Characterization of Kpni Interspersed, Repetitive DNA Sequence Families and Their Association With the Nuclear Matrix

The KpnI, 1.2 and 1.5 kb families of interspersed repetitive DNAs from the African green monkey genome were isolated and characterized. Each family contains three populations of segments based on their sequence lengths and susceptibility to cleavage by the restriction enzymes KpnI and RsaI. The first population contains the smallest segments which are susceptible to both KpnI and RsaI cleavage and have fragment lengths of 1.2 kb (1.2 kb family) and 1.5 kb (1.5 kb family) respectively. The members in this population are referred to as KpnI-sensitive segments. The second population contains longer segments (\u3e 2 kb) which represent fusions of members from different families. The fusion sequences are cleaved by KpnI at their termini but lack internal KpnI sites at the junctions that join the individual component members. The third population contains members from each family that are cleaved occasionally by KpnI (KpnI-resistant segments) and remained linked to the bulk of the high molecular weight DNA. KpnI 1.2 kb, 1.5 kb and KpnI-resistant populations were isolated and analyzed for the presence of internal RsaI sites. All members from both populations were cleaved by RsaI into a simple series of low molecular weight fragments. Some members from both the KpnI-sensitive and the KpnI-resistant populations were found to contain internal RsaI sites. Other members from both populations lacked internal RsaI sites. Genomic KpnI 1.2 kb segments were cloned and two recombinants pBK(1.2)14 and pBK(1.2)39 identified. The partial nucleotide sequence of clone Kpn(1.2)14 was determined. The sequence content of KpnI 1.2 and 1.5 kb families in DNA fragments that anchor DNA loops to the nuclear matrix (att-DNA) was also studied. The relative sequence content of both 1.2 and 1.5 kb families was found to be impoverished when compared to their content in total nuclear DNA. However, members in each family were found to be present in detectable amounts. The association of KpnI 1.2 and 1.5 kb family sequences with the nuclear matrix was also demonstrated by metrizamide gradient centrifugation of nuclear matrix complexes. The results suggest that some KpnI 1.2 and 1.5 kb segments are differentially associated with nuclear proteins

Nature and modulation of the higher order chromatin fibre

The bulk of eukaryotic cellular DNA is compacted into chromatin, a nucleoprotein complex that is responsible for packaging DNA into the nucleus and regulating gene transcription. The chromatin fibre is a dynamic structure which is able to accommodate the many complex processes which occur simultaneously in a living cell. The fundamental building blocks of the lower order chromatin fibre have been studied extensively, providing us with a detailed understanding of the structures present; much is known about how these structures are modulated to allow processes like gene transcription and replication to occur in an organised fashion. In contrast to our detailed knowledge of the fundamental building blocks of chromatin, little is known about the conformation of the higher order chromatin fibre. This lack of understanding is due, predominantly, to the inaccessibility of the higher order fibre for study, and that much of the research to date has considered the conformation of the higher order fibre to be uniform. In this project I have analysed and modulated the higher order chromatin fibre to assess the role this fibre plays in the regulation of cellular processes.The conformation of the higher order chromatin fibre is often thought to change during the differentiation of cells. To study this alteration in conformation I have undertaken a detailed analysis of the higher order chromatin fibre from cells with different differentiation potentials and during their differentiation process. Using a hydrodynamic sedimentation approach to assess the conformation of the chromatin fibre I was unable to find any differences in its conformation. However, I have found that these chromatin fibres do have inherent differences in their nuclease sensitivities, suggesting that although the overall conformation of the fibres are similar, there are chromatin- related differences between cells with various differentiation potentials.To study the uniformity of the higher order chromatin fibre at different chromosomal locations I have analysed the chromatin structure found at centromeres to determine whether there is an alteration in the conformation of the chromatin fibre, which might affect the function of centromeres. My results clearly show that in mouse and human cells the chromatin fibre found at inner centromeric regions is more compact than the chromatin fibre present at outer centromeric regions, and in turn this is more compact than the bulk chromatin fibre. To determine the molecular basis for this, I have analysed acetylated centromeric heterochromatin from embryonic stem (ES) cells and heterochromatin associated with undermethylated centromeric DNA from F9 cells. My results demonstrate that this special chromatin architecture found at centromeres appears to be independent of histone acetylation and DNA methylation.To establish whether an alteration in the chromatin conformation will alter a cell's differentiation potential I have expressed histone H5, a replacement linker histone normally found in nucleated erythrocytes, in pluripotential ES cells. My results show that the constitutive expression of H5 in ES cells causes substantial cell death. I have therefore constructed a regulated, tetracycline based, histone H5 expression system in ES cells, but I was unable to express H5 in a controlled manner to investigate the underlying chromatin structure of these cells. In addition, I expressed histone H5 DNA -binding mutants in ES cells which also caused substantial cell death. I was therefore unable to determine whether the cellular phenotype obtained from expressing H5 in ES cells was due to an alteration in chromatin structure or a non- specific effect from expressing a positively charged molecule. As a first step towards studying the expression of linker histones in living cells and during development, I constructed and analysed a green fluorescent protein (GFP)- histone H5 fusion. As for histone H5, the GFP -H5 fusion protein is correctly expressed in a variety of cell types, but is lethal to cells when expressed at high levels for longer periods of time

Poly(glutamic acid) promoted assembly of nucleosome cores on the histone gene quintet of psammechinus miliaris

Bibliography: leaves 194-217.This thesis investigates whether DNA and histones contain sufficient information to direct nucleosome cores into specific positions. The "in vitro" assembly of nucleosome cores promoted by poly(glutamic acid) has been optimized with respect to rate and yield. This was achieved by paying attention to the purity of the core constituents and in particular by the use of histones in their octameric form. The suitability of a number of octamer purification protocols, to produce pure undenatured histone octamers, has been investigated and the methodology improved. The particles assembled on random DNA have been found to be indistinguishable from native nucleosome cores by the following criteria: Their S value on sucrose gradient centrifugation, resistance to Micrococcal nuclease digestion, DNase I digestion patterns, DNase I digestion kinetics at the susceptible sites, electronmicroscopic appearance, hi stone content and electrophoretic mobility. Cores were also assembled on unique DNA, namely the intact h22 histone quintet of Psammechinus miliaris. Low resolution mapping, by indirect endlabelling of polycores assembled on the quintet, did not reveal any preferred sites of assembly. To investigate the core associated DNA at single base pair resolution, a series of fragments, excised from the H2A-Hl and the Hl-H4 spacer areas, were inserted into pGV403 plasmids. These plasmids can be strand specifically end-labelled with the Klenow fragment at the two different Tth 111 I excision sites utilised to isolate the propagated insert. On the free linearised DNA a complex digestion pattern is produced due to the sequence specificities of Micrococcal nuclease and DNase I. When cores are assembled on this DNA the digestion pattern is changed. This pattern reveals two preferential frames of assembly and indicates that in the remainder of the fragments cores are assembled, randomly, or in a number of overlapping frames. It is concluded that the DNA fragments investigated and the hi stone octamer contain enough structural information to influence the positions occupied by some nucleosome cores. The implications of these findings are discussed

Nucleosomal organisation over the ovine β-lactoglobulin gene

The genetic material of all higher organisms from yeast to mammals is organised in the cell nucleus as a nucleoprotein complex called chromatin. The fundamental repeating unit of chromatin, which covers nearly the entire DNA, is the nucleosome. Each one comprises eight highly conserved protein subunits that sequester approximately 146bp of DNA. Nucleosomes facilitate the highly condensed packaging of DNA, most obvious in metaphase chromosomes, and also permit non-histone protein factors access to the DNA in order to facilitate DNA replication, transcription and repair.For temporally and spatially specific gene activation to occur, chromatin remodeling factors, transcription factors and RNA polymerase and its associated factors must act in concert with the underlying nucleosome environment to effect transcription. In some instances, this has shown to be a complex relationship. Nucleosomes are stably positioned over transcription factor binding sites in some genes. This can prevent access and therefore repress gene activation. In other genes, a positioned nucleosome is required to wrap up DNA between separate transcription factor binding sites. Bringing the sites together allows the binding factors to act cooperatively in initiating transcription. Therefore, nucleosomes that are positioned over a specific DNA sequence can have an instrumental role in gene regulation.To date, there have only been limited studies on the nucleosomal organisation of genes in their natural environment. The majority of these studies have concentrated on short regions of positioned nucleosomes spanning either repetitive DNA or the promoter regions of specific genes. However, nucleosome positioning over an entire gene domain may have a significant impact on its regulation and compaction. I have mapped the nucleosomal organisation over lOkb of a tissue specific, temporally regulated gene using the enzymatic probe, micrococcal nuclease and the chemical probe, cuprous phenanthroline. The ovine p-lactoglobulin (BLG) gene studied has a well characterised developmental profile, a minimal transcriptional domain and has been used extensively as an expression cassette in transgenic animals to drive heterologous gene transcriptionWhen the gene is inactive, in the liver, it displays a tightly defined array of positioned nucleosomes that modulate between two specific phases over the gene domain. A similar, less tightly defined array is present when the gene is active, in the mammary gland, except over the promoter and actively transcribing regions. The same arrays arc present over the BLG promoter region in transgenic mice in both active and inactive states. A monomer extension reaction provides in vitro evidence of the positioning signals that are determined by DNA sequence alone. These show an interesting correlation with the in vivo results.A number of other milk protein genes have a similar pattern of key transcription factor binding sites over their promoter regions. If the nucleosome positions were conserved in these genes, with respect to these binding sites, it might suggest a role for positioned nucleosomes in their regulation. A total of three genes, each in two different organisms, have been analysed to test for a correlation

Human centromere repositioning activates transcription and opens chromatin fibre structure

Human centromeres appear as constrictions on mitotic chromosomes and form a platform for kinetochore assembly in mitosis. Biophysical experiments led to a suggestion that repetitive DNA at centromeric regions form a compact scaffold necessary for function, but this was revised when neocentromeres were discovered on non-repetitive DNA. To test whether centromeres have a special chromatin structure we have analysed the architecture of a neocentromere. Centromere repositioning is accompanied by RNA polymerase II recruitment and active transcription to form a decompacted, negatively supercoiled domain enriched in ‘open’ chromatin fibres. In contrast, centromerisation causes a spreading of repressive epigenetic marks to surrounding regions, delimited by H3K27me3 polycomb boundaries and divergent genes. This flanking domain is transcriptionally silent and partially remodelled to form ‘compact’ chromatin, similar to satellite-containing DNA sequences, and exhibits genomic instability. We suggest transcription disrupts chromatin to provide a foundation for kinetochore formation whilst compact pericentromeric heterochromatin generates mechanical rigidity