RE1 silencing transcription factor (REST) negatively regulates ALL1-fused from chromosome 1q (AF1q) gene transcription
© Hu et al. 2015
Received: 2 March 2015
Accepted: 27 August 2015
Published: 5 September 2015
ALL1-fused from chromosome 1q (AF1q), originally considered as an oncogenic factor, has been implicated in neuronal development; however, its upstream regulatory mechanisms in neural system remained elusive.
Our study showed that REST (RE1 silencing transcription factor), a key transcription factor in neurodevelopment, could down-regulate the gene expression of AF1q. The promoter assay identified a neuron-restrictive silencer element at −383 to −363 bp of human AF1q promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (CHIP) confirmed the binding of REST to the NRSE in AF1q gene promoter. Additionally, the negative correlation between the expression levels of Af1q and Rest in mice neurodevelopment supported the negative regulation of AF1q by REST and the potential functions of AF1q in neurodevelopment.
These results demonstrate that REST regulates AF1q gene transcription through directly binding to a NRSE at −383 to −363 bp of AF1q promoter.
The AF1q gene was initially identified as a mixed lineage leukaemia (MLL) fusion partner from an infant acute myelomonocytic leukemia patient carrying the t(1;11)(q21;q23) translocation , which encoded a highly conserved 90 amino acid residues protein containing a MLLT11 motif and a nuclear export signal  with no similarity to other known proteins . An increase in AF1q mRNA levels had been shown in leukemic and immature hematopoietic cells . MiR-29b can regulate AF1Q gene expression and lower expression of miR-29b was associated with poor overall survival of acute myeloid leukemia . AF1q had been regarded as an oncogenic factor involved in thyroid oncocytic tumors, breast cancer and testicular germ cell tumors [4–8], though its function had not been well characterized. A recent study showed that AF1Q interacted with T cell factor 7 of Wnt signaling pathway to regulate CD44 and promoted breast cancer metastasis . In addition to the proposed oncogenic role, AF1q had been reported to play an important role in the development of neurons in the peripheral and central nervous systems . The mouse Af1q, homologue of human AF1q, was found to be significantly up-regulated during the neuronal production from neural stem/progenitor cells . AF1q was differentially expressed during neuronal differentiation , but the underlying regulatory mechanisms in neurodevelopment were unknown.
REST (RE1 silencing transcription factor) regulates embryonic and neural stem cells by affecting their derivatives [12–14] and participating in the self-renewal of neural stem cells  via regulating the transcription of target genes by binding to a 21-bp DNA element, which is called RE1-binding site/neuron-restrictive silencer element (RE1/NRSE) during neurodevelopment . It was found that the expression of REST was decreased in cultured neurospheres derived from fetal Down syndrome (DS) brain  and in the brains of DS mouse models . REST was decreased in Alzheimer’s disease [10, 18]. REST, is expressed throughout early development  and acts as transcriptional silencer or activator, which is essential for the regulation of target genes during neuronal development [20, 21]. Downregulation of REST during neurogenesis is necessary for proper neuronal differentiation, while overexpression of REST in differentiating neurons interferes in neuronal gene expression and causes axon guidance errors . REST modulated the expression of genes that were critical in normal neuronal functions including neurotransmitter receptors, synaptic proteins, and ion channels proteins [15, 16]. Recent studies have demonstrated that in contrast to the role of REST in the repression of Rest target genes in in vitro cultured neuronal cells, as well as in non-neuronal cells outside of the brain, the CoRest binding site of Rest is dispensable for embryonic neurogenesis in vivo .
In the present study, we demonstrated that AF1q transcription could be down-regulated by REST. We also identified AF1q promoter sequence and demonstrated that REST was a key transcriptional factor participating in AF1q down-regulation via a NRSE site at −383 to −363 bp of human AF1q promoter. Furthermore, our study suggested that the expression of REST and AF1q were negatively correlated during neurodevelopment, implying that AF1Q may be involved in neurodevelopment.
REST inhibited human AF1q gene transcription
To further clarify the molecular mechanism of AF1q gene transcription, we cloned 1811-bp fragment located in the 5′-flanking region of the human AF1q gene (Fig. 1d) into promoterless vector pGL3Basic. TSS represents the first base on exon 1 from Ensemble transcript ENST00000368921, which is 2802-bp upstream of translation start codon. Thus, the expression of luciferase activity in cells transfected with this plasmid depended on the insertion and proper orientation of a functional promoter upstream of the firefly luciferase gene. Plasmid pAF1q promoter (−1349 to +462 bp) was transfected into HEK293 cells, and luciferase activity was measured by a luminometer to reflect AF1q promoter activity. Plasmid pAF1q promoter transfected cells had significantly higher luciferase activity compared with controls (0.9445 ± 0.2401 compared with 0.08008 ± 0.006239 relative luciferase units, p < 0.05; Fig. 1e, f), indicating that the 1.8-kb fragment contains the functional promoter region of the human AF1q gene.
To investigate whether AF1q gene transcription could be regulated by REST, we cotransfected HEK293 cells with REST expression plasmid and pAF1q promoter, and examined the promoter activity of human AF1q gene. A series of different mass ratio of REST and AF1q promoter were selected and screened for modulatory effects on dual-luciferase assay. The transfected mass ratio of REST to AF1q promoter was 1:3, 1:1, 3:1 and 5:1, respectively. The REST protein expression was determined by Western blotting (Fig. 1g). Dual-luciferase assay showed that REST overexpression decreased AF1q promoter activity and the effects depended on the mass ratio of REST to AF1q promoter. We could see that the AF1Q promoter activity was reduce by 19.34 ± 8.499, 43.59 ± 6.122, 53.24 ± 6.251, and 44.58 ± 7.531 % with increased expression of REST (p < 0.05; Fig. 1h). These results indicated that the transcription of human AF1q gene could be down regulated by REST.
Identification of the functional NRSE site in AF1q gene promoter
To confirm that the NRSE-AF1q site verified in vitro can actually bind to REST in vivo, ChIP was employed to pulldown the REST bound DNA. The anti-REST antibody actually immunoprecipitated REST protein as showed in Fig. 3c. Anti-RNA Polymerase II IP was used as positive control and IgG IP was used as negative control for ChIP. Purified DNA was then analyzed by PCR using Control Primers specific for the GAPDH promoter (Fig. 3d). PCR product was observed in the anti-RNA Polymerase II ChIP (lane 2, Fig. 3d), but not in the IgG ChIP (lane 3, Fig. 3d). ChIP-PCR results showed that REST antibody effectively immunoprecipitated the AF1q-NRSE site (lane 2, Fig. 3e). And the ChIP band was greatly reduced in cells with REST knocked down (lane 5, Fig. 3e). AF1q-NRSE PCR product was also observed in the anti-RNA Polymerase II ChIP (lane 6, Fig. 3e) and not in the IgG ChIP (lane 3, Fig. 3e). AF1q promoter specific DNA was also observed in the Input (lane 1, Fig. 3e). Taken together, these data indicated that the NRSE site, corresponding to AF1q promoter −383 to −363 bp, was responsible for the down-regulation of AF1q gene by REST.
Af1q expression was negatively correlated with Rest in mice neurodevelopment
Our study here showed that REST, a key transcription factor in neurodevelopment, can down-regulate the gene expression of AF1q through directly binding to a NRSE at −383 to −363 bp of AF1q promoter. AF1q, originally considered as an oncogenic factor, is highly expressed in normal hematopoietic tissues, leukemic cell lines and neuronal cells in central nervous system [1, 10, 11, 23]. We can see that the promoter activity in pAF1q promoter is higher than the promoter activity in pAF1qAluc, indicating the region of −1349 to −449 bp containing binding sites for some activators. The decrease of inhibitory effect by REST on pAF1QAluc suggested that the region of −1349 to −449 bp contained binding sites for REST cofactors.
Our study here elucidated the regulatory mechanism of AF1q by REST. REST, acting as transcriptional silencer or activator, was essential for the regulation of target genes during neuronal development [20, 21]. REST is required to repress the expression of neuronal genes in undifferentiated neuronal tissue. Expression of REST was highest in embryonic stem cells, but it was decreased while ESCs were differentiated into neuronal stem cells, and it was at low level in mature adult neuronal cells . In addition to participate in neurogenesis, Rest also mediated the interactions between neuron and glia, which was associated with synaptic and neuronal network plasticity and homeostasis in the brain [24, 25]. All these indicated that REST is a key transcription factor in neurogenesis. As a target gene of REST, how AF1q functions in neurogenesis remains elusive. It will be interesting to examine the function of AF1q in neural stem cells and neuronal differentiation. It was reported that REST expression is a protective factor in aging and is decreased in neurodegenerative diseases such as Alzheimer’s disease . It will be interesting to check the expression level of AF1q in some neurodegenerative diseases such as Alzheimer’s disease.
Overexpression of REST has been found in human medulloblastomas, glioblastoma and neuroblastomas , in which REST acted as an oncogene to maintain the stem character of neural cells . REST can also act as a tumor suppressor in carcinomas including lung, breast and colon . Though AF1q is regard as an oncogene, the expression level of AF1q is unknown in these cancers. It will be interesting to examine the expression of AF1Q in these cancers associated with REST dysfunction.
In summary, the current study provides a molecular model for REST in negative regulation of AF1q promoter activity and mRNA expression. These results will help to better understand the role of AF1q gene in in neural stem cells and neuronal differentiation.
The 5′-upstream region of human AF1q gene (−1349 to +462 bp, pAF1q-promoter) was obtained by PCR of genomic DNA isolated from BAC-human-rp11 using a pair of primers (5′-CGGCTAGCAGGTCTCCACCCTGTCCCTGC-3′ and 5′-CCCTCGAGTTCCCTCCACCCAGCTCTGGTC-3′). The first base of the first exon is referred as bp +1. Then they were cloned into pGL3basic vector (Promega, Madison, WI) containing firefly luciferase reporter gene. Primers used to generate a series of promoter deletion plasmids were as follows: forward primers, 5′-CGGCTAGCGTCAGGAGTTCCAGACCAGC-3′ (pAF1qAluc), 5′-CGGCTAGCTGTAATCCCAGCTACTTGGG-3′ (pAF1qBluc), and reverse primers: 5′-CCCTCGAGCAGAAATGGCCTTGTTCTCT-3′. The pAF1qNRSEmut was constructed from pAF1QAluc in which the NRSE site 5′-TTAGCTGGGCGTGGTGGCGGA-3′ was mutated to 5′-TTAGCTGGaaGTctgGaaGGA-3′. All the constructs were verified by sequencing and restriction enzyme digestions. Human REST siRNA was generated using pSuper vector. The target sequence for human REST siRNA is GCTACAATACTAATCGATA. The two strands of RESTsuper sense (5′-gatccccGCTACAATACTAATCGATAttcaagagaTATCGATTAGTATTGTAGCttttta) and RESTsuper antisense (5′-agcttaaaaaGCTACAATACTAATCGATAtctcttgaaTATCGATTAGTATTGTAGCggg) were annealed and inserted into pSuper plasmid to generate the pSuper-REST construct. The vectors pREST were generated as previously described .
Cell culture, transfection and dual-luciferase assay
HEK293 cells were cultured as previously described . All cells were maintained at 37 °C in an incubator containing 5 % CO2. All transfections were carried out with lipofectamine™2000 transfection reagent (Invitrogen) according to the manufacturer’s instructions. Luciferase activity was determined as previously described .
Total RNA was isolated from mouse brains or cells using TRIzol reagent (Invitrogen). The mRNA of REST and AF1q was quantified by TOYOBO R SYBR Green gene Expression Analysis kit (TOYOBO, Japan). Primers for real-time quantitative and semi-quantitative PCR were as follows: human AF1q (270 bp), forward, 5′-CCGCTCGAGGCCACCATGAGGGACCCTGTGAG-3′, and reverse, 5′-GGGGTACCGAGCAAGTCCAGTTCGAAG-3′; human REST (137 bp), forward, 5′-ACTCATCACGGAGAACGCCC-3′, and reverse, 5′-GAGGCCACATAACTGCACTG-3′; mouse Rest (244 bp), forward, 5′-CGAGTCTCAGGAAATTGATGA-3′, and reverse, 5′-GCCGTTACCCACTCACTAATAC-3′; human and mouse β-actin (141 bp), forward, 5′-GACAGGATGCAGAAGGAGAT-3′, and reverse, 5′-TGATCCACATCTGCTGGAAGGT-3′. All animal protocols were approved by Shandong University Institutional Animal Care and Use Committee and by the Institutional Ethics Committee on Animal Research of Qilu Hospital.
EMSA and ChIP
The sense sequences of AF1qNRSE, consensus NRSE and mutant AF1qNRSE were 5′-AAAGATTAGCTGGGCGTGGTGGCGGATGCCTGTA, 5′-TTCAGCACCACGGACAGCGCC, 5′-AAAGATTAGCTGGAAGTCTGGAAGGATGCCTGTA. EMSA and ChIP were performed as previously described . Chip-PCR was performed using the DNA reversed from the cross-linked complex with a pair of primers (NRSE-AF1q:5′-GCCTCCGGTTGTACCACT-3′ and 5′-AGCGATTCTCCTGCCTCA-3′). Control primer specific for human GAPDH were 5′-TACTAGCGGTTTTACGGGCG-3′ and 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′.
Western blot analysis
Western blot was performed as previously described . Anti-REST antibody was from Millipore (#DAM15).
All experiments were repeated three to five times. In figures one representative picture was shown; quantifications were from three or five independent experiments. The values represent the mean ± SEM. The data were evaluated for statistical significance with Student’s t test analysis.
YH participated in drafting of the manuscript and carried out the promoter assays, EMSA and ChIP assays. QS, CZ and QS helped YH in promoter construct cloning and bioinformatic analysis. XS designed the experiment and wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by National Natural Science Foundation of China Grant 81171030.
Compliance with ethical guidelines
Competing interests The authors declares that they have no competing interests.
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