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A Repressor Complex, AP4 Transcription Factor and Geminin, Negatively Regulates Expression of Target Genes in Nonneuronal Cells

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A Repressor Complex, AP4 Transcription Factor and Geminin, Negatively Regulates Expression of Target Genes in Nonneuronal Cells A repressor complex, AP4 transcription factor and geminin, negatively regulates expression of target genes in nonneuronal cells Mi-Young Kim*, Byung Chul Jeong*, Ji Hee Lee*, Hae Jin Kee*, Hyun Kook*, Nack Sung Kim*, Yoon Ha Kim†, Jong-Keun Kim*, Kyu Youn Ahn*, and Kyung Keun Kim*‡ *Research Institute of Medical Sciences and Medical Research Center for Gene Regulation, †Department of Obstetrics and Gynecology, Chonnam National University Medical School, Kwangju 501-190, South Korea Edited by Thomas Maniatis, Harvard University, Cambridge, MA, and approved July 5, 2006 (received for review March 9, 2006) The transcription of neuron-specific genes must be repressed in sequences required to direct temporal brain-specific expression in nonneuronal cells. REST͞NRSF is a transcription factor that restricts vivo. However, no functional NRSE motif was found within 5 kb of the expression of many neuronal genes through interaction with the PAHX-AP1 promoter (Fig. 7, which is published as supporting the neuron-restrictive silencer element at the promoter level. information on the PNAS web site), which indicates that elements PAHX-AP1 is a neuronal gene that is developmentally up-regulated distinct from the NRSE must be present for neuron-specific tran- in the adult mouse brain but that has no functional NRSE motif in scriptional up-regulation of PAHX-AP1 in the adult brain. its 5؅ upstream sequence. Here, we report that the transcription Identification of NRSE-like sequences that repress transcription factor AP4 and the corepressor geminin form a functional complex and binding repressor proteins is important because these se- in which SMRT and histone deacetylase 3 are recruited. The quences may represent common regulatory elements involved in functional complex represses PAHX-AP1 expression in nonneuro- the transcriptional control of other neuronal genes. In this regard, nal cells and participates in regulating the developmental expres- the PAHX-AP1 gene can provide an attractive model for revealing sion of PAHX-AP1 in the brain. This complex also serves as a other NRSE-like sequences. Previously, we identified a putative transcriptional repressor of DYRK1A, a candidate gene for Down’s repressive element (PRE) in the PAHX-AP1 promoter sequence syndrome. Furthermore, compared with that in normal fetal brain, that is suspected to be required for directing brain-specific expres- the expression of AP4 and geminin is reduced in Down’s syndrome sion (10), and we hypothesized that PRE-binding proteins may fetal brain at 20 weeks of gestation age, at which time premature participate in the negative regulation of PAHX-AP1 expression in overexpression of dual-specificity tyrosine-phosphorylated and brain at the promoter level. regulated kinase 1A (DYRK1A) is observed. Our findings indicate In the present study, we screened for PRE-binding proteins to that AP4 and geminin act as a previously undescribed repressor elucidate the responsible molecular mechanisms and potential ͞ complex distinct from REST NRSF to negatively regulate the ex- implications of this PRE-binding transcription factor in the regu- pression of target genes in nonneuronal cells and suggest that the latory control of brain-specific gene expression. Here, we report AP4–geminin complex may contribute to suppressing the preco- that activator protein 4 (AP4) is a PRE-binding transcription factor cious expression of target genes in fetal brain. and that AP4 and the corepressor geminin (Gem) form a previously uncharacterized functional complex to repress target gene expres- he expression of neuronal genes in neural tissues is regulated by sion in nonneuronal cells. The AP4–Gem complex may provide a Tactivator and repressor systems that provide the proper tran- second mechanism for silencing neuronal genes in nonneuronal scriptional pattern (1, 2). One of these systems, interaction of the cells that operates in parallel to the previously reported REST͞ neuron-restrictive silencer element (NRSE) with repressor element neuron-restrictive silencer factor (NRSF). 1-silencing transcription factor (REST, also known as neuron- restrictive silencer factor or NRSF), mediates the repression of Results several neuronal genes in nonneuronal cells, such as type II sodium AP4 Is a Transcriptional Repressor of PAHX-AP1, and Gem Interacts channel, SCG10, and synapsin I. REST acts on promoters that carry with AP4 to Form a Complex. To search for PRE-binding proteins, the NRSE sequence to repress transcription, which is thought to be we used the yeast one-hybrid assay and obtained several positive a general mechanism for the control of neuron-specific gene clones, two of which simultaneously expressed the transcription expression (3–5). However, the transcriptional regulatory mecha- factor AP4 and Gem; the others contained AP4 only. AP4 is a nisms required to direct the temporal expression of brain-specific ubiquitously expressed transcription factor that binds to the DNA genes are not fully understood. consensus sequence 5Ј-CAGCTG-3Ј and a helix–loop–helix protein Phytanoyl-CoA ␣-hydroxylase-associated protein 1 (PAHX- that contains two distinctive leucine-repeat elements, LR1 and LR2 AP1) was isolated as a novel neuron-specific protein that interacts (11). Gem is a bifunctional protein that acts in embryonic neural- with Refsum disease gene product (PAHX) (6) and the cytoplasmic ization (12) and in replication licensing in the cell cycle (13). We region of brain-specific angiogenesis inhibitor 1, a seven-span found that in vitro translated AP4-V5 (Fig. 1A Left) or native AP4 transmembrane protein (7). Refsum disease is an autosomal reces- sive disorder of lipid metabolism; retinitis pigmentosa and periph- eral neuropathy are major clinical findings (8). PAHX-AP1 is Conflict of interest statement: No conflicts declared. involved in the developmental regulation of the photoreceptor’s This paper was submitted directly (Track II) to the PNAS office. function (9) and is weakly expressed in most embryonic tissues, but its expression pattern changes dramatically after birth when it is Freely available online through the PNAS open access option. specifically expressed in the brain in a developmentally up- Abbreviations: AP4, activator protein 4; DS, Down’s syndrome; DYRK1A, dual-specificity tyrosine-phosphorylated and regulated kinase 1A; Gem, geminin; HDAC3, histone deacetyl- regulated pattern (6). Studies in transgenic (TG) mice showed that ase 3; NRSE, neuron-restrictive silencer element; PAHX-AP1, phytanoyl-CoA ␣-hydroxylase- the 5-kb region of 5Ј PAHX-AP1 gene is sufficient to direct the associated protein 1; PRE, putative repressive element; REST, repressor element 1-silencing developmental expression of a reporter gene in the brain only, transcription factor; TG, transgenic; TSA, trichostatin A. especially neuronal cells, in a pattern similar to that of endogenous ‡To whom correspondence should be addressed. E-mail: [email protected]. PAHX-AP1 (10), which indicates that the 5-kb region contains the © 2006 by The National Academy of Sciences of the USA 13074–13079 ͉ PNAS ͉ August 29, 2006 ͉ vol. 103 ͉ no. 35 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0601915103 Downloaded by guest on September 29, 2021 Fig. 1. AP4 is a transcriptional repressor of the PAHX-AP1 promoter, and the C-terminal domain of Gem binds to the leucine-repeat elements (LR1 and LR2) on AP4. (A) AP4 binds to the PAHX-AP1 PRE, but Gem does not. (Left) Labeled PAHX- AP1 PRE oligonucleotide was incubated with in vitro- translated vector (lane 2), in vitro-translated AP4-V5 (lanes 3–7), or Gem (lane 8). Lane 1 is probe only. AP4-V5 was incubated with probe alone (lane 3), excess cold probe (lanes 4 and 5), anti-V5 antibody (lane 6), or IgG (lane 7). The shifted band due to AP4 binding with PRE (thin arrow) and the supershifted band (thick arrow) are indicated. The dashed arrow indicates nonspecific bands. (Right) Labeled WT (lanes 1 and 2) or mutant PAHX-AP1 PRE oligonucleotide (lanes 3 and 4) was incubated with AP4-V5 (lanes 2 and 4). (B) Interaction of AP4 with Gem. HEK293T cells were immunoprecipitated with anti-AP4 antibody and then blotted with anti-Gem an- tibody (Upper) or reciprocally immunoprecipitated and probed (Lower). Input is HEK293T cells. (C) ChIP assay showing that AP4 and Gem bind to the AP4-binding site on the murine PAHX-AP1 promoter. (D) Increasing doses of AP4 were tran- siently cotransfected with WT PAHX-AP1 promoter reporter (open bars) or AP4-binding site-mutated PAHX-AP1 promoter reporter (filled bars) in HEK293T cells. After 48 h, cell extracts were prepared and assayed for luciferase activity. Note that the basal value of mutated PAHX-AP1 promoter reporter was larger than that of WT PAHX-AP1 promoter reporter. Values are mean Ϯ SEM (n ϭ 3). (E) Various deletion constructs of AP4 were pulled down by whole Gem (Upper) or the C-terminal domain of Gem (Lower) and analyzed by immunoblotting. Input is in vitro-translated Gem. (F) HEK293T cells were tran- siently transfected with various AP4 deletion constructs, including bHLH LR-V5, LR PQ-V5, PQ V5, and AP4 full-V5. V5 immunoprecipitation (IP) was performed, CELL BIOLOGY and Gem was analyzed by immunoblotting. Input is HEK293T cells. in nuclear extract (Fig. 8, which is published as supporting infor- loop–helix LR-V5 (Fig. 2A). This result indicates that the LR- mation on the PNAS web site) binds specifically to WT PAHX-AP1 containing Gem-binding domain of AP4 is important for the PRE but not to mutant PRE oligonucleotide (Fig. 1A Right). transcriptional repressor activity of AP4 on the PAHX-AP1 pro- Unexpectedly, however, Gem did not bind to the PRE (Fig. 1A and moter. In HEK293T cells, cotransfection with increasing doses of 8) but interacted with AP4 (Fig. 1B). Binding of endogenous AP4 Gem with a PAHX-AP1 promoter reporter did not substantially to the AP4-binding site on PAHX-AP1 promoter sequence was affect luciferase activity, whereas addition of AP4 (30 ng) dose- confirmed by ChIP (Fig.
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