Changjin Lee

Srg3, a Mouse Homolog of Yeast SWI3, Is Essential for Early Embryogenesis and Involved in Brain Development

ABSTRACT Srg3 (SWI3-related gene product) is a mouse homolog of yeast SWI3,Drosophila melanogaster MOIRA (also named MOR/BAP155), and human BAF155 and is known as a core subunit of SWI/SNF complex. This complex is involved in the chromatin remodeling required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. We generated mice with a null mutation in theSrg3 locus to examine its function in vivo. Homozygous mutants develop in the early implantation stage but undergo rapid degeneration thereafter. An in vitro outgrowth study revealed that mutant blastocysts hatch, adhere, and form a layer of trophoblast giant cells, but the inner cell mass degenerates after prolonged culture. Interestingly, about 20% of heterozygous mutant embryos display defects in brain development with abnormal organization of the brain, a condition known as exencephaly. Histological examination suggests that exencephaly is caused by the failure in neural fold elevation, resulting in severe brain malformation. Our findings demonstrate that Srg3 is essential for early embryogenesis and plays an important role in the brain development of mice.

SRG3 Interacts Directly with the Major Components of the SWI/SNF Chromatin Remodeling Complex and Protects Them from Proteasomal Degradation

The mammalian SWI/SNF complex is an evolutionarily conserved ATP-dependent chromatin remodeling complex that consists of nine or more components. SRG3, a murine homologue of yeast SWI3, Drosophila MOIRA, and human BAF155, is a core component of the murine SWI/SNF complex required for the regulation of transcriptional processes associated with development, cellular differentiation, and proliferation. Here we report that SRG3 interacts directly with other components of the mammalian SWI/SNF complex such as SNF5, BRG1, and BAF60a. The SWIRM domain and the SANT domain were required for SRG3-SNF5 and SRG3-BRG1 interactions, respectively. In addition, SRG3 stabilized SNF5, BRG1, and BAF60a by attenuating their proteasomal degradation, suggesting its general role in the stabilization of the SWI/SNF complex. Such a stabilization effect of SRG3 was not only observed in the in vitro cell system, but also in cells isolated from SRG3 transgenic mice or knock-out mice haploinsufficient for the Srg3 gene. Taken together, these results suggest the critical role of SRG3 in the post-transcriptional stabilization of the major components of the SWI/SNF complex.

Effect of fiber type on postmortem proteolysis in longissimus muscle of Landrace and Korean native black pigs.

The current study was conducted to characterize objective meat quality, fiber type and their relations to postmortem proteolysis in longissimus muscle of Landrace and Korean native black (KNP) pigs. Longissimus muscles from each 10 market-weighted male pigs were removed after conventional slaughtering and chilling procedures, and aged for 1 or 7 days at 4°C to determine WB-shear force, objective meat color, proportion of myosin heavy chain I (MyHC I), intramuscular fat content and rate of proteolysis by a proteomics approach. KNP had a significantly (p<0.05) higher content of MyHC I, and that concurred with greatly (p<0.05) higher intramuscular fat content and Hunter a(∗) value, and significantly (p<0.05) lower drip loss than those seen in Landrace. One-dimension SDS-PAGE indicated that GAPDH, troponin I and creatine kinase were prominent proteolytic products during chiller ageing. By applying a gel-based proteome analysis, 26 proteins were identified, which showed different degradation properties during ageing between the breeds. Biopsied sample revealed that myosin regulatory light chain 2, myosin light chain isotype v/sb, fatty acid-binding protein and albumin were expressed at a greatly higher level for KNP, but their relation to fiber type (or genetic background) is unclear. It was particular noticeable that different actin isoforms showed various degradation behavior during ageing time.

SRG3, a core component of mouse SWI/SNF complex, is essential for extra-embryonic vascular development

The SWI/SNF chromatin-remodeling complex functions as a transcriptional regulator and plays a significant role in cell proliferation, differentiation and embryonic development. SRG3, a homologue of human BAF155, is a core component of the mouse SWI/SNF chromatin remodeling complex. Mutant mice deficient in Srg3 expression are peri-implantation lethal. To investigate the role of SRG3 in the post-implantation stage, we generated SRG3 transgene-rescued (Srg3-/-Tg+) embryos by inducing exogenous gene expression. These Srg3-/-Tg+ embryos overcame early embryonic lethality and extended the life span until mid-gestation. However, the embryos displayed significant defects in blood vessel formation and fetal circulation within the yolk sac around embryonic day 10.5, which led to developmental retardation and death. We found that SRG3 expression was absent in the visceral endoderm of Srg3-/-Tg+ yolk sacs, while SRG3 was normally expressed in wild-type embryos. In addition, expression of angiogenesis-related genes, including Angiopoietin1, Tie2, EphrinB2, Ihh and Notch1, was markedly reduced in Srg3-/-Tg+ yolk sacs. During normal angiogenesis, maturation of the visceral endoderm development is observed in the yolk sac. However, in Srg3-/-Tg+ yolk sacs, the visceral endoderm did not develop normally. Our results indicate that SRG3 is required for angiogenesis and visceral endoderm development in the yolk sac.

CD7 expression and galectin-1-induced apoptosis of immature thymocytes are directly regulated by NF-κB upon T-cell activation

CD7, one of the galectin-1 receptors, has crucial roles in galectin-1-mediated apoptosis of activated T-cells and T-lymphoma progression in peripheral tissues. In this study, we showed that CD7 promoter activity was increased by NF-kappaB and that this activity was synergistic when Sp1 was co-expressed in the immature T-cell line L7. Site-directed mutagenesis analysis of the CD7 promoter indicated that NF-kappaB specifically bound to the NF-kappaE2 site in cooperation with Sp1. Overexpression of E12 or Twist2 proteins negatively regulated NF-kappaB-mediated activity of the CD7 proximal promoter. In addition, CD7 expression was down-regulated by treatment with the p38 MAPK inhibitor SB20358, or the MSK1 inhibitor H-89. These signaling pathway inhibitors prevented galectin-1-mediated apoptosis of immature T-cells. From these results, we concluded that the regulation of CD7 gene expression through NF-kappaB activation induced by TCR/CD28 might have significant implications for T-cell homeostasis.

The SWI/SNF-like BAF Complex Is Essential for Early B Cell Development

During the process of B cell development, transcription factors, such as E2A and Ebf1, have been known to play key roles. Although transcription factors and chromatin regulators work in concert to direct the expression of B lineage-specific genes, little is known about the involvement of regulators for chromatin structure during B lymphopoiesis. In this article, we show that deletion of Srg3/mBaf155, a scaffold subunit of the SWI/SNF-like BAF complex, in the hematopoietic lineage caused defects at both the common lymphoid progenitor stage and the transition from pre–pro-B to early pro-B cells due to failures in the expression of B lineage-specific genes, such as Ebf1 and Il7ra, and their downstream target genes. Moreover, mice that were deficient in the expression of Brg1, a subunit of the complex with ATPase activity, also showed defects in early B cell development. We also found that the expression of Ebf1 and Il7ra is directly regulated by the SWI/SNF-like BAF complex. Thus, our results suggest that the SWI/SNF-like BAF complex facilitates early B cell development by regulating the expression of B lineage-specific genes.

DNA Aptamers against the Receptor Binding Region of Hemagglutinin Prevent Avian Influenza Viral Infection

The entrance of influenza virus into host cells is facilitated by the attachment of the globular region of viral hemagglutinin to the sialic acid receptors on host cell surfaces. In this study, we have cloned the cDNA fragment encoding the entire globular region (residues 101–257) of hemagglutinin of the H9N2 type avian influenza virus (A/ck/Korea/ms96/96). The protein segment (denoted as the H9 peptide), which was expressed and purified in E. coli, was used for the immunization of BALB/c mice to obtain the anti-H9 antiserum. To identify specific DNA aptamers with high affinity to H9 peptide, we conducted the SELEX method; 19 aptamers were newly isolated. A random mixture of these aptamers showed an increased level of binding affinity to the H9 peptide. The sequence alignment analysis of these aptamers revealed that 6 aptamers have highly conserved consensus sequences. Among these, aptamer C7 showed the highest similarity to the consensus sequences. Therefore, based on the C7 aptamer, we synthesized a new modified aptamer designated as C7-35M. This new aptamer showed strong binding capability to the viral particles. Furthermore, it could prevent MDCK cells from viral infection by strong binding to the viral particles. These results suggest that our aptamers can recognize the hemagglutinin protein of avian influenza virus and inhibit the binding of the virus to target receptors required for the penetration of host cells.

The Enhancement of Regression Rate of Hybrid Rocket Fuel by Various Methods

Hybrid rocket has been spotlighted recently by its excellent safety in combustion and comparable thrust level although lower density specific impulse and charging efficiency. Thus, many researches have been focused to find the way to increase regression rate. One of the popular methods to increase regression rate is to use a swirl flow of oxidizer. The effect of swirl flow is to extend the residence time of oxidizer in the propellant grain. Meanwhile, it is well known to solid propellant society that the embedded metal wires can increase the burning rate of solid propellant by increasing heat transfer to solid fuel. Thus, metal wires may cause the same effect in hybrid fuel by enhancing the regression rate of solid. In this study, a series of experimental tests is designed to investigate the enhancement of regression rate of solid fuel by embedded metal wires. And fuel grain configuration is adopted to simply increase the burning surface area and to try to induce swirl flow. PMMA with gaseous oxygen is the solid fuel for investigation. Test results show that embedded metal wires are not effective method in enhancing regression rate. Only 3-4% increases in regression rate are observed. However, fuel port configuration yields higher burning performance up to several ten percentages increase in regression rate. And pitch number total impulse can be design variables in determining overall regression rate.

E2A/HEB and Id3 proteins control the sensitivity to glucocorticoid-induced apoptosis in thymocytes by regulating the SRG3 expression.

The E protein family transcription factors encoded by the E2A and HEB genes are known to play critical roles in the coordinate regulation of lymphocyte development. Previous studies have shown that T cell receptor (TCR) signals rapidly induce Id3, a dominant negative antagonist of E2A activity and allow thymocytes to survive selection events in the thymus. Here we show that SRG3 acts as a novel downstream target of E2A/HeLa E box-binding (HEB) complex and modulates glucocorticoid (GC) susceptibility in thymocytes in response to TCR signals. We have identified a putative E box element in the SRG3 promoter that is required for optimal promoter activity. The transcription factors E2A and HEB specifically associate with the E box element. Moreover, E2A-HEB heterodimers cooperated to activate SRG3 transcription, which was inhibited by the expression of Id proteins. TCR-mediated signals rapidly induced Id3 via MEK/ERK activation and thereby kept the E2A/HEB complex from binding to the E box element in the SRG3 promoter. Retroviral transduction of Id3 also repressed the SRG3 expression by inhibiting the E box binding activity of the E2A/HEB complex. Intriguingly, enforced Id3 expression conferred thymocyte resistance to GCs, which could be overcome by the overexpression of SRG3. Taken together, these results suggest that Id3 may enhance the viability of immature thymocytes by at least rendering them resistant to GCs through SRG3 down-regulation.

Twist2 regulates CD7 expression and galectin-1-induced apoptosis in mature T-cells

In the periphery, a galectin-1 receptor, CD7, plays crucial roles in galectin-1-mediated apoptosis of activated T-cells as well as progression of T-lymphoma. Previously, we demonstrated that NF-κB downregulated CD7 gene expression through the p38 MAPK pathway in developing immature thymocytes. However, its regulatory pathway is not well understood in functional mature T-cells. Here, we show that CD7 expression was downregulated by Twist2 in Jurkat cells, a human acute T-cell lymphoma cell line, and in EL4 cells, a mature murine T-cell lymphoma cell line. Furthermore, ectopic expression of Twist2 in Jurkat cells reduced galectin-1-induced apoptosis. While full-length Twist2 decreased CD7 promoter activity, a C-terminal deletion form of Twist2 reversed its inhibition, suggesting an important role of the C-terminus in CD7 regulation. In addition, CD7 expression was enhanced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate, which indicates that Twist2 might be one of candidate factors involved in histone deacetylation. Based on these results, we conclude that upregulation of Twist2 increases the resistance to galectin-1-mediated-apoptosis, which may have significant implications for the progression of some T-cells into tumors such as Sezary cells.