Analysis of promyelocytic leukemia in human embryonic carcinoma stem cells during retinoic acid-induced neural differentiation

Background: Promyelocytic leukemia protein [PML] is a tumor suppressor protein that is involved in myeloid cell differentiation in response to retinoic acid [RA]. In addition, RA acts as a natural morphogen in neural development

Objectives: This study aimed to examine PML gene expression in different stages of in vitro neural differentiation of NT2 cells, and to investigate the possible role of PML in pluripotency and/or neural development

Materials and Methods: RA was used as a neural inducer for in vitro neural differentiation of NT2 cells. During this process PML mRNA and protein levels were assessed by quantitative real time RT-PCR [QRT-PCR] and Immunoblotting, respectively. Furthermore bisulfite sequencing PCR [BSP] was used to assess PML promoter methylation in NT2 cells and NT2 derived neuronal precursor cells [NT2.NPCs]

Results: QRT-PCR results showed that, PML had maximum expression with significant differences in NT2 derived neuronal precursor cells relative to NT2 cells and NT2 derived neural cells [NT2.NCs]. Numerous isoforms of PML with different intensities appeared in immunoblots of pluripotent NT2 cells, NT2.NPCs, and NT2.NCs. Furthermore, the methylation of the PML promoter in NT2.NCs was 2.6 percent lower than NT2 cell

Conclusions: The observed differences in PML expression in different cellular stages possibly could be attributed to the fact that PML in each developmental state might be involved in different cell signaling machinery and different functions. The appearance of different PML isoforms with more intensity in neural progenitor cells; may suggest apossible role for this protein in neural development

Constructing a mouse Oct4 promoter/EGFP vector, as a whole-cellular reporter to monitor the pluripotent state of cells

The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter ystem is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein [EGFP] was controlled by the mouse Oct-4 promoter. In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs

Characterization and functional assessment of mouse PPAR[gamma]1 promoter

Peroxisome Proliferator Activated Receptor gamma [PPAR[gamma]], a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPAR[gamma] gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPAR[gamma]1 promoter region. Thus, expression pattern of PPAR[gamma]1 isoform is due to the potential transcription factors that could influence its promoter activity. PPAR[gamma], Retinoid X Receptor [RXR] and Vitamin D Receptor [VDR], as nuclear receptors could influence PPAR[gamma] gene expression pattern during several differentiation processes. During neural differentiation, PPAR[gamma]1 isoform expression reaches to maximal level at neural precursor cell formation. A vast computational analysis was carried out to reveal the PPAR[gamma]1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPAR[gamma]1 promoter was assessed in different cell lines. Results indicated that Rosiglitazone increased PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body [EB] formation. Furthermore vitamin D reduced PPAR[gamma]1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPAR[gamma]1 isoform promoter. Also VDR/RXR heterodimers may decrease PPAR[gamma] expression through binding to its promoter