Study Of Transcription Regulation Of Human LRP5 Gene And KLF15 Gene

Part 1. Regulation of human LRP5 gene transcriptionLRP5 (low-density lipoprotein receptor related protein 5), located on human chromosome 11ql3, contains 23 exons encoding a 1615 amino acid single-pass transmembrane receptor that belongs to the low density lipoprotein (LDL) receptor superfamily. LRP5 is expressed in multiple adult and embryonic tissues with strongest expression occurring in the live. In bone, it is expressed by osteoblasts of the endosteal and trabecular bone surfaces but not osteoclasts.LRP5 regulates diverse developmental processes in embryogenesis and maintains physiological homeostasis in maturity. Loss-of-function mutation in LRP5 can result in osteoporosis, and gain-of-function mutation leads to high bone mass (HBM), indicating that LRP5 plays an important role in bone formation. Such role is through the Wnt signaling pathway. In addition to these roles, the LRP5 is also required for normal cholesterol and glucose metabolism.Despite lots of effort has been done in study of function of LRP5, the molecular regulation of LRP5 expression remains unclear. Therefore, we studied the transcriptional regulation of LRP5 and got the following results:1. Determination of the transcriptional start site of LRP5 gene. Using primer extension, we identified the transcriptional start site of LRP5 gene, and found that the transcriptional start site is located at the 114bp upstream of the translational start site,40 nucleotides upstream of the 5'end of the published cDNA sequence (GenBank accession no. NM002335).2. In order to identify putative transcriptional factor binding sites, we analyzed a 2495bp region upstream of translational start site of LRP5 using the programs Matlnspector (www.genomatix.de/products/MatInspector/) and AliBaba 2.1 (www.gene-regulation.com/pub/programs.html), and failed to find any canonical TATA box or CAAT-like sequence. However, several potential transcription factor binding motifs were identified in the promoter region close to transcription start site, including Sp1, KLF15, CDE, MAZ, and ZBPF. Using luciferase reporter gene, we tested the transcriptional activity of this fragment in U2OS cells and HEK293 cells, and found this region has promoter activity.3. To determine the minimal region required for basal activity of the promoter, a series of deletion constructs were generated and designated as pWT-2015, pWT-1594, pWT-1442, pWT-1309, pWT-1167, pWT-1085, pWT-953, pWT-359, pWT-219, pWT-72, pWT-53 and pWT-40 based on their variable 5'end. These constructs were tested for their ability to drive the luciferase reporter gene in transiently transfected U2OS and HEK293 cells. Similar trends were observed in these two cell lines, suggesting that cell-specific element may not be present in those sequences. Comparing the expression activity of different deletion constructs, the proximal promoter has been located at-72 and-53.4. With software analysis, two overlapped transcriptional factor binding sites, one for KLF15 and the other for Sp1, were found in-72 to-53. To determine the contribution of the Spl and/or KLF15 sites to the promoter activity, constructs with either single or double mutant reporters were generated. The luciferase activity assay showed both KLF15 and Spl binding sites between-72 and-53 of human LRP5 promoter contributes to the basal activity of human LRP5 transcription.5. It is found in EMSA that wild type probe containing the region-72 to-53 can specificly binds to nuclear proteins. Unlabeled wild type probe can effectively abolish the complex of labeled probe and nuclear protein, while unlabeled mutant probe can't, indicating that the KLF15 and Spl sites can specificly bind to nuclear proteins. In ChIP analysis using anti-Sp1 and anti-KLF15 antibodies, DNA fragment from-72 to-53 can be detected from co-immunoprecipitated chromatin complex. This result further confirmed this region can specificly bind to KLF15 and Sp1.6. By co-transfecting Drosophila SL2 cells lacking KLF15 and Spl proteins with pPac-KLF15 and pPac-Spl, it is found that the increase of LRP5 promoter activity by Spl or KLF15 relies on the existence of corresponding binding sites in-72 to-53.The foregoing study from different aspects, including the binding sites of Spl and KLF15, transcription factor and their binding, proved the Spl and KLF15 binding sites in-72 to-53 of LRP5 gene are essential for its promoter activity.Part 2. KLF15 regulates the transcription of endogenous LRP5Kruppel-like factors (KLFs), a subclass of the zinc-finger family of transcriptional regulators, can function as transcriptional activators and/or repressors depending on promoter context. In the first part, we demonstrated that both KLF15 and Sp1 functions as a positive regulatory element and activates the human LRP5 promoter. C2C12 cell line, a muscle satellite cell line, can differentiate into myotubes, adipocytes and osteoblasts under different condition. Using quantitative PCR and Western blotting, the expression of KLF15 and its regulation of transcription of LRP5 under different differentiating condition were detected, and the results are as following.1. C2C12 cell matained in DMEM medium with 2% horse serum was induced to the typical morphology of multinucleated myotube. Myotube-specific myogenin gene was dramaticlly upregulated during myogenic differentiation as determined by real-time PCR. During myogenic differentiation, both mRNA and protein of KLF15 were significantly increased. Consequently, LRP5 transcription was significantly upregulated.2. C2C12 cells matained in the adipogenic medium containg 5μM rosiglitazone became adipose-like morphology and contain lipid droplet which can be stained with Oil red O. The successful induction was confirmed by the significant upregulation of aP2 mRNA, an adipose-sepcific marker, during the adipogenic differentiation. The KLF15 expression was significantly upregulated at both mRNA and protein levels. LRP5 mRNA level was also increased during the adipogenic differentiation.3. C2C12 matained in osteogenic differentiation medium was induced to differentiate toward osteoblast. On day 24 after treatment, the mineralized nodules were detected by Alizarin red staining. The ALP mRNA, osteoblast-specific gene, was upregulated during the differentiation process. Both mRNA and protein levels of KLF15 gene were significantly induced during differentiation. Consequently, LRP5 mRNA was significantly upregulated.Taken together, inducing C2C12 cells to differentiate toward myogenis, adipogenis and osteogenis can alter the expression of KLF15 and LRP5. These further confirmed that LRP5 is the target gene of KLF15 and KLF15 can activate the transcription of LRP5 gene.Part 3. Regulation of human KLF15 gene transcriptionKLF15 can activate, as well as repress, gene transcription. The results of last two parts all showed KLF15 can be upregulated during cell differentiation and activate the transcription of LRP5 gene. However, the regulation of transcription of KLF15 is still unknown. Therefore, in this part we studied the regulation mechanism of KLF15 transcription.1. Using the programs MatInspector (www.genomatix.de/products/MatInspector/) and AliBaba 2.1 (www.gene-regulation.com/pub/programs.html) to analyze the promoter sequence of KLF15 gene, we failed to find any canonical TATA box. However, multiple potential transcription factor binding motifs were identified, including five GC-boxes that can bind to Spl, as well as USF1, CREB, Smad3, Smad4, AP2, and NYF sites.2. Serial deletion constructs with luciferase reporter gene, pGL3-1804, pGL3-1505, pGL3-863, pGL3-792, pGL3-514, pGL3-217, pGL3-189, pGL3-80, pGL3-45 and pGL3-5, were generated, and luciferase activity were measured after transient transfecting these constructs into HEK293 cells. According to the luciferase activity of these deletion constructs, it is found that the proximal transcription regulator is located in-189 to-80 and-80 to-45.3. Four GC boxes, designated as GC1, GC2, GC3 and GC4, were found within-189 to-80 by software analysis. Through site-direct mutagenesis, we found GC2 and GC3 play an important role in regulation of KLF15 transcription. The effect of GC1 is not so strong, but might become important after GC2 or GC3 mutated.4. There are two partially overlapping sites, one E-box consensus sequence and one CREB binding sits within-80 to-45. After deletion and mutation, E-box was found as an essential element that maintains the promoter activity of human KLF15 gene. The possibility of effect of CREB on promoter activity was excluded using RNA interference of CREB.5. EMSA results showed that GC boxs located in-189 to-155 can form specific DNA-protein complex with nuclear protein. Unlabeled wild type probe can effectively abolish this complex, whereas the unlabeled mutant probe can't. ChIP results also confirmed that Spl protein can specificly bind to this region.6. Also with EMSA was E-Box found to be able to form specific DNA-protein complex. Unlabeled wild type probe can effectively abolish this complex, whereas the unlabeled mutant probe can't. ChIP results also confirmed that transcription factor USF1 can specificly bind to this region.In summary, this study adopting different approaches, such as deletion and mutation analysis, EMSA, ChIP, to prove that USF1 and Spl can bind to E-box in-80 to-45 and GC-box in-189 to-155 in KLF15 promoter, respectively, thus regulate the transcription of KLF15 gene.