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Research Pathology Medical Biology Research Epigenetic Editing
University Medical Center Groningen

Student projects

Awake the sleeping; gene-specific upregulation of (tumor-suppressor) genes via epigenetic editing

(M.Sc. Marloes de Groote)

Introduction

Numerous diseases are associated with aberrant gene expression profiles. Epigenetic factors like DNA methylation and histone modifications play an important role in controlling gene expression. In contrast to genetic mutations, epigenetic abnormalities are reversible and thus provide nice targets for therapeutic intervention. Moreover, without active interference, epigenetic marks are stable and will be inherited to daughter cells.

Making use of these characteristics of epigenetics, a number of epigenetic drugs have been designed to reverse aberrant DNA methylation and histone modification patterns. The disadvantage of these epigenetic drugs is that they work genome-wide, thereby increasing the risk of side-effects, and their effects are only transient.

Therefore, we set out to actively change epigenetic marks of specific genes only, through epigenetic editing. Fusion of an epigenetic enzyme to an engineered gene-specific DNA binding domain, like a zinc finger protein, will result in gene-specific and sustained alteration of the epigenetic mark. Upregulation of gene-expression in this manner makes sure that all splice variants are expressed in their natural ratios and from the natural promoter. Moreover, downregulation via epigenetic editing has been shown to be permanent.

In consultation with the supervisor, input of the student is highly appreciated.

Possible student projects:

Characterisation of epigenetic marks on ICAM promoter in ovarian cancer cells in order to identify effectors for re-expression of the gene

To obtain knowledge on the epigenetic regulation of ICAM in ovarian cancer, the student will screen different ovarian cancer cell lines for the epigenetic marks present on the promoter of ICAM.

Targeted DNA demethylation making use of Zinc Fingers fused to potential DNA demethylases

Since there is no clarity yet about which enzyme is responsible for active DNA demethylation in mammals, the effect of candidate enzymes will be screened upon fusion to zinc finger proteins in human cancer cell lines.

Targeted histone modification making use of Zinc Fingers fused to known histone modifying enzymes

To investigate whether it is possible to place histone modifications associated with gene activation on a predetermined site in the genome, well known histone modifying enzymes capable of placing these activating histone marks will be tested in human cell lines.

Techniques:

Molecular cloning

Transfection/Retroviral transduction

qRT-PCR

FACS/Western blot

Methylation specific PCR/Bisulfite sequencing

ChIP

References

Smith, A. E., Hurd, P. J., Bannister, A. J., Kouzarides, T., & Ford, K. G. (2008). Heritable gene repression through the action of a directed DNA methyltransferase at a chromosomal locus. The Journal of Biological Chemistry, 283(15), 9878-9885.

Snowden, A. W., Gregory, P. D., Case, C. C., & Pabo, C. O. (2002). Gene-specific targeting of H3K9 methylation is sufficient for initiating repression in vivo. Current Biology : CB, 12(24), 2159-2166.

Wu, S. C., & Zhang, Y. (2010). Active DNA demethylation: Many roads lead to rome. Nature Reviews.Molecular Cell Biology, 11(9), 607-620.

Mysterious cross-talk: (cross)-linking collagen biosynthesis to cancer

Contact: Prof. Dr. M.G. Rots (m.g.rots med.umcg.nl), R.A.F. Gjaltema, MSc ( r.a.f.gjaltema med.umcg.nl )

Introduction

This project is part of the Dutch Top Institute NIRM (Netherlands Institute for Regenerative Medicine). Our interests lie in the triangle relation between inflammation, fibrosis and cancer. In this case we focus on the enzyme Lysyl hydroxylase 2b (LH2b) which is predominantly expressed in extracellular matrix-producing cells such as fibroblasts. In these cells LH2b has a crucial role in initiating the first steps of pyridinoline cross-linking of collagen molecules, which is necessary for appropriate biomechanical stability of tissues. In fibrosis however, increased LH2b expression results in an accumulation of pyridinoline cross-linked collagen that leads to “scarring” of tissues and eventually failure of organ function. Additionally to fibrosis, LH2b is upregulated in various types of cancer such as cervical cancer, breast cancer and glaucoma. Since cancer cells do not produce vast amount of cross-linked collagens, the benefit of increased expression of LH2b in tumors is currently unknown. (See references at the bottom for more background information)

Aim

In this specific project we like to identify the presence and function of LH2b in various types and stages of cancer and whether depletion of this enzyme (by siRNA knockdown) or overexpression has effects on the growth and invasiveness of cancer cells. Furthermore, we want to identify how LH2b is regulated on a (epi)genetic level in cancer cells, in order to find novel targets to interfere with LH2b expression.

Possible student projects

Functional characterisation of LH2b in various types of cancer

In this subproject the main focus is on the function of LH2b in cancer. What is the role of LH2b in cancer and what happens to cancer cells when the expression is increased or decreased?

Methods :

siRNA mediated knockdown of LH2b in high expressing cells and overexpression of LH2b in low expressing cells; determine the effects on cell phenotype Gene expression analysis with quantitative-RT-PCR Identifying protein expression by western blotting of cell lines or Immunohistochemistry on patient material

Regulatory principles behind LH2b expression in cancer

In this subproject we switch our focus to a more (epi)genetic level. Herein we like to zoom in to the regulatory principles of LH2b in various types of cancer (cells). Is there an epigenetic state that maintains LH2b expression and does this resemble regulatory methods comparable to a cells in a (pro)fibrotic environment?

Methods:

Identify transcription factor binding and specific histone modifications by chromatin immunoprecipitations.

Bisulfite sequencing or methylation specific PCR on DNA from cancer cells to identify methylated DNA regions around the gene encoding LH2b

References

Chang HY , Sneddon JB , Alizadeh AA , Sood R , West RB , Montgomery K , Chi JT , van de Rijn M , Botstein D , Brown PO . Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS Biol. 2004 Feb;2(2):E7. Epub 2004 Jan 13.

Dong S , Nutt CL , Betensky RA , Stemmer-Rachamimov AO , Denko NC , Ligon KL , Rowitch DH , Louis DN . Histology-based expression profiling yields novel prognostic markers in human glioblastoma. J Neuropathol Exp Neurol. 2005 Nov;64(11):948-55.

Rajkumar T , Sabitha K , Vijayalakshmi N , Shirley S , Bose MV , Gopal G , Selvaluxmy G . Identification and validation of genes involved in cervical tumourigenesis. BMC Cancer. 2011 Feb 22;11:80.

Laatst gewijzigd:14 maart 2013 10:34