[Proteomic analysis of LIMD1-binding proteins]

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Acknowledgement

I would take this opportunity to thank my research supervisor, family and friends for their support and guidance without which this research would not have been possible.

DECLARATION

I [type your full first names and surname here], declare that the contents of this dissertation represent my own unaided work, and that the dissertation has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the University.

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Abstract

LIMD1 is a tumour suppressor gene (TSG) down regulated in not, vert, similar80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1.

Table of Contents

CHAPTER 1: INTRODUCTION6

Structure of LIM Protien9

Function10

Interaction10

CHAPTER 2: METHODOLOGY12

Promoter mapping analysis12

LIMD1 hypoxic response12

Bioinformatic analysis14

Point mutagenesis of IR514

siRNA aimed at depletion and qRT-PCR analysis15

PU.1 subcloning16

Results19

The CpG Island inside the LIMD1 promoter comprises both affirmative and contradictory regulatory elements19

An Ets family agreement sequence is conserved between mammals inside the LIMD1 promoter21

Mutation inside the Ets binding domain agreement decreases transcription by 90%22

ChIP investigation of IR5 inside the LIMD1 promoter23

PU.1 and the PU.1 Ets domain solely join to the IR5 DNA agreement in vitro24

PU.1 siRNA aimed at depletion outcomes in decrease of LIMD1 expression25

Generation of anti-LIMD1 antibodies27

Cell cycle guideline and phosphorylation of LIMD127

Cellular localization of LIMD1 and colocalization with vinculin29

Staining protocol30

Evaluation of staining30

Absence of atomic LIMD1 staining correlates with a decline in persevering survival34

CHAPTER 3: DISCUSSION AND ANALYSIS37

CHAPTER 5: CONCLUSION52

REFERENCES57

REFERENCES57

Chapter 1: Introduction

Lim domains encompassing protein 1 (LIMD1) is a bona fide  tumour suppressor gene (TSG) encoded at chromosome 3p21.3, a district that routinely undergoes homozygous deletion, decrease of heterozygosity and epigenetic calming in numerous carcinomas, the most revised being lung. Experimentally the proficiency of the A549 lung cancerous infection cell line to pattern lung metastases in a mouse form is considerably decreased upon steady sign of LIMD1.  Limd1next term-/- mice are predisposed to chemical-induced lung adenocarcinomas and genetic inactivation of  Limd1 in mice heterozygous for oncogenic K-Ras (G12D) talks markedly expanded tumour initiation, advancement, and mortality. In corroboration with the mouse form, LIMD1 protein sign is decreased in 80% of lung squamous cell and adeno-carcinomas in 50% of head and neck squamous cell carcinomas (HNSCC). In childhood acute lymphoblastic leukaemias 20% of trials have chromosomal deletions at the 3p21. locus with LIMD1 decrease flagged as a likely origin of tumour formation. A decrease in LIMD1 sign is furthermore correlated with poor prognosis and survival rates in breast ...