Analysis of Id-1 and Twist-1 Regulation in Bone Development

1. Analysis of Id-1 and Twist-1 Regulation in Bone Development Anna E. Muñoz Cal State University, Los Angeles-City of Hope Cancer Collaborative April 7, 2008

2. Outline • Introduction • Helix-Loop-Helix proteins • Id-1 and Twist-1 • Human stem cells • Study model system • Significance of Study • Cell line preliminary results • Collaborative research project • Project overview • Acknowledgements

3. Helix Loop Helix Proteins • Various helix-loop-helix (HLH) proteins play a key role in the regulation of cellular growth and differentiation • Basic HLH, bHLH, proteins include a basic DNA binding domain • MyoD (directs muscle development) and TWIST-1 • HLH proteins lack the basic domain • Id proteins do not bind DNA

4. Id-1 • Belongs to the Id protein family (Id-1, 2, 3, 4) • HLH protein • Inhibitor of differentiation • Preferentially dimerizes with bHLH proteins • Acts in a dominant negative fashion • Prevents bHLH proteins from forming dimers with other bHLH proteins • Prevents bHLH proteins from binding DNA • Is differentially regulated during differentiation of mesenchymal stem cells to different cell types

5. Twist-1 • bHLH transcription factor • Homodimer or heterodimer with other bHLH proteins (i.e. E proteins) • Regulates cell movement and mesoderm development during early embryogenesis (i.e. bone and muscle) • Twist-1 has both positive and negative functions regulating mesenchymal cell differentiation • Binds to a conserved E-box sequence (CANNTG) on the promoter region that activates or inhibits transcription of a target gene

6. Id bHLH bHLH HLH Proteins +1 E-Box

7. Stem Cells • Unspecialized cells • ability to self regenerate • ability to differentiate into other cells http://stemcells.nih.gov/info/scireport/chapter5.asp

8. Mesenchymal Stem Cells • Also known as “bone marrow stromal cells” • Capacity to differentiate along myogenic, chondrogenic, osteogenic, and adipogenic lineages www.worldhealthspecialists.org/stemCellBasics.asp

9. Study’s Model System • Normal human cells undergo a limited number of cell divisions in culture • Enter senescence, a non-dividing state • Telomere shortening has been linked to cellular senescence • Retroviral transduction of human telomerase reverse transcriptase (hTERT) • Maintains telomere length • Extends life span • Dr. Glackin’s lab at COH created a human fetal mesenchymal stem cell line that has been immortalized by the hTERT gene, hfMSC-SK-hTERT cell line Mol Biol Cell, 2005, 16:1491-1499

10. Significance of Study • Different members of the Id family are overexpressed in different tumor types • Abnormally high expression of Twist-1 in cancer cells has been associated with metastasis • Invasive breast cancer • Twist-1 overexpression prevents normal bone and muscle development • The molecular basis of mechanisms that induce the differentiated osteoblastic phenotype is poorly understood

11. Cell line Preliminary results

12. Experimental Methods • Cell culture experiment performed by Dr. Glackin in 2007 • To determine the expression of Id-1, Id-2, Twist-1, Dermo-1 and bone markers during the differentiation of hfMSC-SK-hTERT cell line to bone.

13. Experimental Methods • Cells were grown in expansion medium • Alpha-Minimal Essential Medium supplemented with Fetal bovine serum, penicillin, streptomycin, L-glutamine, and ascorbic acid 2- phosphate. • Differentiation was induced by changing medium conditions. • Expansion medium was supplemented with dexamethasone, sodium pyruvate, hepes, and inorganic phosphate to induce differentiation to bone. • Differentiation was carried out for 28 days • RNA was collected at days 2, 4, 7, 14, 21, and 28 • Expression of the genes listed above along with bone marker genes was measured by real time RT PCR

14. Experimental Methods DAY 2 DAY 4 DAY 7 DAY 14 DAY 21 DAY 28 OSTEOGENIC ADIPOGENIC MYOGENIC OSTEOGENIC OSTEOGENIC ADIPOGENIC ADIPOGENIC MYOGENIC MYOGENIC Diff Media Control Media OSTEOGENIC OSTEOGENIC ADIPOGENIC ADIPOGENIC MYOGENIC MYOGENIC Diff Media Control Media OSTEOGENIC ADIPOGENIC MYOGENIC

15. MSC differentiation to bone Unpublished preliminary data collected by Dr. Glackin, 2007

16. Twist-1 and Id-1 Expression in Osteogenic Differentiation of MSCs Id-1 ????????????? Twitst-1 Osteocyte MSC Osteoprogenitor Preosteoblast Osteoblast Bone Cell Lining

17. Cancer collaborative Research project

18. Research Goals • To compare the regulation of Id-1 and Twist-1 in the hfMSC-hTERT cell line throughout its osteogenic differentiation. • To identify and analyze the regulatory features of the Id-1 and Twist-1 promoters that contribute to the development of MSCs to osteoblasts.

19. Id-1 and Twist-1 Regulation in MSC-hTERT line • To compare Id-1 and Twist-1 regulation Change medium every 3 days Obtain mRNA from cells at various time points Grow cells in maintenance medium Perform Quantitative PCR Introduce osteogenic medium to promote differentiation Maintain cells at 70%-80% confluency Analyze Id-1 and Twist-1 expression

20. Human Id-1 and Twist-1 promoter constructs • Make Id-1 and Twist-1 promoter/reporter constructs • To study transcriptional regulation of Twist-1 and Id-1 in differentiating MSCs Grow and isolate sufficient quantity luciferase reporter vector Isolate genomic DNA from fhMSC-SK-hTERT cells Bioinformatic analysis of human Id-1 and Twist-1 Clone Id-1 and Twist-1 upstream regions via PCR Ligate promoters into vectors Design primers for human Id-1 and Twist-1 upstream regions Transform fhMSC-SK-hTERT cells Grow cells and differentiate Perform luciferase assays

21. Acknowledgements • CSULA-COH Cancer Collaborative Program • NIH grant • Dr. Sharp, Cal State LA • Laura Martinez, Sharp Lab • Dr. Glackin, City of Hope • Shan Li, Glackin Lab • Joyce Ho, Cal State LA Collaborating student

22. Thank You