Histone H1 Subtypes and phosphorylation in cell life and death

.................................................................................................. 10 POPULÄRVETENSKAPLIG SAMMANFATTNING............................. 12 PAPERS IN THE PRESENT THESIS ....................................................... 15 ABBREVIATIONS ....................................................................................... 16 INTRODUCTION......................................................................................... 17 THE ORGANISATION OF DNA INTO CHROMATIN ........................................... 17 DNA and the chromosomes ....................................................................................... 17 The nucleosome ......................................................................................................... 18 Chromosome structure ............................................................................................... 20 REGULATION OF THE CHROMATIN STRUCTURE-REGULATION OF GENES........ 21 THE STRUCTURAL ROLE AND PHYSIOLOGICAL FUNCTION OF HISTONE H1 .... 22 Chromatin structure.................................................................................................... 22 Gene regulation .......................................................................................................... 22 Inhibition of replication.............................................................................................. 23 Apoptosis.................................................................................................................... 23 THE STRUCTURE OF HISTONE H1.................................................................. 25 HISTONE H1 GENES, REGULATION AND EXPRESSION .................................... 29 HISTONE H1 SUBTYPES................................................................................. 30 THE SIGNIFICANCE OF MULTIPLE H1 SUBTYPES ............................................ 34 Sequence conservation ............................................................................................... 34 Gene regulation .......................................................................................................... 34 Knock-out experiments .............................................................................................. 34 Affinity for DNA and chromatin................................................................................ 35 Distribution in chromatin ........................................................................................... 35 POSTTRANSLATIONAL MODIFICATIONS OF H1 HISTONES .............................. 36 PHOSPHORYLATION OF HISTONE H1 ............................................................. 38 Significance of H1 phosphorylation........................................................................... 38 Histone H1 phosphorylating and dephosphorylating enzymes.................................. 38 H1 phosphorylation in gene regulation ...................................................................... 40 Histone H1 phosphorylation and chromatin condensation ........................................ 40 H1 phosphorylation in the cell cycle.......................................................................... 41 Histone H1 phosphorylation in apoptosis .................................................................. 43 Histone H1 phosphorylation pattern in malignant transformation............................. 44 AIMS OF THE THESIS............................................................................... 45 MATERIAL AND METHODS.................................................................... 47 HISTONE H1 EXTRACTION ............................................................................ 48 SEPARATION OF H1 HISTONES AND THEIR PHOSPHORYLATED VARIANTS...... 50 Reversed Phase-High Performance Liquid Chromatography (RP-HPLC) of H1 histones ........................................................................................................................... 50 Hydrophilic Interaction Liquid Chromatography (HILIC) ........................................ 51 High Performance Capillary Electrophoresis (HPCE)............................................... 51 GENETIC ANALYSIS ...................................................................................... 52 DNA-samples............................................................................................................. 52 PCR amplification of H1 genes or gene fragments.................................................... 53 DNA sequencing ........................................................................................................ 54 Detection of sequence variations ............................................................................... 54 Restriction Fragment Length Polymorphism (RFLP) analysis ......................................... 54 Denaturing HPLC (Wave) ................................................................................................ 55 FLOW CYTOMETRY ....................................................................................... 57 Cell cycle distribution ................................................................................................ 58 Cell tracing ................................................................................................................. 60 Apoptosis detection.................................................................................................... 60 Cell Sorting using FACS Aria Special order system ................................................. 63 CELL CULTURE ............................................................................................. 64 Jurkat .......................................................................................................................... 64 T cell activation.......................................................................................................... 64 Ag1523....................................................................................................................... 65 IMMUNOCYTOCHEMISTRY ............................................................................ 65 RESULTS AND DISCUSSION ................................................................... 67 THE EXISTENCE OF H1 MICROSEQUENCE VARIANTS ..................................... 67 HISTONE H1 AND H1 PHOSPHORYLATION IN APOPTOSIS............................... 68 HISTONE H1 SUBTYPE DISTRIBUTION DURING CELL DIVISION....................... 70 HISTONE H1 PHOSPHORYLATION IN THE CELL CYCLE................................... 72 CONCLUSIONS............................................................................................ 75 FUTURE ASPECTS ..................................................................................... 77 ACKNOWLEDGEMENTS.......................................................................... 79 REFERENCES .............................................................................................. 84 PAPERS ......................................................................................................... 99