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