| 1. |
- Gréen, Anna, 1973-
(author)
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Histone H1 : Subtypes and phosphorylation in cell life and death
- 2009
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Doctoral thesis (other academic/artistic)abstract
- The genetic information of a human diploid cell is contained within approximately 2 metres of linear DNA. The DNA molecules are compacted and organized in various ways to fit inside the cell nucleus. Various kinds of histones are involved in this compaction. One of these histones, histone H1 is the topic of the present thesis. In addition to its structural role, H1 histones have been implicated in various processes, for example gene regulation and inhibition of chromatin replication.H1 histones, also termed linker histones, are relatively conserved proteins, and the various subtypes seem to have different and important functions even though redundancy between the subtypes has been demonstrated. Despite the sequence conservation of H1 subtypes, two sequence variations were detected within the H1.2 and H1.4 subtypes using hydrophilic interaction liquid chromatographic separation of H1 proteins from K562 and Raji cell lines in Paper I in the present thesis. The variations were confirmed by genetic analysis, and the H1.2 sequence variation was also found in genomic DNA of normal blood donors, in an allele frequency of 6.8%. The H1.4 sequence variation was concluded to be Raji specific. The significance of H1 microsequence variants is unclear, since the physiological function of H1 histones remains to be established.H1 histones can be phosphorylated at multiple sites. Changes in H1 phosphorylation has been detected in apoptosis, the cell cycle, gene regulation, mitotic chromatin condensation and malignant transformation. Contradictory data have been obtained on H1 phosphorylation in apoptosis, and many results indicate that H1 dephosphorylation occurs during apoptosis. We and others hypothesized that cell cycle effects by the apoptosis inducers may have affected previous studies. In Paper II, the H1 phosphorylation pattern was investigated in early apoptosis in Jurkat cells, taking cell cycle effects into account. In receptor-mediated apoptosis, apoptosis occurs with a mainly preserved phosphorylation pattern, while Camptothecin induced apoptosis results in rapid dephosphorylation of H1 subtypes, demonstrating that H1 dephosphorylation is not a general event in apoptosis, but may occur upon apoptosis induction via the mitochondrial pathway. The dephosphorylation may also be a result of early cell cycle effects or signalling.Therefore, the H1 phosphorylation pattern in the cell cycle of normal activated T cells was investigated in Paper IV in this thesis. Some studies, which have been made using cancer cell lines from various species and cell synchronization, have indicated a sequential addition of phosphate groupsacross the cell cycle. Normal T cells and cell sorting by flow cytometry were used to circumvent side-effects from cell synchronization. The data demonstrate that a pattern with phosphorylated serines is established in late G1/early S phase, with some additional phosphorylation occurring during S, and further up-phosphorylation seems to occur during mitosis. Malignant transformation may lead to an altered G1 H1 phosphorylation pattern, as was demonstrated using sorted Jurkat T lymphoblastoid cells.During mitosis, certain H1 subtypes may be relocated to the cytoplasm. In Paper III, the location of histones H1.2, H1.3 and H1.5 during mitosis was investigated. Histone H1.3 was detected in cell nuclei in all mitotic stages, while H1.2 was detected in the nucleus during prophase and telophase, and primarily in the cytoplasm during metaphase and early anaphase. H1.5 was located mostly to chromatin during prophase and telophase, and to both chromatin and cytoplasm during metaphase and anaphase. Phosphorylated H1 was located in chromatin in prophase, and in both chromatin and cytoplasm during metaphase, anaphase and telophase, indicating that the mechanism for a possible H1 subtype relocation to the cytoplasm is phosphorylation.In conclusion, data obtained during this thesis work suggest that H1 histones and their phosphorylation may participate in the regulation of events in the cell cycle, such as S-phase progression and mitosis, possibly through altered interactions with chromatin, and/or by partial or complete removal of subtypes or phosphorylated variants from chromatin.
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| 2. |
- Bergström, Rosita, 1978-
(author)
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Epigenetic Regulation of Replication Timing and Signal Transduction
- 2008
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Doctoral thesis (other academic/artistic)abstract
- Upon fertilization the paternal and maternal genomes unite, giving rise to the embryo, with its unique genetic code. All cells in the human body are derived from the fertilized ovum: hence they all contain (with a few exceptions) the same genetic composition. However, by selective processes, genes are turned on and off in an adaptable, and cell type-specific, manner. The aim of this thesis is to investigate how signals coming from outside the cell and epigenetic factors residing in the cell nucleus, cooperate to control gene expression. The transforming growth factor-β (TGF-β) superfamily consists of around 30 cytokines, which are essential for accurate gene regulation during embryonic development and adult life. Among these are the ligands TGF-β1 and bone morphogenetic (BMP) -7, which interact with diverse plasma membrane receptors, but signal via partly the same Smad proteins. Smad4 is essential to achieve TGF-β-dependent responses. We observed that by regulating transcription factors such as Id2 and Id3 in a specific manner, TGF-β1 and BMP-7 achieve distinct physiological responses. Moreover, we demonstrate that CTCF, an insulator protein regulating higher order chromatin conformation, is able to direct transcription by recruiting RNA polymerase II to its target sites. This is the first mechanistic explanation of how an insulator protein can direct transcription, and reveals a link between epigenetic modifications and classical regulators of transcription. We also detected that DNA loci occupied by CTCF replicate late. The timing of replication is a crucial determinant of gene activity. Genes replicating early tend to be active, whereas genes replicating late often are silenced. Thus, CTCF can regulate transcription at several levels. Finally, we detected a substantial cross-talk between CTCF and TGF-β signaling. This is the first time that a direct interplay between a signal transduction pathway and the chromatin insulator CTCF is demonstrated.
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| 3. |
- Holmgren, Claes
(author)
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Epigenetic Regulation of the H19 Chromatin Insulator in Development and Disease
- 2003
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Doctoral thesis (other academic/artistic)abstract
- The coordinated regulation of gene expression must be tightly controlled for normal development to occur. In mammals, this issue is further complicated by the requirement of both the maternal and paternal genomes for normal development, reflecting the fact that a subset of genes are monoallelically expressed depending on parental inheritance, a phenomenon known as genomic imprinting. The imprinted H19 and Igf2 genes are often considered as paradigms of genomic imprinting, since their monoallelic expression patterns are coordinated via a short stretch of sequence upstream of H19, known as the imprinting control region (ICR). This region is differentially methylated, with specific CpG methylation on the paternal allele. It is shown here that the ICR harbours several maternal-specific hypersensitive sites, located in linker regions between positioned nucleosomes. Furthermore, this region functions as an orientation-dependent insulator, that binds the chromatin insulator factor CTCF. The hypothesis that the methylation status of the ICR dictates the activity of the Igf2 gene 90 kb further upstream was confirmed by the demonstration that the insulator function is lost when the ICR is CpG methylated. The ICR has previously been shown to act as a silencer when positioned in a promotor-proximal position. The cause of this silencing was shown to be distance-dependent, suggesting that the silencing features of the ICR depend on a chromatin conformation that renders adjacent sequences inaccessible to the RNA polymerase. These data issue a cautionary note with respect to the interpretation of silencer functions. In several forms of cancer, the normally silent maternal IGF2 gene is expressed, possibly as a result of loss of insulator function at the ICR. The utilisation of CTCF target-sites was analysed in different tumours, and was shown to be highly variable. Methylation analysis showed that potential loss of insulator function and gain of methylation at the maternal ICR did not always correlate with biallelic expression of IGF2. Further investigations uncovered a novel mechanism, in which the activation of the IGF2 promoter was independent of insulator function in some cancers. This thesis shows that the regulation of the imprinted state of Igf2 depends on the formation of an epigenetically regulated chromatin insulator, and that the loss of IGF2 imprinting in human cancer can be attributed to several mechanisms, including a novel mechanism that neutralises chromatin insulator function.
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| 4. |
- Whitehead, Joanne, 1974-
(author)
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Genomic Imprinting in Development and Evolution
- 2004
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Doctoral thesis (other academic/artistic)abstract
- Genetic information is encoded by the linear sequence of the DNA double helix, while epigenetic information is overlayed as the packaging of DNA and associated proteins into the chromatin structure. Variations in chromatin structure play a vital role in establishing and maintaining patterns of gene expression during differentiation and development of higher eukaryotes, and disruption of this epigenetic gene regulation can lead to cancer. Mammals display an epigenetic phenomenon known as genomic imprinting, which provides an ideal model system for the study of epigenetics. Genes subject to genomic imprinting are differentially expressed within a single cell depending on the parental origin of the chromosome. Imprinting of the maternally expressed H19 gene and the adjacent paternally expressed Igf2 gene is regulated by the chromatin insulator protein CTCF. The studies presented in this thesis aim to investigate the functional mechanisms of CTCF-dependent gene regulation at the H19/Igf2 locus and at numerous other target sites throughout the genome. We have investigated the role of CTCF and a related protein BORIS in establishing the maternal to paternal imprint transition in chromatin structure at the H19/Igf2 locus in the male germline. We have developed novel microarray based methods to identify and characterize numerous new CTCF target sites throughout the mouse genome. We have shown that CTCF acts as part of the RNA polymerase II complex. We have identified the post-translational modification by addition of ADP-ribose polymers to CTCF, and demonstrated that this modification regulates its insulating ability. The results of these studies of CTCF-dependent epigenetic gene regulation are discussed in light of the evolution of genomic imprinting and chromatin insulators, and a novel role for poly ADP-ribosylation of CTCF in the progression of cancer is proposed.
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| 5. |
- Yu, Yang, 1970-
(author)
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Molecular Mechanisms Underlying Abnormal Placentation in the Mouse
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Placental development can be disturbed by various factors, such as mutation of specific genes or maternal diabetes. Our previous work on interspecies hybrid placental dysplasia (IHPD) and two additional models of placental hyperplasia, cloned mice and Esx1 mutants, showed that many genes are deregulated in placental dysplasia. Two of these candidate placentation genes, Cpe and Lhx3, were further studied. We performed in situ hybridization to determine their spatio-temporal expression in the placentas and placental phenotypes were analyzed in mutant mice. Our results showed that the placental phenotype in Cpe mutant mice mimics some IHPD phenotypes. Deregulated expression of Cpe and Cpd, a functionally equivalent gene, prior to the manifestation of the IHPD phenotype, indicated that Cpe and Cpd are potentially causative genes in IHPD. Lhx3 mutants lacked any placental phenotype. Deletion of Lhx3 and Lhx4 together caused an inconsistent placental phenotype which did not affect placental lipid transport function or expression of Lhx3/Lhx4 target genes. Down regulation of Lhx3/Lhx4 did not rescue the placental phenotype of AT24 mice and hence could be excluded as causative genes in IHPD. Analysis of placental development in diabetic mice showed that severe maternal diabetes resulted in fetal intrauterine growth restriction (IUGR) without any change in placental weight and lipid transport function. The diabetic placentas however exhibited abnormal morphology. Gene expression profiling identified some genes that might contribute to diabetic pathology. In another study, it was found that the heterochromatin protein CBX1 is required for normal placentation, as deletion of the gene caused consistent spongiotrophoblast and labyrinthine phenotypes. Gene expression profiling and spatio-temporal expression analysis showed that several genes with known function in placental development were deregulated in the Cbx1 null placenta.
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