MiT Family Transcriptional Factors in Immune Cell Functions

Kim, Seongryong,Song, Hyun-Sup,Yu, Jihyun,Kim, You-Me Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.5

The microphthalmia-associated transcription factor family (MiT family) proteins are evolutionarily conserved transcription factors that perform many essential biological functions. In mammals, the MiT family consists of MITF (microphthalmia-associated transcription factor or melanocyte-inducing transcription factor), TFEB (transcription factor EB), TFE3 (transcription factor E3), and TFEC (transcription factor EC). These transcriptional factors belong to the basic helix-loop-helix-leucine zipper (bHLH-LZ) transcription factor family and bind the E-box DNA motifs in the promoter regions of target genes to enhance transcription. The best studied functions of MiT proteins include lysosome biogenesis and autophagy induction. In addition, they modulate cellular metabolism, mitochondria dynamics, and various stress responses. The control of nuclear localization via phosphorylation and dephosphorylation serves as the primary regulatory mechanism for MiT family proteins, and several kinases and phosphatases have been identified to directly determine the transcriptional activities of MiT proteins. In different immune cell types, each MiT family member is shown to play distinct or redundant roles and we expect that there is far more to learn about their functions and regulatory mechanisms in host defense and inflammatory responses.

Localization for Transcription Factors in the Stage of Early Embryo Development in Porcine

Joo Bin Lee,Tao Lin,Hyeon Yeong Shin,Jae Eun Lee,Jung won Kang,So Yeon Kim,Dong Il Jin 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Embryo development is the most important time in the life of a multicellular organism because the limited cells of the embryo produce all the cells of the adult body. And transcription factors are transcriptional regulatory proteins that specifically bind to the transcriptional regulatory region DNA and activate or inhibit the transcription of that gene. It is change in the stage of embryo development that positions and levels of expression of transcription factors. Therefore, we know that transcription factors conduct vary crucial role in the stage of embryo development. So, if we could identify clearly changes of transcription factors, it can be very helpful in understanding the mechanism of embryo development. We experimented in transcription factors from GV oocyte to blastocyst how are expressed in porcine. We focused on especially pluripotency genes, which play an important role in early embryo development. So we conducted to immuno- staining using antibodies of Cdx2, Oct4, Sox2, Nanog, and E-Cadherin. Unfortunately we used mouse’s antibody because pig`s antibody against transcription factors did not exist. We checked five transcription factors (Cdx2, Oct4, Sox2, Nanog and E-Cadherin) in early embryo development in porcine. We found different expression patterns between mouse and pig in some stages. Therefore, we need to experiment that different patterns of transcription factors play a role in species, and why occur.

NMDA투여에 의한 transcription factor (Egr-1, C-Jun, JunB, FosB)의 발현 변화 양상

하종수(Jong-Su Ha),김재화(Jae-Wha Kim),송재찬(Jae-Chan Song) 한국생명과학회 2015 생명과학회지 Vol.25 No.9

신경과흥분은 신경세포의 수지돌기 말단부에 있는 흥분성 수용체에 대한 과도한 자극에 의해서 신경세포가 손상을 받는 현상으로 transcription factor의 발현을 유도하여 통증을 유발하는 자극, 학습, 발작, 흥분, 신경변성, 저산소성 국소빈혈, 뇌신경손상, 신경절제, 약제내성 등의 원인이 된다. 신경과흥분은 정상농도 이상의 NMDA에 의해서도 유발되는데 본 논문에서는 mouse의 복강으로 과량의 NMDA를 투여하여 소뇌에서 RT-PCR 방법으로 Inducible transcription factors (Egr-1, c-jun, JunB, FosB) mRNAs의 상대적 발현량을 비교하였다. NMDA를 투여한 군에서 inducible transcription factors (Egr-1, C-Jun, JunB, FosB)가 투여량과 시간의 경과에 따라 다양한 발현의 변화를 보였으며, NMDA투여 후 일정한 시간에서 투여한 양에 대한 변화는 체중 g 당 5 μg의 NMDA투여한 경우에 현저한 변화가 나타났다. 조사한 transcription factor 중에서 JunB의 발현 변화가 다른 transcription factor보다 두드러지게 나타났다. NMDA 투여량이 일정할 때 투여 후 경과 시간에 따른 발현양상은 투여 후 24시간이 경과한 후에 발현의 변화가 두드러지게 증가하는 경향을 나타내었고 대부분 이 48시간 경과 후 발현이 최고치에 도달하였다. 이러한 결과는 과흥분이 유도된 소뇌에서의 유전자 발현의 변화를 2D-gel 또는 microarray와 같은 방법을 이용하여 세포 내의 전체 단백질 혹은 유전자의 변화를 관찰함으로써 NMDA 수용체의 과흥분에 의한 뇌세포의 사멸에 관련된 기전을 밝힐 수 있는 좋은 자료가 될 수 있을 것으로 기대된다. Glutamate is one of the principle transmitters in the CNS. Ionotropic receptors of glutamate, selectively activated by N-methyl-D-aspartate (NMDA), play an important role in the processes of cell development, learning, memory, and etc. On the other hand, many studies discovered that over-activation of glutamate receptors leads to neurodegeneration and are known to be implicated in major areas of brain pathology. Any sustained effect of a transient NMDA receptor activation is likely to involve signaling to the nucleus and to trigger coordinated changes in gene expression. Classically, a set of immediate-early genes are induced first; some of genes are by themselves transcription factors that control expression of other target genes. This study provides understanding of changes of inducible transcription factors mRNA levels with RT-PCR by inducing over-activation of NMDA receptor with intraperitoneal NMDA injection. The experimental conditions were varied by 1, 5, 25, and 125 g/ of body weight NMDA and measured transcription factors mRNA levels are Egr-1, c-Jun, JunB, and FosB. Based on result obtained, inducible transcription factors mRNA in NMDA injection to mice with 5 g/body weight showed the greatest change. And ITF mRNA showed greatest change 24 hr after injection. The expression level of JunB mRNA was markedly changed. Up to the present days, no study clearly understood how ITF mRNA affected the apoptosis of purkinje cells in the cerebellum. The current study improves the understanding of the mechanism of apoptosis of purkinje cells in the cerebellum.

Temporal Regulation of Ovine Interferon-tau Gene by the Transcription Factor Eomesodermin in the Peri-Implantation Period

Min-Su Kim,Hyun-Joo Lim,Ji Hwan Lee,Tae Young Hur,Jun Kyu Son 한국동물생명공학회(구 한국동물번식학회) 2019 Journal of Animal Reproduction and Biotechnology Vol.34 No.4

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and ‘EOMES transcription factor mRNAs’ were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

Temporal Regulation of Ovine Interferon-tau Gene by the Transcription Factor Eomesodermin in the Peri-Implantation Period

김민수,임현주,이지환,허태영,손준규 사단법인 한국동물생명공학회 2019 한국동물생명공학회지 Vol.34 No.4

Interferon tau (IFNT) regulation, an anti-luteolytic factor produced by conceptuses of the ruminant ungulates, is essential for the maintenance of early pregnancy, but a definitive mechanism for its temporal transcription has not been elucidated. We and others have observed the T-box protein eomesodermin (EOMES) exhibited high mRNA expression in the ovine embryonic trophectoderm; thus, both caudal-relatedhomeobox-2 (CDX2) and EOMES coexist during the early stages of conceptus development. Objective of this study was to examine the effect of EOMES on ovine IFNT gene transcription when evaluated with CDX2, ETS2 and AP1 transcription factors implicated in the control of cell differentiation in the trophectoderm. In this study, quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis between ovine trophoblast cells was initially performed, finding that transcription factors CDX2 and ‘EOMES transcription factor mRNAs’ were specific to trophectoderm cells. These mRNAs were also found in days 15, 17, and 21 ovine conceptuses. Furthermore, human choriocarcinoma JEG3 cells (trophoblast cell line) were cotransfected with an ovine IFNT (-654bp)-luciferase reporter (-654-oIFNT-Luc) construct and several transcription factor expression plasmids. Cotransfection of the reporter construct with CDX2, ETS2 and AP1 increased transcription of -654-oIFNT-Luc by about 11-fold compared with transfection of the construct alone. When cells were initially transfected with EOMES followed by transfection with CDX2, ETS2 and/or AP1, the expression of -654-oIFNT-Luc was decreased. Also, EOMES factor inhibited the stimulatory activity of CDX2 alone. These results suggest that when conceptuses attach to the uterine epithelium, ovine IFNT gene transcription is down-regulated by an increase of EOMES factor expression in the attached ovine trophoblast cells.

Localization of CDX2, OCT4, SOX2, NANOG, and E-CADHERIN in the Stage of Early Embryo Development

Joo Bin Lee,Tao Lin,Reza K. Oqani,Hyeon Yeong Shin,Jae Eun Lee,Jung won Kang,So Yeon Kim,Dong Il Jin 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Transcription factors play a very important role in the development of embryo. It is different for each growth stage that position and expression level of the transcription factor. Also, it is different pattern in species of mammals. So we thought that if we could well know the expression of the transcription factor in the stage of embryo development, it would be more benefit in embryo culture system. Because the stages of early embryo development are basically from oocyte to blastocyst, we checked transcription factors from oocyte to blastocyst how are expressed in pig. We focused on pluripotency genes, which play an important role in early embryo development, among the transcription factors. So we were conducted to immuno-staining using antibodies of CDX2, OCT4, SOX2, NANOG, and E-CADHERIN. Unfortunately we used mouse`s antibody because pig`s antibody against transcription factors did not exist. We identified five transcription factors (CDX2, OCT4, E-CADHERIN, NANOG, and SOX2) in early development of oocyte embryo development in pig. Our results showed not only similar patterns but also different patterns between pig and mouse. Therefore, we have to investigate that different patterns of transcription factors play a role in pigs, and why come out.

Targeted inactivation of transcription factors by overexpression of their truncated forms in plants

Seo, Pil Joon,Hong, Shin‐,Young,Ryu, Jae Yong,Jeong, Eun‐,Young,Kim, Sang‐,Gyu,Baldwin, Ian T.,Park, Chung‐,Mo Blackwell Publishing Ltd 2012 The Plant journal Vol.72 No.1

<P><B>Summary</B></P><P>Transcription factors are central constituents of gene regulatory networks that control diverse aspects of plant development and environmental adaptability. Therefore they have been explored for decades as primary targets for agricultural biotechnology. A gene of interest can readily be introduced into many crop plants, whereas targeted gene inactivation is practically difficult in many cases. Here, we developed an artificial small interfering peptide (a‐siPEP) approach, which is based on overexpression of specific protein domains, and evaluated its application for the targeted inactivation of transcription factors in the dicot model, Arabidopsis, and monocot model, <I>Brachypodium</I>. We designed potential a‐siPEPs of two representative MADS box transcription factors, SUPPRESSOR OF OVEREXPRESSOR OF CONSTANS 1 (SOC1) and AGAMOUS (AG), and a MYB transcription factor, LATE ELONGATED HYPOCOTYL (LHY). Transgenic plants overproducing the a‐siPEPs displayed phenotypes comparable to those of gene‐deficient mutants. The a‐siPEPs attenuate nuclear import and DNA‐binding of target transcription factors. Our data demonstrate that the a‐siPEP tool is an efficient genetic means of inactivating specific transcription factors in plants.</P>

Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR

Kim, Yea ,Woon,Kim, AeRi Portland Press Ltd. 2017 Bioscience reports Vol.37 No.4

<P>Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here, we applied the CRISPR (clustered, regularly interspaced, short palindromic repeat)/spCas9 (CRISPR-associated protein 9) system to mutate the binding motifs of transcription factors. Binding motifs for erythroid-specific transcription factors were mutated in the locus control region (LCR) hypersensitive sites (HSs) of the human β-globin locus. Guide RNAs targetting binding motifs were cloned into lentiviral CRISPR vector containing the <I>spCas9</I> gene, and transduced into MEL/ch11 cells carrying human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR (qPCR) in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs (PAMs). These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors.</P>

전사 인자의 결합되는 위치의 통계적 모형화에 관한 연구

황성희,손인석 한국자료분석학회 2009 Journal of the Korean Data Analysis Society Vol.11 No.6

To predict over-represented transcription factors from a set of known co-regulated gene promoters will provide insight into the mechanism that underlies the interaction among the co-regulated genes. Several statistical methods have been used to identify of over- represented transcription factors in a set of coexpressed genes. There has been no report on the systematic comparison of these methods in identifying over-represented transcription factors. In this study, we extensively compare fisher-exact test, Chi-square test, hyper- geometric method, and Audic-Claverie method using both two microarray data and two transcription factor regulated data. We also provide the guidelines for finding the most appropriate statistical methods under various situations. Through our comparison study, Audic-Claverie method has the best power. 상호 발현되는 유전자들은 공통적인 조절 기작을 수행할 것으로 보고 이러한 상호 발현되는 유전자들에서 over-represented TFBS를 선택하는 것은 생물학 연구에 중요한 역할을 할 수 있다. 비표적 유전자들에 비해 표적 유전자들에서 통계적으로 유의하게 많이 포함하고 있는 특정 전사 인자들을 선택함으로써 이 전사 인자들로부터 over-represented TFBS를 발견할 수 있다. 상호 발현되는 유전자들에서 over-represented TFBS를 선택하는 몇 가지 통계 검정 방법들이 사용되어오고 있으며, 이러한 방법들을 비교한 연구는 보고된 적이 없다. 본 연구에서는 over-represented TFBS를 선택하기 위해 사용되고 있는 피셔의 정확 검정, 카이제곱 검정, 초기하 방법과 Audic- Claverie 방법에 대해 두 개의 마이크로어레이 자료와 두 개의 전사 인자(transcription factor) 자료를 이용하여 비교하고 또한 다양한 상황에서 가장 적절한 방법을 선택하는 가이드라인을 제시하고자 한다. 우리의 비교 연구에서 Audic-Claverie 방법이 가장 좋은 검정력을 보였다.

Nuclear Import and DNA Binding of the ZHD5 Transcription Factor Is Modulated by a Competitive Peptide Inhibitor in <i>Arabidopsis</i>

Hong, Shin-Young,Kim, Ok-Kyoung,Kim, Sang-Gyu,Yang, Moon-Sik,Park, Chung-Mo American Society for Biochemistry and Molecular Bi 2011 The Journal of biological chemistry Vol.286 No.2

<P>Competitive inhibition of transcription factors by small proteins is an intriguing component of gene regulatory networks in both animals and plants. The small interfering proteins possess limited sequence homologies to specific transcription factors but lack one or more protein motifs required for transcription factor activities. They interfere with the activities of transcription factors, such as DNA binding and transcriptional activation, by forming nonfunctional heterodimers. A potential example is the <I>Arabidopsis</I> MIF1 (mini zinc finger 1) protein consisting of 101 residues. It has a zinc finger domain but lacks other protein motifs normally present in transcription factors. In this work, we show that MIF1 and its functional homologues physically interact with a group of zinc finger homeodomain (ZHD) transcription factors, such as ZHD5, that regulate floral architecture and leaf development. Gel mobility shift assays revealed that MIF1 blocks the DNA binding activity of ZHD5 homodimers by competitively forming MIF1-ZHD5 heterodimers. Accordingly, the transcriptional activation activity of ZHD5 was significantly suppressed by MIF1 coexpressed transiently in <I>Arabidopsis</I> protoplasts. Notably, MIF1 also prevents ZHD5 from nuclear localization. Although ZHD5 was localized exclusively in the nucleus, it was scattered throughout the cytoplasm when MIF1 was coexpressed. Transgenic plants overexpressing the <I>ZHD5</I> gene (35S:<I>ZHD5</I>) exhibited accelerated growth with larger leaves. Consistent with the negative regulation of ZHD5 by MIF1, the 35S:<I>ZHD5</I> phenotypes were diminished by MIF1 coexpression. These observations indicate that MIF1 regulates the ZHD5 activities in a dual step manner: nuclear import and DNA binding.</P>