- Research article
- Open Access
Developmental expression profile of the yy2 gene in mice
© Drews et al; licensee BioMed Central Ltd. 2009
- Received: 18 March 2009
- Accepted: 28 July 2009
- Published: 28 July 2009
The transcription factor Yin Yang 2 (YY2) shares a structural and functional highly homologue DNA-binding domain with the ubiquitously expressed YY1 protein, which has been implicated in regulating fundamental biological processes. However, the biological relevance of YY2 has not been identified yet.
Towards the understanding of YY2 biology, we analyzed in detail the expression pattern of yy2 in various organs during embryonic and postnatal mouse development till adulthood. Thereby, a constant yy2 level was detected in heart and lung tissue, whereas in different brain regions yy2 expression was dynamically regulated. Interestingly, in any analyzed tissue neither the homologue yy1 nor the mbtps2 gene showed changes in mRNA expression levels like yy2, although the intronless yy2 gene is located within the mbtps2 locus.
Furthermore, we detected yy1, yy2, and mbtps2 mRNA in primary mouse neurons, microglia cells, and astrocytes. In comparison to yy2 and mbtps2, yy1 revealed the highest expression level in all cell types. Again, only yy2 showed significantly altered gene expression levels among the cell types. Higher yy2 expression levels were detected in microglia cells and astrocytes than in primary neurons.
Yy2 expression in the heart and lung is constitutively expressed during embryogenesis and in adult mice. For the first time, developmental changes of yy2 transcription became obvious in various areas of the brain. This suggests that yy2 is involved in developmental gene regulation.
- Primary Neuron
- Fundamental Biological Process
- Primary Mouse Brain
- Mouse Cortical Primary Culture
- Postnatal Mouse Development
The biology of the transcription factor Yin Yang 2 (YY2) is not well characterized yet, but may be of particular interest due to its similarities to the ubiquitously expressed YY1, which has been implicated in fundamental biological processes such as DNA-replication, cell cycle regulation and organogenesis [1–5]. YY2 is a zinc finger protein that shares 56% sequence homology with YY1 . Especially, the C-terminal DNA-binding zinc finger is highly conserved [6–8] and mediates a similar binding specificity to the 5'-(A/c/g)(A/t)NATG(G/a/t)(C/a)(G/c/t)-3' DNA consensus motif [8, 9], suggesting synergistic or competitive functions, if both factors act within the same cell . The biological significance of yy1 is highlighted by early embryonic lethality of mice with homozygous yy1 deletion. In addition, mice with heterozygous yy1 ablation show growth retardation and defects in neurulation .
Analysis of yy2 ablation has not been reported, yet. The intron lacking yy2 gene is extraordinarily positioned between exons 5 and 6 of another X-chromosomally located gene encoding mbtps2 (membrane bound transcription factor protease site 2). First expression analyses by in situ hybridization in testis, ovaries and brain from adult mice suggested a shared control of yy2 and mbtps2 gene activities . However, our data indicate that the upstream region of the human YY2 gene mediates significant promoter activity independently from MBTPS2 .
To gain more information on the biology of yy2, we established whole mount in situ hybridization. In parallel, real-time PCR was used to quantify yy2 mRNA expression in various organs of developing and adult mice more precisely. Herein, we demonstrate that the yy2 mRNA expression pattern differs from that of yy1 and mbtsp2. Importantly, yy2 expression underlies significant changes during development, particularly in various areas of the brain.
Expression of yy2in mouse embryos
Quantitative analysis of yy2expression in heart and lung
Quantitative analysis of yy2expression in the brain
Quantitative analysis of yy2expression in neurons, astrocytes and microglia
Herein, for the first time we provide data implying that the transcription factor yy2 possess a functional role during development, as it has already been shown for its famous and well characterized homologue yy1 [3, 12, 13]. Since, yy2 is broadly, possibly ubiquitously expressed in embryonic mice (Figure 1), we conducted extensive real-time PCR analyses indicating a tissue-specific expression pattern that is in part developmentally regulated. Thereby, our experiments were especially focused on the cardiopulmonary and central nervous system.
By analyzing different primary cell types isolated from the murine brain, we found differences of yy2 expression as well, whereas yy1 levels showed no significant changes. In contrast to microglia cells and astrocytes, neurons express almost no yy2. The neuronal data suggest that yy2 could be required for proliferation, since neurons, in contrast to astrocytes and microglia cells, cannot divide anymore. Ongoing experiments may verify this hypothesis.
Comparing all real-time PCR results it becomes obvious that the expression of yy2 and mbtps2, respectively, is individually regulated depending upon the analyzed organ, tissue or cell type. While both genes seem to be co-regulated via the same promoter in heart and lung as well as in microglia and astrocytes, however, the data from primary neurons and all three brain tissues indicate an alternative transcriptional activity. Currently, two different possibilities of yy2 and mbtps2 gene regulation are discussed: (a) both genes are under the control of the mbtps2 promoter, and (b) yy2 transcription is mediated by its own adjacent promoter, located upstream of its coding sequence, a model that we favour according to own data on the regulation of the human YY2 gene [7, 11]. Of note, only the yy2 promoter shows a significant sensitivity against DNA-(de)methylation, strengthening the implication of yy2 in developmental processes. Our observations presented in this manuscript emphasize that in fact both promoter regions seem to be utilized in a tissue- or cell type-specific manner.
The results from the real-time PCR experiments confirm that yy2 is differentially expressed in the lung, heart and brain, indicating a stringent spatial regulation and function. Precisely, we show a significant change of yy2 expression during the development and maturation of the neocortex and cerebellum. Within the central nervous system, yy2 is predominantly expressed in astrocytes and microglia cells.
C57/Bl6 mice were mated, and embryos/pups were collected at defined time points (morning of vaginal plug was considered as embryonic day E0.5). The protocol for animal use was approved by the Institutional Review Board (T 0167/08).
Preparation of primary neurons
Serum-free preparation of mouse cortical primary cultures was performed with E18 (+/- 0.5 days) mouse embryos as previously described . After removal of meninges, entire cortices were mechanically dissociated in HBSS buffer (w/o Ca2+ and Mg2+), with trypsin (0.25%) for 10 min at 37°C. Trypsinization was stopped by adding minimum essential medium (Gibco-Invitrogen; Karlsruhe, Germany). Medium was supplemented with 0.6% glucose, 10% horse serum (Gibco), and penicillin and streptomycin. Further mechanical dissociation was performed by adding of DNAseI (0.06%). Cells were cultured on poly-L-lysine-coated dishes at a density of 75.000 cells/cm2. After three hours in vitro, medium was changed to Neurobasal A medium (Gibco) supplemented with 2% B27 (Gibco), 0.5 mM glutamine, and penicillin and streptomycin for 12 days. Purity of neuronal cell preparation was tested by real-time PCR [see additional file 3].
Preparation and purification of astrocytes and microglial cells
For astrocyte and microglia preparation, cerebra from mouse pups aged P1 to P3 were prepared and the meninges, including the pia mater and arachnoid, were carefully removed and the cerebrum was washed in DMEM (4.5 g/l glucose, 200 mM glutamine, pyrovate) containing 10% fetal calf serum and 100 U/ml penicillin/streptomycin. Following careful homogenization with a fire-polished Pasteur pipette, a 10 min trypsin/DNAse (1.25%/2 U) incubation followed, and digestion was stopped by adding the same volume culture medium (DMEM containing 4.5 g/l glucose, 200 mM glutamine, pyrovate, 10% fetal calf serum, and 100 U/ml penicillin/streptomycin). Dissociated astrocytes were plated on poly-L-lysine (10 μg/ml)-coated dishes. Every third day, dishes were shaken for 10 min at 37°C and afterwards rinsed three times with warm PBS to remove microglial cells. They were then resuspended with a fire-polished Pasteur pipette, and replated in culture medium at low density. The suspended microglial cells were collected and replated in astroglial culture medium containing 2% fetal calf serum. Purity of both cell types was tested by real-time PCR [see additional file 3].
Whole-mount in situ expression analysis of yy2mRNA
For in situ hybridization, a yy2 probe was generated by cloning a yy2-specific 200 bp fragment (nt 11 to 200) derived from the murine coding sequence [GenBank: EF688658] into pBluescript SK (-) plasmid (Fermentas; St. Leon-Rot, Germany). For digoxygenin (DIG)-labeling, 12 μl of linearized plasmid (EcoRI: anti-sense or BamHI: sense) were combined with 2 μl 10-fold buffer (Roche; Mannheim, Germany), 1 μl RNAsin, 2 μl T3 (for anti-sense synthesis) or T7 (for sense synthesis) RNA polymerase and 2 μl labeling mix containing 7.1 μl ATP, 7.1 μl CTP, 7.1 μl GTP, 4.6 μl UTP, 20.5 μl RNase free water, and 25 μl DIG-11-UTP (Roche; Cat.-No.: 1209256) in order to conduct in vitro transcription for 2 h (37°C). Afterwards, the freshly prepared probe was diluted by adding 70 μl RNase free water and 10 μl 0.1 M DTT. The embryos were dissected in ice-cold PBS, followed by overnight fixation in 4% PFA, 3 washes in PBS with 1% Tween 20 (PBT) for 5 minutes, and dehydration in rising methanol concentrations. Next, specimens were rehydrated with decreasing methanol concentrations. For increasing permeability and signal detection, embryos were incubated in 6% H2O2 for 1 h and subsequently treated with proteinase K for 15 min with 3 washing steps (5 min each) in PBT. Digestion was stopped in 2 mg/ml glycine in PBT for 20 min and 2 washing steps in PBT followed by post fixation in 4% PFA (in PBT) for 20 min at room temperature. PFA was removed by 3 washing steps in PBT. After incubation in hybridization solution containing 50% deionized formamide, 5× SSC [pH 4.5], 1% SDS, 50 μg/ml tRNA and 50 μg/ml heparin at 65°C for 1 h, 1–2 μl denaturized DIG-labeled probes (2 min at 90°C) were added and hybridized at 65°C overnight. Finally, embryos were washed 3 times for 1 h in solution I (50% formamide, 5 × SSC [pH 4.5], 1% SDS) at 65°C followed by 3 washing steps for 1 hour in solution II (50% formamide, 2 × SSC [pH 4.5], 1% Tween 20) at 60°C and washing 3 times in TBST for 5 min each. Before the embryos were treated with anti-DIG antibody (1:2000) in 1% sheep serum/TBST overnight at 4°C, we conducted a blocking step in 10% heat-inactivated sheep serum/TBST with 0.1% blocking reagent (Roche; Cat.-No.: 11112589001) for 1 h at room temperature. After performing post-antibody washes (3 times 10 min and 5 times 1 h in TBST), samples were hybridized in fresh prepared NTMT [pH 9.5] solution containing 100 mM NaCl, 100 mM Tris-Cl (pH 9.5), 50 mM MgCl2 and 0.1% Tween 20. For signal detection, NTMT solution was removed and reaction mix (NTMT with 125 μg/ml BCIP and 250 μg/ml NBT) was added. To stop the staining process, samples were washed 3 times in PBT, followed by post-fixation in 4% PFA. The embryos were analyzed with a Leica MZ 9.5 stereo microscope (Wetzlar, Germany).
Preparation of cDNA
Mouse organs were immediately snap-frozen following their collection from 3 sets of 6 animals of each age. Primary mouse brain cells from 3 independent preparations of 3 pregnant animals or mouse organs were homogenized in TRIzol reagent (Invitrogen), and the total RNA was purified according the TRIzol protocol. RNA concentrations were determined by using a UV-Visible Spectrophotometer (Biomate 3 spectrometer, Fisher Scientific, Waltham, MA, USA). A High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA) was used to generate total cDNA for the real-time PCR from 5 μg total RNA from each sample as per the manufacturer's recommendations.
Real-time PCR (TaqMan)
TaqMan cDNA samples were prepared as described. 5'-FAM-labeled probes and appropriate primer pairs for detection of murine yy2 (probe: 6FAM-cagcctgttcttcagctatgggatcttctt-BBQ; primer: 5'-gggtgacaaacagtgggagc-3' (forward) and 5'-ggatcagaaagatcaatgccaggt-3' (reverse)), yy1 (probe: 6FAM-agggtctgagaggtcaatgccaggt-BBQ; primer: 5'-atgaaacagtggttgaagagcagatc-3' (forward) and 5'-caagctattgttcttggagcatcatc-3'(reverse)) and mbtps2 (probe: 6FAM-tgtcccgttactaatgtgcaagattggaa-BBQ; primer: 5'-ggagaccttgtcactcatctacagga-3' (forward) and 5'-gtcgtttgtatgctctaactgggaag-3' (reverse)) were designed and synthesized by TIB MOLBIOL (Berlin, Germany). β-actin (TaqMan Gene Expression Assays; Cat.-No.: ACTB 4352664-0602004) was purchased from Applied Biosystems. For all PCR reactions, 1 μl of cDNA was added to 10 μl TaqMan 2× Universal PCR Mastermix Mix (Applied Biosystems), 1 μl of each primer [20 μM], 1 μl 5'-FAM-labeled probe [0.3 μM] and 8 μl deionized H2O. All reactions were performed in duplicates and all three transcripts (within each set of specimens) were always analyzed within the same experiment. Amplification and fluorescence detection was conducted with the i-Cycler Multicolor Real-Time PCR Detection System (Bio-Rad; Munich, Germany). The fluorescence threshold value was calculated by using the iCycle iQ Optical System Software, version 3.1. yy2, yy1 and mbtps2 values were normalized against β-actin.
Statistical significances were determined by using the one-way analysis of variance (ANOVA). Bonferroni's multiple comparison procedure was used to discriminate, which means were different from others. A p-value < 0.05 was considered to be significant.
DD was supported by a fellowship from the Sonnenfeld-Stiftung, Berlin. Funding was provided by the Verein für Frühgeborene Kinder am Virchow-Klinikum e. V., Berlin. DD has cooperated with Prof. Constance Scharff (Department of Biology, Chemistry and Pharmacy, Freie Universität Berlin).
The authors thank Bettina Brokowski, Rike Dannenberg and Justus Goyn for preparation of cDNA samples as well as Angelika Zwirner and Jan Csupor for stimulating discussions. DD acknowledges Sandy von Salisch for guidance in TaqMan real-time PCR and Dr. Malte Cremer for supporting statistical analysis. We also thank all members of our laboratories for their support and critical discussion of the data.
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