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One form of protein regulation is accomplished by post-translational modification (PTM). In order to test the importance one type of PTM, methylation, in chromosome segregation, we inhibited protein methylation for brief periods in G2 using the general methylation inhibitor adenosine dialdehyde (AdOx). Inhibiting methylation solely in late G2 leads to mitotic defects. We observed that several methylated histone residues; H3K9me3, H4K20me3 and H4K20me1, are predominantly affected by AdOx in G2. We show both that the kinetochore proteins are not affected and that the mitotic checkpoint is intact. Further, we observed structural defects and chromosome misalignment in mitotic cells. These results indicate that methylation events during late G2 operate to maintain and ensure the structural integrity of pericentromeric heterochromatin prior to mitosis. These results suggest that pericentromeric heterochromatin is required for the proper sensing of kinetochore tension and inactivation of the mitotic checkpoint. / Experimental Oncology

This dissertation examines the interaction of the linker histone with DNA and with nucleosomes. The first goal of the project was to characterize the interaction of the linker histone with DNA. Three factors previously reported to influence the linker histone's interaction with DNA were examined: ratio of linker histone to DNA sites of binding, monovalent ions in the local environment, and conformation of the DNA molecules. Evidence obtained through gel mobility shift assays demonstrates the strong preference by the linker histone for DNA with superhelical torsion, i.e., supercoiling, and the negative cooperative mode of binding that the linker histone exhibits in association with supercoiled DNA. The second part of the dissertation examines the location of linker histone binding on the nucleosome, and documents the pronounced tendency of the linker histone to bind to two DNA duplex strands. A preparation of homogeneous nucleosome core particles, consisting of a defined 238 base pair DNA fragment and the core histone octamer positioned precisely on this DNA, was used as a substrate for the UV-induced crosslinking of the linker histone to the DNA of this nucleosome. By site-specific labeling of a single site on the DNA of the nucleosome, the linker histone was observed crosslinked at that labeled site, confirming that the linker histone binds at the pseudo-dyad axis of the nucleosome. This evidence was used to support a model of linker histone binding to the nucleosome that invokes the association of the linker histone with no fewer than two duplex strands of DNA of the nucleosome. / Graduation date: 2004

Herpes simplex virus type 1 (HSV-1) genomes associate with histones in unstable nucleosomes during lytic infections. Nucleosome core particles are 146 base pairs of DNA wrapped around a histone octamer of two molecules of each H2A, H2B, H3, and H4. Histone H1 binds to nucleosomes at DNA entry and exit points. Association with histones is proposed to regulate HSV-1 gene expression. Consistently, HSV-1 transcription transactivators disrupt chromatin and HSV-1 strains mutant in these transactivators are replication impaired or transcriptionally inactive. HSV-1 genomes have dynamic associations with histones. The genomes are not associated with histones in capsids, and input genomes are delivered to nuclear domains depleted of histones. Later during infection, HSV-1 genomes again occupy nuclear domains depleted of histones. Histone synthesis is inhibited during infection and the total level of nuclear histones remains relatively constant. It is therefore unlikely that the histones that first bind to HSV-1 genomes are newly synthesized. The source of the histones that associate with HSV-1 genomes has yet to be addressed. Histones in cellular chromatin normally disassociate, diffuse through the nucleus, and re-associate at different sites. I propose that histones are mobilized from domains of cellular chromatin to those domains containing HSV-1 genomes in cellular attempts to silence HSV-1 gene expression. I additionally propose that HSV-1 further mobilizes histones to counteract such silencing attempts. My hypothesis is that histones are mobilized during HSV-1 infection. In this thesis, I show that linker and core histones are mobilized during HSV-1 infection. Such mobilization results in increases to their free (not bound to chromatin) pools. Linker and core histones were mobilized even when HSV-1 proteins were not expressed, mobilization that likely reflects cellular responses to infection. Histone mobilization was enhanced when HSV-1 IE or E proteins were expressed. This enhanced mobilization was independent of HSV-1 DNA replication and late proteins. Core histones H2B and H3.3 were differentially mobilized, suggesting that different mechanisms may mobilize histones during HSV-1 infection. My discovery of histone mobilization reveals a novel consequence of cell-virus interactions that addresses a previously unexplained aspect of HSV-1 infection.

Thèse de doctorat : Biologie cellulaire et moléculaire des plantes : Strasbourg 1 : 2008. / Titre provenant de l'écran-titre. Bibliogr. 16 p.. Notes bibliogr.. Index.

Thesis (Ph.D.)--Drexel University, 2009. / Includes abstract. Includes bibliographical references (leaves 187-201).

Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 155-169). Also available in print.

Altered histone modifications are known to be observed in cancer cells. Acetylation of histone H4 (acetyl-H4) occurs reversibly on its amino-terminal end at four lysines positions 5, 8, 12 and 16 by Histone Acetyltransferase (HATs). Acetyl-H4 is responsible for a complex set of post-translational modifications that regulate the accessibility of DNA, transcription activation and DNA repair processes. Acetyl-H4 can also acetylate non-histone proteins which eventually control numerous cell signal pathways such as 53BP1, BRCA, AKT. Accumulated evidence showed that P13K, AKT controls the survival signaling pathway and is crucial in developing in drug resistance. This study investigates the global level of all lysine sites of acetyl-H4, its association with p-AKT as well as its prognostic significance in breast cancer. The expression levels of acetyl-H4 were assessed by immunohistochemistry on 102 cases of breast cancer from Queen Mary Hospital in Hong Kong using tissue microarray technology. Nuclear expression of acetyl-H4 was scored and SPSS used for statistical analysis. p-AKT expression data previously obtained in our laboratory (Chen et al.) was also retrieved for correlation with nuclear acetyl-H4 scores. Nuclear acetyl-H4 scores were analyzed for association with a) various clinico-pathological parameters, b) luminal subtypes (ER-and PR-positive), HER-2-positive and triple negative breast cancer, c) p-AKT in breast cancer and d) patient survival by Kaplan-Meier analysis and Cox-regression. By Pearson correlation test, we observed high acetyl-H4 expression was significantly associated with PR-positive breast cancer but not correlated with other clinical parameters and phenotypes of breast cancers (p>0.05). High acetyl-H4 expression was not correlated with p-AKT activity (p=0.84), although it showed inverse correlation with high nuclear p-AKT score. By Kaplan-Meier and Cox-regression analysis, high nuclear acetyl-H4 expression was significantly correlated with poor disease-specific survival in invasive ductal carcinoma (p=0.008 and 0.017 respectively). Multivariate Cox regression analysis confirmed high acetyl-H4 expression (p=0.047) when analyzed together with lymph node involvement (p=0.032) and T-stage (p=0.008) was an independent predictor of poor disease-specific survival in invasive ductal carcinoma. Our results suggest that high acetyl-H4 expression is significantly associated with PR-positive phenotype and lower disease-free survival. The expression of acetyl-H4 was not correlated with p-AKT. Acetyl-H4 may be a potential biomarker to predict poor disease-specific survival for invasive ductal carcinoma. Further investigation is needed for possible use as therapeutic targeting for breast cancer. / published_or_final_version / Pathology / Master / Master of Medical Sciences

Post-translational modifications of histone proteins serve as one of the key epigenetic regulatory mechanisms in the development of organisms. It is well-known that methylation on histone lysine residues is an important epigenetic modification for the transcriptional regulation of normal hematopoiesis and leukemogenesis. JARID1B, a member of the JARID1 histone H3 lysine 4 (H3K4) demethylases, was found essential for the self-renewal of both embryonic stem cell and melanoma stem-like cell, and was involved in regulating genes, such as Egr1, Bmi-1 and p27, during embryo development. In addition, JARID1B is involved in the differentiation of neural cells and macrophages. Although JARID1B is believed to have important functions in stem cell biology, its role in hematopoiesis and leukemogenesis has not been systematically studied. We therefore examined the expression profile of JARID1B in different hematopoietic lineage cells. We observed an up-regulation of JARID1B in differentiated hematopoietic cells by comparing with hematopoietic stem cells and progenitor cells, suggesting that the enhanced cellular level of JARID1B is associated with hematopoietic lineage commitment. Interestingly, JARID1B expression is generally low in human leukemia cell lines and in CML (Chronic Myeloid leukemia) patient samples compared to 〖CD34〗^+ cord blood cells and normal peripheral white blood cells, which indicates the down-regulation of JARID1B is associated with leukemia development. We further modulated the expression of JARID1B in human leukemia cell lines, K562 and SEM, and in mouse hematopoietic stem/progenitor cells (〖Lin〗^-/〖Sca〗^(-1+)/c-〖Kit〗^+, LSK cells). We found that knockdown of Jarid1b in LSK cells did not alter their cell-cycle pattern. However, total colony formation number was reduced in serial re-plating assays, suggesting Jarid1b is required for the maintenance of colony-forming ability and self-renewal property. Knockdown of JARID1B in K562 cells did not change their cell proliferation and cell-cycle pattern, but did consistently inhibit their colony-forming ability during serial re-plating assays. On the other hand, overexpression of JARID1B in K562 and SEM leukemic cells inhibited cell proliferation and colony formation, but with no significant changes on cell-cycle patterns. Furthermore, apoptosis staining did not show any correlations between JARID1B overexpression and apoptosis. Previously, JARID1A, another JARID1 family member, was found as a fusion partner in AML (Acute Myeloid Leukemia); its third PHD domain, which locates at C-terminus, is associated with leukemogenesis. By amino acid sequence alignment, the differences between JARID1A and JARID1B protein are mainly occurred at their C-terminal regions, after the second PHD domain. Therefore, GST fusion protein pull-down experiments for this region was performed. Preliminary results showed that the C-terminus of JARID1B protein interacts with proteins of RNA transcriptional machinery complex. However, further investigation is needed to demonstrate these interactions are directly associated with JARID1B inhibitory effects on gene expression. To conclude, our results suggest that JARID1B plays an essential role in the biology of hematopoietic stem cells and leukemic cells. Investigation on its interacting partners and downstream target genes would lead us a detailed understanding of JARID1B function in hematopoietic cells. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy