SIMULACIÓN DEL MODELO Y ANALISIS DE RESULTADOS

2D 2-dimensional

3D 3-dimensional

Ab Antibody

BAC Bacterial Artificial Chromosome

Bp Base pair

BrdU 5-bromo-2’-deoxyuridine

BSA Bovine Serum Albumine

CCD Charge-Coupled Device

CENP-A,-B,-C Centromeric Proteins -A,-B,-C

ChIP Chromatin Immunoprecipitation

cm centimeter

CT Chromosome Territory

DAPI 4’,6-diamidino-2-phenylindole

Dig Digoxigenine

DNMT DNA Methyl Transferase

d H2O de-ionized water

dd H2O ultrapure water

DMEM Dulbecco’s Modified Eagle Medium

DNA Deoxyribo-Nucleic Acid

Dnase Deoxyribo-nuclease

DOP-PCR Degenerate Oligonucleotid Primer-Polymerase Chain Reaction

dUTP deoxyuridine-triphosphate

EDTA Ethylendiamintetraacetat

EtOH Ethanol

EZH Enhancer of Zeste

FA Formaldehyde

FCS Fetal Calf Serum

FISH Fluorescence In Situ Hybridization

FITC Fluorescein-isothiocyanat

HAT Histone Acetyl Transferase

HDAC Histone Deacetylase

h.c.a. highly cross adsorbed

HEPES N-2-hydroxyethylpiperazin-N’-2-ethanesulfonic acid

HFb Human Fibroblasts

HMT Histone Methyl-Transferase

HSA Homo Sapiens Autosome

HOX Homeobox genes

HP-1 Heterochromatin Protein-1

IF Immunofluorescence

kb kilobase

LINE/SINE Long/Short Interspersed Nuclear Element

MCF-7 Mammary Carcinoma Fibroblasts

MEF Mouse Embryonic Fibroblasts

Mb Megabase

ON Over Night

PBS Phosphate-Buffered Saline

PEV Position Effect Variegation

PcG Polycomb Group

PSF Point Spread Function

RAC Radial Autocorrelation Function

RNA Ribonucleic Acid

RPMI cell culture medium (Roswell Park Memorial Institute)

Rpm Rotations per minute

RT Room Temperature

SAM S-Adenosyl-Methionine

SET1/SET2 Proteins containing a SET-domain

SSC Sodium chloride Sodium Citrate

SUV 39 Suppressor of Variegation

Tris Tri(hydroxymethyl)aminomethane Triton X-100 Octylphenoldecaethylenglycolether Tween 20 Polyoxyethylensorbitanmonolaurat

Xa/Xi active X-chromosome/inactivated X-chromosome

wt wildtype

Xic X inactivation centre

9 Table of Figures

Figures

Fig. 1 Structural features of the IC-CT model -4-

Fig. 2 The packing of DNA and nucleosomes to higher chromatin folding levels -6-

Fig. 3 Lysine methylation sites on histones H3 and H4 -13- Fig. 4 Histone methyltransferases and their substrates -13- Fig. 5 Partitioning of chromatin by histone lysine methylation -14-

Fig. 6 A schematic representation of the HP1 protein -17-

Fig. 7 Transmission images of DLD-1 cells -31- Fig. 8 BACs and paints of HSA #12 tested on metaphase spreads -41-

Fig. 9 Peptide competition assay: “normal” antibody pattern -44-

Fig. 10 Peptide competition assay: incubation with specific peptides -44- Fig. 11 Peptide competition assay: incubation with similar peptides -45- Fig. 12 Point spread function measured on the Leica SP2 -56-

Fig. 13 The impact of deconvolution on image restoration -59-

Fig. 14 H3K27me3 staining before and after deconolution (magnification) -60-

Fig. 15 Deconvolution facilitates threshold-setting -61- Fig. 16 Test for the consistency of co-localization analysis over a range of thresholds -62-

Fig. 17 Results of co-localization analysis delineated in a scatterplot -65- Fig. 18 Path in Image J to calculate Manders coefficients out of two channels -66- Fig. 19 Histone lysine methylation patterns of H3K4me3, H3K9me1 and H4K20me1

together with centromeres -70-

Fig. 20 Histone lysine methylation patterns of H3K9me3, H3K27me3 and H4K20me3

together with centromeres -72-

Fig. 21 Quantitative overlap-assessment of centromeres and different histone

lysine methylation sites -73- Fig. 22 Spatial relation between different histone lysine methylation sites and nascent RNA -75-

Fig. 23 3D reconstructions of H4K20me3 staining patterns incycling and quiescentcells -76-

Fig. 24 Comparison of different lysine methylation sites in cycling and quiescent cells

by RAC analysis -77- Fig. 25 Comparison of differently methylated histone sites by double immunostaining -80- Fig. 26 Distinct nuclear zones formed by histone methylation sites -82-

Fig. 27 Cell viability in the presence of different Chaetocin concentrations -84- Fig. 28 Replication labeling in control and Chaetocin treated cells -85-

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Fig. 29 Comparison of tubulin, B23, SC-35 and H3K9me3 anibody staining in untreated

cells and three days after Chaetocin incubation -86- Fig. 30 Confocal mid-sections of a HFb nucleus three days after Chaetocin treatment

and in control cells -87-

Fig. 31 H3K9me3 and DAPI pattern formation changes in HFbs but not in the cancer

cell-lines MCF-7 and DLD-1 -89- Fig. 32 Distribution of HP1-alpha in HFbs, DLD-1 and MCF-7 cells before and

after Chaetocin treatment -91-

Fig. 33 No reorganization of chromatin occurs after Chaetocin rescue -93- Fig. 34 3D-reconstruction of objects with a size of 500 and 1000 voxel -94- Fig. 35 Evaluation of H3K9me3 pattern size in HFbs before and after Chaetocin application -95-

Fig. 36 Evaluation of H3K9me3 pattern size in MCF-7 cells before and after Chaetocin

application -96-

Fig. 37 Evaluation of H3K9me3 pattern size in DLD-1 cells before and after Chaetocin

application -97-

Fig. 38 Ideogramms of human chromosomes HSA #18, 19 -98- Fig. 39 Immuno-FISH delineating H3K27me3 and H3K9me3 together withHSA #18 and 19 -99- Fig. 40 Immuno-FISH delineating H3K4me3 together withHSA #18 and 19 -100-

Fig. 41 Linescan through a X-chromosome territory -101-

Fig. 42 Ideogramms of human chromosomes HSA #12 and X -102- Fig. 43 Immuno-FISH of gene-rich/poor DNA segments of HSA #12 with H3K4me3 and of

highly expressed/repressed genes of HSA #12 with H3K4me3 and H3K27me3 -104-

Fig. 44 Immuno-FISH delineating X-territories and X-specific genes together with H3K27me3 -106- Fig. 45 The interexperimental differences of histone methylation sites -123-

Tables

Table 1 Properties of different types of chromatin -7/8- Table 2 Flow chart of different approaches for a new immuno-FISH protocol -48-

Table 3 Parameters for Zeiss LSM 410 -53/54- Table 4 Parameters for Leica SP2 -54- Table 5 General parameters for Huygens deconvolution program -57/58-

Table 6 Flow chart of the evaluation procedure after Chaetocin treatment -69-