a role in maintaining the pluripotency of stem cells.

ASXL1 represses retinoic acid receptor-mediated transcription through associating with HP1 and LSD1.

We previously suggested that ASXL1 (additional sex comb-like 1) functions as either a coactivator or corepressor for the retinoid receptors retinoic acid receptor (RAR) and retinoid X receptor in a cell type-specific manner. Here, we provide clues toward the mechanism underlying ASXL1-mediated repression. Transfection assays in HEK293 or H1299 cells indicated that ASXL1 alone possessing autonomous transcriptional repression activity significantly represses RAR- or retinoid X receptor-dependent transcriptional activation, and the N-terminal portion of ASXL1 is responsible for the repression. Amino acid sequence analysis identified a consensus HP1 (heterochromatin protein 1)-binding site (HP1 box, PXVXL) in that region. Systematic in vitro and in vivo assays revealed that the HP1 box in ASXL1 is critical for the interaction with the chromoshadow domain of HP1. Transcription assays with HP1 box deletion or HP1alpha knockdown indicated that HP1alpha is required for ASXL1-mediated repression. Furthermore, we found a direct interaction of ASXL1 with histone H3 demethylase LSD1 through the N-terminal region nearby the HP1-binding site. ASXL1 binding to LSD1 was greatly increased by HP1alpha, resulting in the formation of a ternary complex. LSD1 cooperates with ASXL1 in transcriptional repression, presumably by removing H3K4 methylation, an active histone mark, but not H3K9 methylation, a repressive histone mark recognized by HP1. This possibility was supported by chromatin immunoprecipitation assays followed by ASXL1 overexpression or knockdown. Overall, this study provides the first evidence that ASXL1 cooperates with HP1 to modulate LSD1 activity, leading to a change in histone H3 methylation and thereby RAR repression.

Clinical and molecular characterizations of novel POU3F4 mutations reveal that DFN3 is due to null function of POU3F4 protein.

X-linked deafness type 3 (DFN3), the most prevalent X-linked form of hereditary deafness, is caused by mutations in the POU3F4 locus, which encodes a member of the POU family of transcription factors. Despite numerous reports on clinical evaluations and genetic analyses describing novel POU3F4 mutations, little is known about how such mutations affect normal functions of the POU3F4 protein and cause inner ear malformations and deafness. Here we describe three novel mutations of the POU3F4 gene and their clinical characterizations in three Korean families carrying deafness segregating at the DFN3 locus. The three mutations cause a substitution (p.Arg329Pro) or a deletion (p.Ser310del) of highly conserved amino acid residues in the POU homeodomain or a truncation that eliminates both DNA-binding domains (p.Ala116fs). In an attempt to better understand the molecular mechanisms underlying their inner ear defects, we examined the behavior of the normal and mutant forms of the POU3F4 protein in C3H/10T1/2 mesodermal cells. Protein modeling as well as in vitro assays demonstrated that these mutations are detrimental to the tertiary structure of the POU3F4 protein and severely affect its ability to bind DNA. All three mutated POU3F4 proteins failed to transactivate expression of a reporter gene. In addition, all three failed to inhibit the transcriptional activity of wild-type proteins when both wild-type and mutant proteins were coexpressed. Since most of the mutations reported for DFN3 thus far are associated with regions that encode the DNA binding domains of POU3F4, our results strongly suggest that the deafness in DFN3 patients is largely due to the null function of POU3F4.

Membrane transducing activity of recombinant Hoxc8 protein and its possible application as a gene delivery vector.

In order to examine whether the Hoxc8 protein can deliver nucleic acid into mammalian cells, we designed several Hoxc8-derived recombinant proteins to be synthesized as glutathione S-transferase (GST) fused forms in E. coli (GST-Hoxc8(1-242), containing a full length of Hoxc8; GST-Hoxc8(152-242), possessing a deletion of the acidic N-terminus of Hoxc8; GST-Hoxc8(149-208), which contained the homeodomain only). After labeling these proteins with Oregon 488, we examined their membrane transduction ability under the fluorescence microscope and verified that all three proteins showed similar transduction efficiency. The ability of the proteins to form in vitro protein-DNA complexes was analyzed on agarose gel; both GST-Hoxc8(1-242) and GST-Hoxc8(149-208) formed complexes. In contrast, the GST-Hoxc8(152-242) protein did not form a complex. The GST-Hoxc8(149-208) protein formed a complex with DNA at a mass ratio of 1ú1 (DNAúprotein), and GST-Hoxc8(1-242) formed a complex at a mass ratio of 1ú5. When the DNA (pDsRed1-C1) and protein complexes were added to culture media containing mammalian cells, the cells uptook the complexes, which was indicated by red fluorescence expression under the fluorescent microscope. These results indicate that recombinant Hoxc8 derivatives that harbor a homeodomain are able to traverse the mammalian cellular membrane. DNA that is bound to the recombinant derivatives can be carried across the membrane as well. This process could be applied in the development of a useful delivery vector for gene therapy in the future.

Foxc2 is a common mediator of insulin and transforming growth factor beta signaling to regulate plasminogen activator inhibitor type I gene expression.

Elevated plasma levels of plasminogen activator inhibitor type I (PAI-1), a significant risk factor of ischemic heart disease, are associated with insulin resistance in which insulin and transforming growth factor (TGF)-beta play a pivotal role in regulating PAI-1 production. Forkhead transcription factor FOXC2 is an important regulator of insulin resistance. However, the underlying molecular mechanisms to link FOXC2 to PAI-1 levels in insulin resistance remain to be elucidated. Here, we demonstrate that Foxc2 is a common transcriptional activator of insulin and TGF-beta signaling to directly regulate PAI-1 expression via 2 distinct target sites, an insulin response element (IRE) and a novel forkhead-binding element (FBE), adjacent to a Smad-binding site. We found that in adipocytes and endothelial cells Foxc2 mediates insulin action competing with another Forkhead protein, FOXO1, via the insulin response element, and simultaneously cooperate with the TGF-beta/Smad pathway to transactivate PAI-1. Importantly, Foxc2 haploinsufficiency in mice significantly attenuates TGF-beta1-induced PAI-1 expression in the cardiovascular system and adipose tissue. Taken together, we propose that Foxc2 is a key molecule to regulate PAI-1 gene expression.

Expression of the Nd1 gene is down-regulated by doxorubicin at post-transcriptional level.

Doxorubicin is an anti-neoplastic agent with cardiotoxicity as a side effect. We previously demonstrated that doxorubicin treatment of mice resulted in a selective decrease in expression of the Nd1 gene, which encoded a new kelch family actin binding protein in the heart. Here we show that doxorubicin treatment also reduced the Nd1 expression in various organs of mice and cultured cell lines. The treatment of Nd1-transgenic mice and Nd1-transfectants also selectively reduced levels of the exogenous Nd1 mRNAs, whose expression was under the control of various promoters. Furthermore, the doxorubicin-induced reduction of Nd1 mRNA expression in NIH3T3 cells was inhibited by treatment of these cells with cycloheximide. Thus, the doxorubicin treatment may specifically reduce the stability of Nd1 mRNA.

Overexpression of Nd1-s, a variant form of new kelch family protein, perturbs the cell cycle progression of fibroblasts.

The murine Nd1 gene encodes two forms of protein, Nd1-L and Nd1-S, both of which share the BTB/POZ domain, but Nd1-S lacks the kelch repeats. Although Nd1-L ubiquitously expresses, localizes in the cytoplasm and functions as a stabilizer of actin filaments, expression and function of Nd1-S were unknown. Here we show that Nd1-S were expressed in all tissues examined and localized in the nucleus as a speckled-like pattern. Furthermore, overexpression of Nd1-S perturbed cell growth of NIH3T3 cells at the G1/S phase of the cell cycle. These results suggest that Nd1-S may play a role in cell cycle progression in the nucleus.

Protective role of Nd1 in doxorubicin-induced cardiotoxicity.

OBJECTIVE: The Ndl gene, which encodes a novel kelch family protein, is expressed ubiquitously in mouse tissues. In vitro studies suggest that Ndl protein, which binds to actin filaments, functions as a cytoskeletal stabilizer. In order to elucidate a physiological function of Ndl in vivo, we generated Nd1-deficient (Ndl-/-) mice. METHODS: We developed Nd1-/- mice by standard gene targeting technique. Cardiac function was studied in wild type and Nd1-/- mice. RESULTS: Nd1-/- mice were viable and no gross anatomical abnormality was observed after birth. When mouse embryonic fibroblasts were cultured in the presence of cytochalasin D or doxorubicin, the number of apoptotic cells in the Nd1-/- cell culture was larger that that in the wild-type cell culture. Furthermore, Nd1-/- mice were sensitive to doxorubicin-induced cardiotoxicity with increased numbers of cardiomyocytes apoptosis. CONCLUSIONS: Although Nd1 is dispensable for normal mice development, Nd1 plays a protective role in doxorubicin-induced cardiotoxic responses.

Hoxc8 represses BMP-induced expression of Smad6.

Proper regulation of bone morphogenetic protein (BMP) signaling is critical for correct patterning and morphogenesis of various tissues and organs. A well known feedback mechanism is a BMP-mediated induction of Smad6, an inhibitor of BMP signaling. Hoxc8, one of the Hox family transcription factors, has also been shown to negatively regulate BMP-mediated gene expression. Here we add another level of Hoxc8 regulation on BMP signaling. Our results show that Hoxc8, when over-expressed in C3H10T1/2 or C2C12 cells, suppressed basal Smad6 promoter activity and its mRNA expression. Activation of Smad6 transcription either by BMP2 treatment or Smad1 over-expression was also abolished by Hoxc8. When chromatin was precipitated from mouse embryos with anti-Smad1 or anti-Hoxc8 antibody, Smad6 promoter sequence was enriched, suggesting that Hoxc8 proteins make complexes with Smad1 in the Smad6 promoter region. Yet, a lack of Hox binding motifs in the Smad6 promoter sequence suggests that instead of directly binding to the DNA, Hoxc8 may regulate Smad6 expression via an indirect mechanism. Our results suggest that the Smad6-mediated negative feedback mechanism on BMP signaling may be balanced by the repression of Smad6 expression by Hoxc8.

In vitro osteoblast differentiation is negatively regulated by Hoxc8.

Hoxc8 has multiple roles in normal skeletal development. In this paper, a MC3T3-E1 subclone 4 osteogenic cell differentiation model was used to examine expression of Hoxc8 at multiple stages of osteogenesis. We found that Hoxc8 expression levels do not change in the early stage but increase in the middle stage and decrease in the late stage of osteogenesis. A knockdown of Hoxc8 by small-interfering RNA transfection in C2C12 cells indicated that Hoxc8 is a negative regulator of osteogenesis. Similarly, expression of Hoxc8 in C2C12 cells decreases alkaline phosphatase levels induced by bone morphogenetic protein-2 (BMP-2). The results of this study showed that Hoxc8 is involved in BMP-2-induced osteogenesis, and osteoblast differentiation in vitro is negatively regulated by Hoxc8, suggesting that Hoxc8 regulation is essential for osteoblast differentiation.

The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development.

Accumulating evidence suggests that in the vertebrate embryo, acquisition of arterial and venous identity is established early by genetic mechanisms, including those regulated by vascular endothelial growth factor (VEGF) and Notch signaling. However, although the COUP-TFII nuclear receptor has recently been shown to regulate vein identity, very little is known about the molecular mechanisms of transcriptional regulation in arterial specification. Here, we show that mouse embryos compound mutant for Foxc1 and Foxc2, two closely related Fox transcription factors, exhibit arteriovenous malformations and lack of induction of arterial markers whereas venous markers such as COUP-TFII are normally expressed, suggesting that mutant endothelial cells fail to acquire an arterial fate. Notably, consistent with this observation, overexpression of Foxc genes in vitro induces expression of arterial markers such as Notch1 and its ligand Delta-like 4 (Dll4), and Foxc1 and Foxc2 directly activate the Dll4 promoter via a Foxc-binding site. Moreover, compound Foxc mutants show a defect in sprouting of lymphatic endothelial cells from veins in early lymphatic development, due to reduced expression of VEGF-C. Taken together, our results demonstrate that Foxc transcription factors are novel regulators of arterial cell specification upstream of Notch signaling and lymphatic sprouting during embryonic development.