Analysis of Id-1 and Twist-1 Regulation in Bone Development
Anna E. MuñozCal State University, Los Angeles-City of Hope Cancer Collaborative
April 7, 2008
Outlineo Introduction
o Helix-Loop-Helix proteinso Id-1 and Twist-1
o Human stem cellso Study model systemo Significance of Study
o Cell line preliminary resultso Collaborative research project
o Project overview
o Acknowledgements
Helix Loop Helix Proteins o Various helix-loop-helix (HLH) proteins play a
key role in the regulation of cellular growth and differentiation
o Basic HLH, bHLH, proteins include a basic DNA binding domain o MyoD (directs muscle development) and
TWIST-1o HLH proteins lack the basic domain
o Id proteins do not bind DNA
Id-1
o Belongs to the Id protein family (Id-1, 2, 3, 4)o HLH proteino Inhibitor of differentiationo Preferentially dimerizes with bHLH proteinso Acts in a dominant negative fashion
o Prevents bHLH proteins from forming dimers with other bHLH proteins
o Prevents bHLH proteins from binding DNA
o Is differentially regulated during differentiation of mesenchymal stem cells to different cell types
Twist-1
o bHLH transcription factor o Homodimer or heterodimer with other bHLH
proteins (i.e. E proteins)o Regulates cell movement and mesoderm
development during early embryogenesis (i.e. bone and muscle)
o Twist-1 has both positive and negative functions regulating mesenchymal cell differentiation
o Binds to a conserved E-box sequence (CANNTG) on the promoter region that activates or inhibits transcription of a target gene
HLH Proteins
bHLHbHLH bHLHbHLHId Id
E-Box
+1
Stem Cells
o Unspecialized cellso ability to self
regenerateo ability to
differentiate into other cells
http://stemcells.nih.gov/info/scireport/chapter5.asp
Mesenchymal Stem Cellso Also known as “bone marrow stromal cells”
o Capacity to differentiate along myogenic, chondrogenic, osteogenic, and adipogenic lineages
www.worldhealthspecialists.org/stemCellBasics.asp
Study’s Model Systemo Normal human cells undergo a limited number of cell
divisions in cultureo Enter senescence, a non-dividing state
o Telomere shortening has been linked to cellular senescence
o Retroviral transduction of human telomerase reverse transcriptase (hTERT)o Maintains telomere lengtho Extends life span
o 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
Significance of Studyo Different members of the Id family are
overexpressed in different tumor typeso Abnormally high expression of Twist-1 in
cancer cells has been associated with metastasis o Invasive breast cancer
o Twist-1 overexpression prevents normal bone and muscle development
o The molecular basis of mechanisms that induce the differentiated osteoblastic phenotype is poorly understood
CELL LINE PRELIMINARY RESULTS
Experimental Methods
o Cell culture experiment performed by Dr. Glackin in 2007
o 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.
Experimental Methodso Cells were grown in expansion medium
o Alpha-Minimal Essential Medium supplemented with Fetal bovine serum, penicillin, streptomycin, L-glutamine, and ascorbic acid 2- phosphate.
o Differentiation was induced by changing medium conditions.o Expansion medium was supplemented with
dexamethasone, sodium pyruvate, hepes, and inorganic phosphate to induce differentiation to bone.
o Differentiation was carried out for 28 dayso RNA was collected at days 2, 4, 7, 14, 21, and 28 o Expression of the genes listed above along with
bone marker genes was measured by real time RT PCR
OSTEOGENIC ADIPOGENIC MYOGENIC
DiffMedia
ControlMedia
DAY 2OSTEOGENIC ADIPOGENIC MYOGENIC
DAY 4
OSTEOGENIC ADIPOGENIC MYOGENIC
DAY 7
OSTEOGENIC ADIPOGENIC MYOGENIC
DiffMedia
ControlMedia
DAY 14
OSTEOGENIC ADIPOGENIC MYOGENIC
DAY 21
OSTEOGENIC ADIPOGENIC MYOGENIC
DAY 28
Experimental Methods
MSC differentiation to bone
Unpublished preliminary data collected by Dr. Glackin, 2007
Twist-1 and Id-1 Expression in Osteogenic Differentiation of MSCs
MSC Osteoprogenitor
OsteoblastPreosteoblast
Osteocyte
Bone Cell Lining
Id-1
?????????????
Twitst-1
CANCER COLLABORATIVE RESEARCH PROJECT
Research Goals
o To compare the regulation of Id-1 and Twist-1 in the hfMSC-hTERT cell line throughout its osteogenic differentiation.
o To identify and analyze the regulatory features of the Id-1 and Twist-1 promoters that contribute to the development of MSCs to osteoblasts.
Id-1 and Twist-1 Regulation in MSC-hTERT line
o To compare Id-1 and Twist-1 regulation
Grow cells in maintenance
medium
Maintain cells at 70%-80% confluency
Obtain mRNA from cells at various time points
Change medium every 3 days
Introduce osteogenic medium to promote
differentiation
Perform Quantitative PCR
Analyze Id-1 and Twist-1 expression
Human Id-1 and Twist-1 promoter constructs
Bioinformatic analysis of human Id-
1 and Twist-1
Design primers for human Id-1 and Twist-
1 upstream regions
Grow and isolate sufficient quantity luciferase reporter
vector
Isolate genomic DNA from fhMSC-SK-hTERT
cells
Clone Id-1 and Twist-1 upstream regions via PCR Ligate promoters
into vectors
Transform fhMSC-SK-hTERT cells
o Make Id-1 and Twist-1 promoter/reporter constructs o To study transcriptional regulation of Twist-1 and
Id-1 in differentiating MSCs
Perform luciferase assays
Grow cells and differentiate
Acknowledgements
o CSULA-COH Cancer Collaborative Programo NIH grant
o Dr. Sharp, Cal State LAo Laura Martinez, Sharp Lab
o Dr. Glackin, City of Hopeo Shan Li, Glackin Lab
o Joyce Ho, Cal State LA Collaborating student
Thank You