No description available.

Chromatin is a complex of genomic DNA, RNA, and associated proteins. Many of the processes that occur on chromatin regulate the accessibility of the genetic material of a cell. The nucleosome is the basic subunit of chromatin, composed of a histone octamer wrapped with approximately 150bp of DNA. Alterations to chromatin structure, including to nucleosomes and their location, underlie global transcriptional diversity. A striking example of this is the so-called "open" chromatin state in pluripotent cells, characterized by loosely bound chromatin proteins and rapid nucleosome turnover, that allows transcriptional flexibility for subsequent differentiation. In contrast, differentiated cells contain compacted chromatin that can selectively block access to DNA and subsequent transcription. Thus, characterizing the physical state of chromatin is important to understanding its regulatory state. Digestion of chromatin with micrococcal nuclease (MNase) and subsequent sequencing of the protected DNA fragments produces a map of nucleosome occupancy. Traditional MNase mapping experiments capture a snapshot of nucleosome occupancy, providing information about nucleosomes that are accessible at the level of digestion used. We analyzed regions of difference in nucleosome occupancy between embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and differentiated cell types using traditional MNase-seq and found that differences in pluripotent and differentiated cells are punctate and correlate with regulatory regions important for pluripotency and development. Further, our analysis shows ESCs and iPSCs to be vastly more similar to each other in their chromatin structure than to the differentiated cells. We then developed a new way of collecting and analyzing MNase-seq data that allows us to determine both nucleosome occupancy as well as the accessibility of DNA to regulatory factors. Our methodology discerns distinct physical states of chromatin and provides novel insights into the accessibility of regulatory regions. Additionally, we present a quantitative metric useful for characterizing local and global regions of the genome that should be useful in future cell type comparisons.

Marked by a defect in the production of the Beta (β)-globin chain that make-up hemoglobin, Beta (β)-thalassemia is the most prevalent form of inherited single-gene disorders in the world. To understand the molecular mechanisms that govern the expression of the β-globin polypeptide encoded by the β-globin locus, we examined closely the enzymes involved in the epigenetic regulation of gene expression through histone 3 lysine 9 mono and di-methylation (H3K9 me1/2). G9a-like protein (GLP), a mammalian methyltransferase involved in the establishment and maintenance of H3K9 me1/2 mark at euchromatin, regions was found to be critical for the full activation of the adult β-globin genes in vivo during Murine erythroleukemia cell line (MEL) differentiation. Though it was initially hypothesized that GLP binding to H3K9 me1/2 mark through its Ankyrin domain was key to its activating function, we found that Flag- GLP ankyrin mutants E817R and W791A unable to bind to the methyl mark, are able to activate β-globin genes as well as their wild-type counterpart. Additionally, this study found that the embryonic gene εγ, known to be re-activated after G9a KD at the mRNA level, was effectively transcribed at the protein level using Triton Urea Acetic acid (TAU) western blots, thereby identifying potential therapeutic applications for treatment for β-thalassemia patients.

During spermatogenesis in rainbow trout, a synchronous development of stem cells → spermatocytes → spermatids → mature sperm occurs, with replacement of the histones in chromatin by protamines. PART A: Histone methylation. Along with histone acetylation and phosphorylation, methylation of specific lysyl residues of histones H3 and H4 can be observed. This histone methylation occurs predominantly in the large diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably has no role in the replacement of histones by protamine. Other labelling experiments suggest that histone H4 methylation is a late event in the cell cycle, occurring after the synthesis, acetylation and deacetylation of histone H4. This methylation may be necessary for histone phosphorylation or chromatin condensation prior to cell division. PART B: Chromatin subunit structure. When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients, or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11S (monomer), 16S (dimer), 22S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (>40 mM NaCl) or divalent cations (≃5 mM) cause subunit precipitation. Chromatin subunits have a protein:DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone H6. There are however no detectable nonhistone chromosomal proteins. Mg⁺⁺ precipitates of the 11S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg⁺⁺ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11S monomers, which are deficient in histone Hi. This histone may crosslink the larger oligomers, resulting in a disordered Mg⁺⁺ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions, and crosslinked by histone Hi. Testis consisting predominantly of early spermatids (meiotic tissue, containing mainly nucleohistone) gives similar yields of DNA fragments and 11S subunits. Later stage testis (protamine has replaced the histones) however, gives no DNA fragments or 11S subunits. This presumably reflects large differences in structure between nucleoprotamine and nucleohistone. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

No description available.

No description available.

Chromatin diminution is defined as the elimination of DNA during the differentiation of early embryonic cells into pre-somatic cells. While it was first observed in the nematode Parascaris equorum, it also been identified in other parasitic nematodes, hagfish and copepods. In the copepod Mesocyclops edax, up to 90% of genomic DNA is eliminated during chromatin diminution. It was previously shown that the eliminated DNA contained highly repetitive heterochromatic sequences. Here, we digested pre- and post-diminution DNA with BamHI and produced small libraries of clones from each. Analyses revealed no decrease in low copy numbered sequences, such as transposable elements. Rather, both libraries are found to be surprisingly similar in all aspects analysed. Further comparison also demonstrated similarity of our libraries with the DNA sequences eliminated from Cyclops kolensis. Consequently, we suggest that M. edax eliminates portions of euchromatic DNA, in addition to the previously characterized satellite sequences.

Chromatin diminution is defined as the elimination of DNA during the differentiation of early embryonic cells into pre-somatic cells. While it was first observed in the nematode Parascaris equorum, it also been identified in other parasitic nematodes, hagfish and copepods. In the copepod Mesocyclops edax, up to 90% of genomic DNA is eliminated during chromatin diminution. It was previously shown that the eliminated DNA contained highly repetitive heterochromatic sequences. Here, we digested pre- and post-diminution DNA with BamHI and produced small libraries of clones from each. Analyses revealed no decrease in low copy numbered sequences, such as transposable elements. Rather, both libraries are found to be surprisingly similar in all aspects analysed. Further comparison also demonstrated similarity of our libraries with the DNA sequences eliminated from Cyclops kolensis. Consequently, we suggest that M. edax eliminates portions of euchromatic DNA, in addition to the previously characterized satellite sequences.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2006. / Härtill 5 uppsatser.