Mechanism mediated by a noncoding RNA, nc886, in the cytotoxicity of a DNA-reactive compound

DNA-reactive compounds are harnessed for cancer chemotherapy. Their genotoxic effects are considered to be the main mechanism for the cytotoxicity to date. Because this mechanism preferentially affects actively proliferating cells, it is postulated that the cytotoxicity is specific to cancer cells. Nonetheless, they do harm normal quiescent cells, suggesting that there are other cytotoxic mechanisms to be uncovered. By employing doxorubicin as a representative DNA-reactive compound, we have discovered a cytotoxic mechanism that involves a cellular noncoding RNA (ncRNA) nc886 and protein kinase R (PKR) that is a proapoptotic protein. nc886 is transcribed by RNA polymerase III (Pol III), binds to PKR, and prevents it from aberrant activation in most normal cells. We have shown here that doxorubicin evicts Pol III from DNA and, thereby, shuts down nc886 transcription. Consequently, the instantaneous depletion of nc886 provokes PKR and leads to apoptosis. In a short-pulse treatment of doxorubicin, these events are the main cause of cytotoxicity preceding the DNA damage response in a 3D culture system as well as the monolayer cultures. By identifying nc886 as a molecular signal for PKR to sense doxorubicin, we have provided an explanation for the conundrum why DNA-damaging drugs can be cytotoxic to quiescent cells that have the competent nc886/PKR pathway.ope

Transgenic overexpression of human LY6K in mice suppresses mature T cell development in the thymus

Lymphocyte antigen 6 family member K (LY6K) is upregulated in a number of types of cancer and promotes tumor cell proliferation and metastasis. In addition, LY6K is involved in tamoxifen resistance in breast cancer. However, the in vivo molecular mechanism of LY6K has not yet been investigated. In the present study, transgenic mice overexpressing human LY6K (hLY6K) were generated using the pMAMneo vector, and the effect of LY6K upregulation in vivo was investigated. A total of 4 transgenic mice were generated, and the gene copy number was examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RT-qPCR demonstrated that mRNA of hLY6K was overexpressed in the thymus and spleen of the transgenic mice compared with wild-type mice. Flow cytometric analysis demonstrated that the proportions of B and T cells in the spleen were similar in wild-type and transgenic mice; however, the proportion of thymic mature T cells decreased in the transgenic mice, while there was an increase in the proportion of naïve T cells. These findings suggest that the overexpression of LY6K suppresses T cell development, and that LY6K is a potential therapeutic target for cancer.ope

Transcription factors Sp1 and Sp3 regulate expression of human ABCG2 gene and chemoresistance phenotype

ABCG2 is a member of the ATP binding cassette (ABC) transmembrane proteins that plays an important role in stem cell biology and drug resistance of cancer cells. In this study, we investigated how expression of human ABCG2 gene is regulated in lung cancer A549 cells. Binding of Sp1 and Sp3 transcription factors to the ABCG2 promoter in vitro and in vivo was elucidated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ABCG2 promoter activity was impaired when Sp1 sites were mutated but was enhanced by overexpression of Sp1 or Sp3 proteins. Knockdown of Sp1 or Sp3 expression by short interfering RNA significantly decreased the expression of ABCG2 mRNA and protein, resulting in attenuated formation of the side population in A549 cells. In addition, Sp1 inhibition in vivo by mithramycin A suppressed the percentage of the side population fraction and sphere forming activities of A549 cells. Moreover, inhibiting Sp1- or Sp3-dependent ABCG2 expression caused chemosensitization to the anticancer drug cisplatin. Collectively, our results demonstrate that Sp1 and Sp3 transcription factors are the primary determinants for activating basal transcription of the ABCG2 gene and play an important role in maintaining the side population phenotype of lung cancer cells.ope

Epigenome mapping highlights chromatin-mediated gene regulation in the protozoan parasite Trichomonas vaginalis

Trichomonas vaginalis is an extracellular flagellated protozoan parasite that causes trichomoniasis, one of the most common non-viral sexually transmitted diseases. To survive and to maintain infection, T. vaginalis adapts to a hostile host environment by regulating gene expression. However, the mechanisms of transcriptional regulation are poorly understood for this parasite. Histone modification has a marked effect on chromatin structure and directs the recruitment of transcriptional machinery, thereby regulating essential cellular processes. In this study, we aimed to outline modes of chromatin-mediated gene regulation in T. vaginalis. Inhibition of histone deacetylase (HDAC) alters global transcriptional responses and induces hyperacetylation of histones and hypermethylation of H3K4. Analysis of the genome of T. vaginalis revealed that a number of enzymes regulate histone modification, suggesting that epigenetic mechanisms are important to controlling gene expression in this organism. Additionally, we describe the genome-wide localization of two histone H3 modifications (H3K4me3 and H3K27Ac), which we found to be positively associated with active gene expression in both steady and dynamic transcriptional states. These results provide the first direct evidence that histone modifications play an essential role in transcriptional regulation of T. vaginalis, and may help guide future epigenetic research into therapeutic intervention strategies against this parasite.ope

Modulation of dendritic cell function by Trichomonas vaginalis-derived secretory products

Trichomoniasis caused by the parasitic protozoan Trichomonas vaginalis is the most common sexually transmitted disease in the world. Dendritic cells are antigen presenting cells that initiate immune responses by directing the activation and differentiation of naïve T cells. In this study, we analyzed the effect of Trichomonas vaginalis-derived Secretory Products on the differentiation and function of dendritic cells. Differentiation of bone marrow-derived dendritic cells in the presence of T. vaginalis-derived Secretory Products resulted in inhibition of lipopolysaccharide-induced maturation of dendritic cells, down-regulation of IL-12, and up-regulation of IL-10. The protein components of T. vaginalis-derived Secretory Products were shown to be responsible for altered function of bone marrow- derived dendritic cells. Chromatin immunoprecipitation assay demonstrated that IL-12 expression was regulated at the chromatin level in T. vaginalis-derived Secretory Productstreated dendritic cells. Our results demonstrated that T. vaginalis- derived Secretory Products modulate the maturation and cytokine production of dendritic cells leading to immune tolerance.ope

(The) role of PKMζ in the nucleus accumbens in the expression of psychostimulantinduced locomotor sensitization

의과학과/석사약물 중독은 약물이 가져다 주는 쾌락적(hedonic)인 보상 효과뿐만 아니라, 이와 짝지어진 어떤 환경적 요소들이 불러 일으키는 조건화된 갈망 반응으로 인해 매우 치료가 어려운 만성 뇌 질환이다. 중독이 형성되는 과정에는 대뇌 보상회로를 구성하는 여러 부위에서 일어나는 LTP(long-term potentiation)와 같은 시냅스 가소성이 중요한 역할을 하며, 한번 형성된 보상 기억은 매우 오래도록 유지된다. PKMζ(protein kinase M ζ)는 LTP와 기억을 지속적으로 유지한다고 알려져 있으므로, 약물 중독이 형성되어 장기간 유지되는 과정에서 어떤 역할을 할 가능성이 높다고 생각된다. 이를 바탕으로, 암페타민에 의해 유도된 행동과민반응 모델에서 중독이 장기간 유지되기 위해 PKMζ가 어떠한 역할을 하고 있는지 밝히고자 하였다. 암페타민(amphetamine; 1 mg/kg, IP) 또는 생리식염수(saline; 1 ml/kg, IP)를 2~3일 간격으로 총 4회 복강 주사를 주고, 1주일 동안 휴식기간을 준 뒤, 다음과 같이 테스트를 진행하였다. PKMζ 저해제인 ZIP(ζ-pseudosubstrate inhibitory peptide; 1.0 or 10 μg/0.5 μl/side) 또는 생리식염수(0.5 μl/side)를 중격측좌핵 내에 국소주입하고 30분 뒤, 암페타민(1 mg/kg, IP) 또는 생리식염수를 복강 주사하여 보행성 활동량을 측정하였다. 암페타민을 반복적으로 준 그룹에서는 테스트할 때 처음으로 암페타민을 받게 된 그룹에 비하여 보행성 활동량이 증가되어 나타나는 반면에, PKMζ의 저해제인 ZIP을 중격측좌핵 내에 주입한 그룹에서는 증가된 보행성 활동량이 저해되어 나타나는 것을 확인할 수 있었다. 그러나, 생리식염수에 사전 노출된 후 암페타민을 처음으로 받게 된 쥐들에서는 ZIP에 의한 보행성 활동량 저해효과를 관찰할 수 없었다. 이 같은 실험조건 하에서 이번에는 western blotting 방법으로 Akt와 GSK의 인산화 정도 변화를 보았을 때, GSK는 그룹간의 차이가 없었으나, Akt는 암페타민에 사전에 노출된 뒤 암페타민을 다시 받은 그룹의 경우 생리식염수를 먼저 국소주입한 그룹보다 ZIP을 먼저 국소주입한 그룹이 threonine 308(Thr308) 부위의 인산화 정도가 현저하게 감소되는 것을 확인할 수 있었다. 이 결과를 종합하여 보면, PKMζ가 암페타민에 의하여 유도된 행동과민반응을 발현하는데 중요한 역할을 하는 것으로 보이며, 이 작용은 Akt Thr308부위에 관련이 있는 것으로 보인다.restrictio

A Study on Characterized Others through Isolated Fantasy : Based on My Works

학위논문(석사) -- 서울대학교대학원 : 미술대학 서양화과, 2022.2. 임자혁.나의 작업은 타자를 바라보는 사회의 시선에 대한 문제의식에서 출발한다. 그 문제의식을 드러내는 일에 내가 쓰는 방법에는 여러 가지가 있다. 비꼬기, 은유, 선언, 울음 터뜨리기, 평정 유지하기 등 수단과 방법을 가리며 그 때의 상황에 따라 다른 방식을 취한다. 또한 타자를 하나의 캐릭터로 상상하는 것에서부터 시작해 그들이 어떤 대화를 나누고 어떤 삶을 사는지에 대한 상상으로 작업의 서사를 만들어낸다. 내가 상상해 낸 괴물인 척하는 소녀와 소녀인 척하는 괴물은 게임이나 만화에서 받은 영향을 숨기지 않으면서 타자의 삶과 타자가 삶을 살아가는 방식, 사회가 그들을 대하는 방식을 토로한다. 그 후에는 비뚤어진 타자를 기반으로 한 이질적인 작품들을 한데 그러모아 유동적이고 가벼운 설치를 한 뒤, 그 속에서 타자들이 사는 환상의 세계에 관한 상상을 이어 나간다. 그곳은 나의 상상력을 기반으로 만들어낸 세상으로, 타자들이 마음껏 공동체로서 기쁨과 슬픔을 누리는 곳이다. 나는 그 세계에서 타자들의 삶과 그들의 존재의미에 관한 실마리, 그들이 자유롭게 살 수 있는 방법을 찾는다. 그 실마리란 양가성에 관한 것이고, 여러 입으로 여러 말을 하는 것이며, 고정되지 못하는 정체성의 고정되지 않아도 됨에 관한 것이다. 그것은 타자의 담론에서 그치는 것이 아니라 오브제로서의 회화, 예술과 삶을 살아가는 방식까지 이어진다. 완성되지 않아도 되는 작품과 그와 이어지는 자유롭고 가벼운 삶의 태도는 나의 예술에서 중요한 자리를 차지하고 있다.My art practice starts from realizing the social views of people. I have several methods for revealing this critical portion of their minds: being sarcastic, metaphoric, making pronouncements, crying, and staying calm. In the end, different ways are applied by any means. Moreover, by starting from conceptualizing others as characters, the narrative is built in my imagination of how they live and communicate. According to my imagination, a girl who pretends to be a monster and a monster who pretends to be a girl represent how people live and how society sees them, not by hiding their influence with a game or cartoon. Then, different works based on these crooked people are collected into one character to be flexible and light, and my imaginary story about the illusion where they live continues. It is the world based on my imagination where everyone enjoys happiness and sadness as much as they want within a community. In this world, I look for people’s lives, clues about the meaning of existence, and a way that they can freely live. This clue is about ambivalence, something about telling different things with different mouths, and how an identity that cannot be fixed does not have to be fixed. It is not finished with a discussion of others but continues with a painting as an object and as a way of art and life. An artwork that does not have to be finished and, in this respect, the free and light attitude of life takes an important position in my art practice.Ⅰ. 서론 1 Ⅱ. 비뚤어진 타자와 그들의 투쟁 3 1. 캐릭터화된 타자 5 1) 괴물인 척하는 소녀 6 2) 소녀인 척하는 괴물 17 2. 비뚤어진 타자의 세계 29 1) 애잔한 환상적 군락 34 2) 깨지기 쉬운 타자의 정원 39 Ⅲ. 즉흥적인 설치와 주도면밀한 게릴라전 41 1. 울타리 허물기 42 2. 게릴라전의 열린 결말 49 Ⅳ. 결론 56석

Epigenetic regulation of RNA polymerase III transcription in early breast tumorigenesis

RNA polymerase III (Pol III) transcribes medium-sized non-coding RNAs (collectively termed Pol III genes). Emerging diverse roles of Pol III genes suggest that individual Pol III genes are exquisitely regulated by transcription and epigenetic factors. Here we report global Pol III expression/methylation profiles and molecular mechanisms of Pol III regulation that have not been as extensively studied, using nc886 as a representative Pol III gene. In a human mammary epithelial cell system that recapitulates early breast tumorigenesis, the fraction of actively transcribed Pol III genes increases reaching a plateau during immortalization. Hyper-methylation of Pol III genes inhibits Pol III binding to DNA via inducing repressed chromatin and is a determinant for the Pol III repertoire. When Pol III genes are hypo-methylated, MYC amplifies their transcription, regardless of its recognition DNA motif. Thus, Pol III expression during tumorigenesis is delineated by methylation and magnified by MYC.restrictio

Dynamic Long-Range Chromatin Interaction Controls Expression of IL-21 in CD4+ T Cells.

IL-21, a pleiotropic cytokine strongly linked with autoimmunity and inflammation, regulates diverse immune responses. IL-21 can be potently induced in CD4(+) T cells by IL-6; however, very little is known about the mechanisms underlying the transcriptional regulation of the Il21 gene at the chromatin level. In this study, we demonstrated that a conserved noncoding sequence located 49 kb upstream of the Il21 gene contains an enhancer element that can upregulate Il21 gene expression in a STAT3- and NFAT-dependent manner. Additionally, we identified enhancer-blocking insulator elements in the Il21 locus, which constitutively bind CTCF and cohesin. In naive CD4(+) T cells, these upstream and downstream CTCF binding sites interact with each other to make a DNA loop; however, the Il21 promoter does not interact with any cis-elements in the Il21 locus. In contrast, stimulation of CD4(+) T cells with IL-6 leads to recruitment of STAT3 to the promoter and novel distal enhancer region. This induces dynamic changes in chromatin configuration, bringing the promoter and the regulatory elements in close spatial proximity. The long-range interaction between the promoter and distal enhancer region was dependent on IL-6/STAT3 signaling pathway but was disrupted in regulatory T cells, where IL-21 expression was repressed. Thus, our work uncovers a novel topological chromatin framework underlying proper transcriptional regulation of the Il21 gene.restrictio