The CCAAT-box transcription factor NF-Y complex mediates neuronal specification in C. elegans

Neuronal specification, NF-Y complex, IL1, nfya-1, C. elegansNeuronal differentiation is coordinated through a cascade of gene expression via inter-actions between trans-acting transcription factors and cis-regulatory elements of their target genes. However, the mechanisms by which transcriptional regulation determines neuronal cell fate are not fully understood. In C. elegans, the IL1 sensory/inter/motor neurons consist of six neurons that are peptidergic as well as glutamatergic. To identify molecular mechanisms by which IL1s are terminally differentiated, I performed mutagenesis screens and isolated nfya-1 mutants, in which flp-3 neuropeptide gene expression is decreased in IL1. nfya-1 encodes nu-clear transcription factor Y alpha subunit, which is highly conserved from yeast to humans. I found that NFYA-1 is expressed in and localized to the nuclei of IL1, and NFYA-1 expres-sion in the IL1 neurons of nfya-1 mutants restores flp-3 expression. Other IL1-expressed genes, including eat-4 vesicular glutamate transporter gene and unc-8 DEG/ENaC cation channel gene and differentially affected in nfya-1 mutants, suggesting that NFYA-1 regulates the expression of distinct terminal differentiation marker genes in IL1. In addition, I found that mutations in other subunits of NF-Y, NFYB-1, and NFYC-1, and a C. elegans paralog, NFYA-2, affect flp-3 expression in IL1. Then I performed promoter analysis of IL1-expressed genes and identified a motif necessary and sufficient for flp-3 expression that is similar to the mammalian CCAAT-box and directly bound by the NF-Y complex. Taken together, my data indicate that NFYA-1 regulates neuronal subtypes specification via directly regulating a set of terminal-differentiation marker genes.신경은 발달 과정 중 특이적 성질을 지닌 특정 신경 세포로 정확히 분화되어야 하는데, 이 과정이 제대로 이루어 지지 않으면 여러 신경과 연관된 질병을 일으키게 된다. 이와 같이 신경은 신경 세포 내 특정한 유전자들이 발현하여야 그 신경 특이적인 분화가 진행되고 유지가 되는데, 이 과정은 trans-작용 (trans-acting)을 하는 전사인자 (transcription factor)와 cis-작용 (cis-regulatory)을 하는 DNA 부분 (motif)이 상호작용하여 표적 유전자 (target genes 혹은 terminal differentiation genes)들을 발현시킴으로써 이루어진다. 그러나 신경 세포의 세포 운명 (cell-fate)을 결정하는 전사조절 과정에 대해서는 아직 잘 밝혀져 있지 않다. 예쁜 꼬마 선충 (Caenorhabditis elegans)은 302개의 신경으로 이루어진 비교적 간단한 모델 동물로써, 신경의 위치, 이름, 특징 및 신경회로에 관한 정보가 상당히 밝혀지고 명명되어 있어 신경 연구에 적합한 생물이다. 본 연구자는 이 예쁜 꼬마 선충의 여러 신경들 중 IL1 이라는 감각/개재/운동 신경을 이용하여 연구를 수행하였다. 이 IL1 신경은 6개의 개별 신경으로 이루어져 있고 펩티드성 (peptidergic) 및 글루탐산성 (glutamatergic) 신경전달물질을 매개로 신호를 전달한다고 알려져 있다. 본 연구자는 신경 세포의 특이적 분화 및 선별화 과정을 연구하기 위해 해당 신경의 표지 유전자인 flp-3 신경펩타이드 (neuropeptide) 유전자를 이용하여 무작위적인 돌연변이 생성 실험을 진행하였다. 그 결과 nfya-1 유전자가 결핍된 돌연변이를 찾았고, 해당 돌연변이에서 flp-3 표지 유전자의 발현이 정상적인 선충과는 달리 그 발현이 일부 약해짐을 확인하였다. 이 nfya-1 유전자는 Nuclear transcription factor Y (NF-Y) 라는 전사 복합체를 구성하는 a 소단위체를 암호화하고 있으며 해당 유전자는 예쁜 꼬마 선충 뿐만 아니라 초파리, 식물, 마우스 및 인간 유전자에도 보존되어 있는 중요 전사인자다. NF-Y 복합체는 여러 생물에서 생명현상을 유지하기 위해 필요한 전사인자이지만, 신경의 분화에 관한 연구는 많지 않았음을 확인하였다. 본 연구자는nfya-1 유전자가 실제로 IL1 신경의 핵에서 발현함을 확인하였고 돌연변이에 정상적인 nfya-1 유전자를 인위적으로 발현시켰을 때 돌연변이에서의 flp-3 유전자 발현형태가 정상으로 돌아오는 것을 확인했다. IL1 신경은 flp-3 말고도 eat-4 (vesicular glutamate transporter gene)와 unc-8 (DEG/ENaC channel gene) 이라는 유전자도 발현하는데, nfya-1 은 이러한 유전자들의 발현을 서로 다르게 조절함을 확인하였다. 이는 nfya-1 유전자가 IL1의 신경말단유전자 (terminal differentiation gene)를 서로 다르게 조절함을 의미한다. 이에 더해 NF-Y 전사복합체가 a, b, c의 세가지 소단위로 구성되어야 활성화된다는 점에 착안하여 nfyb-1, nfyc-1 유전자와 예쁜 꼬마 선충의 동종 상동 유전자 (paralog)인 nfya-2 유전자의 돌연변이들을 이용하여 flp-3, eat-4, unc-8 표지 유전자들의 발현을 확인하였고, NF-Y 전사복합체가 어떻게 IL1 신경의 특이적 분화를 조절하는지를 확인하였다. 또한 IL1 신경의 표지 유저자인 flp-3, eat-4, unc-8의 프로모터 (promoter)를 분석하여 flp-3 표지 유전자의 프로모터 상에 NF-Y 복합체가 결합한다고 알려진 CCAAT-box를 확인하였다. 이는 NFYA-1이 직접적으로 결합하여 flp-3 유전자 발현을 조절함을 시사한다. 이러한 결과들을 통해 본 연구자는 NF-Y 복합체가 신경말단유전자를 서로 다르게 조절하여 신경 하위 유형 (subtype)의 선별화를 유도할 수 있음을 밝혀냈다.Ⅰ. INTRODUCTION 1 1.1 Neuronal specification 1 1.2 Terminal selector 1 1.3 The nervous system of C. elegans 6 1.4 The NF-Y complex 11 1.5 The IL1 neurons 14 1.6 Summary 14 II. MATERIALS AND METHODS 17 III. RESULTS 32 3.1 Expression of a flp-3 in the IL1 neurons is abolished in nfya-1 mutant animals 32 3.2 Expression of the IL1-specific marker genes is differentially affected in nfya-1 mutant animals 41 3.3 nfya-1 is expressed and functions in the IL1 neurons to regulate flp-3 and eat-4 expression 48 3.4 Post-developmental expression of nfya-1 is not sufficient to induce flp-3 expression in the IL1 neurons 54 3.5 Expression of nfya-1 is partially sufficient to induce flp-3 expression in other cell types 54 3.6 Signaling molecules show different flp-3 expression phenotypes 57 3.7 Identification of cis-regulatory motifs of terminally differentiated IL1 markers 61 3.8 nfya-1 and nfya-2 function redundantly together with nfyb-1 and nfyc-1 to regulate the expression of the IL1 markers 69 3.9 nfya-1 expression is autoregulated in the IL1 neurons 76 IV. DISCUSSIONS 78 4.1 Roles of terminal selector in neuronal specification 78 4.2 Roles of conserved transcription factor NF-Y in many organisms 79 4.3 Previous studies about the NF-Y complex in C. elegans 80 4.4 NFYA-1 regulates neuronal subtypes specification 81 V. REFERENCES 84 VI. SUMMARY IN KOREANS 94DoctordCollectio

The CCAAT-box transcription factor, NF-Y complex, mediates the specification of the IL1 neurons in C. elegans

Neuronal differentiation is highly coordinated through a cascade of gene expression, mediated via interactions between trans-acting transcription factors and cis-regulatory elements of their target genes. However, the mechanisms of transcriptional regulation that determine neuronal cell-fate are not fully understood. Here, we show that the nuclear transcription factor Y (NF-Y) subunit, NFYA-1, is necessary and sufficient to express the flp-3 neuropeptide gene in the IL1 neurons of C. elegans. flp-3 expression is decreased in dorsal and lateral, but not ventral IL1s of nfya-1 mutants. The expression of another terminally differentiated gene, eat-4 vesicular glutamate transporter, is abolished, whereas the unc-8 DEG/ENaC gene and pan-neuronal genes are expressed normally in IL1s of nfya-1 mutants. nfya-1 is expressed in and acts in IL1s to regulate flp-3 and eat-4 expression. Ectopic expression of NFYA-1 drives the expression of flp-3 gene in other cell-types. Promoter analysis of IL1-expressed genes results in the identification of several cis-regulatory motifs which are necessary for IL1 expression, including a putative CCAAT-box located in the flp-3 promoter that NFYA-1 directly interacts with. NFYA-1 and NFYA-2, together with NFYB-1 and NFYC-1, exhibit partly or fully redundant roles in the regulation of flp-3 or unc-8 expression, respectively. Taken together, our data indicate that the NF-Y complex regulates neuronal subtype-specification via regulating a set of terminal-differentiation genes. © Korean Society for Biochemistry and Molecular Biology.TRU

Fas-apoptotic inhibitory molecule 2 localizes to the lysosome and facilitates autophagosome-lysosome fusion through the LC3 interaction region motif-dependent interaction with LC3

Fas-apoptotic inhibitory molecule 2 (FAIM2) is a member of the transmembrane BAX inhibitor motif-containing (TMBIM) family. TMBIM family is comprised of six anti-apoptotic proteins that suppress cell death by regulating endoplasmic reticulum Ca2+ homeostasis. Recent studies have implicated two TMBIM proteins, GRINA and BAX Inhibitor-1, in mediating cytoprotection via autophagy. However, whether FAIM2 plays a role in autophagy has been unknown. Here we show that FAIM2 localizes to the lysosomes at basal state and facilitates autophagy through interaction with microtubule-associated protein 1 light chain 3 proteins in human neuroblastoma SH-SY5Y cells. FAIM2 overexpression increased autophagy flux, while autophagy flux was impaired in shRNA-mediated knockdown (shFAIM2) cells, and the impairment was more evident in the presence of rapamycin. In shFAIM2 cells, autophagosome maturation through fusion with lysosomes was impaired, leading to accumulation of autophagosomes. A functional LC3-interacting region motif within FAIM2 was essential for the interaction with LC3 and rescue of autophagy flux in shFAIM2 cells while LC3-binding property of FAIM2 was dispensable for the anti-apoptotic function in response to Fas receptor-mediated apoptosis. Suppression of autophagosome maturation was also observed in a null mutant of Caenorhabditis elegans lacking xbx-6, the ortholog of FAIM2. Our study suggests that FAIM2 is a novel regulator of autophagy mediating autophagosome maturation through the interaction with LC3. © 2019 Federation of American Societies for Experimental Biology.1