551 research outputs found
Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure
We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken α-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the α-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a âguestâ compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited
Relationship of the Surface Structure of Metaphase Chromosomes to the Higher Order Organization of Chromatin Fibers
Scanning electron microscopy (SEM), as well as transmission electron microscopy (TEM), has been utilized to determine how the surface structure of mitotic chromosomes is related to the organization of the 30 nm chromosomal fibers. SEM revealed the surfaces of isolated, HeLa cell chromosomes to possess a knobby substructure with chromosomes prepared for EM in buffers containing 0.5-1.5 mM Mg2+. These projections had substantially greater widths (65-70 nm) than the underlying chromatin fibers. Reducing the Mg ion concentration to 0.05-0.15 mM resulted in the further expansion of the chromosomes, which flattened the chromosomes for SEM so the fibers became the dominant feature of the micrographs. The surface protuberances are interpreted as representing the peripheral tips of radial chromatin loops.
The same procedure of slightly expanding chromosomes by decreasing the Mg2+ concentration in resuspension buffer was also utilized in a TEM, serial sectioning study. Longitudinal sections close to the central chromatid axis showed radially oriented fibers within the planes of the sections. This was replaced by a dot pattern when the longitudinal sections grazed the periphery of the chromatid. Transverse sections displayed more clearly the radial orientation of the fibers.
A consistent picture emerges from applying SEM and TEM that supports the radial loop model for the primary mode of organization of chromatin fibers in metaphase chromosomes
Mechanisms of Regulatory Adaptation in the Evolving Genome
The development from a single cell into a complex organism requires the precise control of gene expression in space and time. To achieve this, the activity of genes is governed by large regulatory chromatin landscapes that when disrupted can cause gene mis-regulation and disease. However, at the same time, the successful modification of these landscapes is thought to be a major driver of phenotypic innovation during evolution. Given the vulnerability of these landscapes in disease settings, it remains largely unknown how their integrity is maintained when novel genes are âsafelyâ incorporated during evolution, which is addressed in this work.
Specifically, here, multiple mechanisms are dissected that adapted the Fat1 regulatory landscape to maintain its integrity while simultaneously incorporating a novel gene, Zfp42, during evolution. First, comparative evolutionary genomics was used to reconstruct the history of the locus (section 1). Second, the three-dimensional chromatin configuration of the locus was examined in relationship to the gene activities using genomics-technologies (HiC, DamID) combined with super resolution microscopy and in silico modeling (section 2). Finally, the mechanisms that adapted the landscape in ESCs (section 3) and embryonic limbs (section 4) for the emergence of Zfp42 were investigated using genome engineering and genomics.
Two tissue-specific mechanisms were identified that enabled the independent activities of Zfp42 and Fat1 despite sharing the same regulatory chromatin landscape: In ESCs, the landscape physically restructures and isolates the genes together with their regulatory information, from one another, thereby allowing their independent regulation. Surprisingly, this restructuring is not driven by the most recognized chromatin structuring force, loop extrusion, but rather by the underlying epigenetic state of chromatin. A different mechanism operates in embryonic mouse limbs where both genes are exposed to the same regulatory information driving Fat1 activation, but surprisingly not Zfp42. The inactivity of Zfp42 cannot be explained by nuclear envelopment attachment nor by enhancer-promoter specificity. Instead, Zfp42 is kept inactive by a highly context-dependent silencing mechanism driven by DNA methylation. As such, Zfp42 is ectopically active and responsive to the surrounding regulatory information when DNA methylation is removed or when the gene is slightly repositioned within its domain.
Combined, we find that 3D-restructuring and context-dependent silencing adapted the Fat1 landscape to integrate Zfp42. More generally, this demonstrates that even single regulatory landscapes harbor an enormous regulatory complexity and, thus can accommodate multiple independently regulated genes. We believe that this has significant consequences for human genetics where similar genomic alterations do not drive disease in patients. This is possible, because additional, yet still unknown, mechanisms control how regulatory information is used in the genome
Effects of trypsin on cellular, chromosomal and DNA damage induced by X-rays
When cells are trypsinized before irradiation, potentiation of
cell killing is seen; this is known as the 'trypsin effect'. The
trypsin effect is re-examined here in the light of experiments in which
enzymatic modifications of DNA in permeabilized cells has become a
powerful experimental tool (Bryant et al, 1978, Ahnstrom and
Bryant,1982; Natarajan et al, 1980; Bryant, 1984, 1985; Natarajan and
Obe, 1984) and where in some cases it is suspected that trypsinization
as part of the technique could significantly alter cell membrane
permeability and chromatin structure (Obe et al, 1985; Obe and Winkel,
1985; Bryant and Christie, 1989).
The trypsin effect was investigated at various cellular levels,
assaying for cell survival (to verify the potentiation), anaphase
chromosomal aberrations, DNA damage and repair and lastly using a
nucleoid assay to investigate the effect of trypsin on DNA-nuclear
matrix interactions. Each of these are considered in separate chapters
as individual studies, then all compared in the final discussion.
A small potentiation effect of X-ray damage on cell killing was
seen when using Chinese Hamster Ovary (CHO) cells but no potentiating
effect was found in the murine Ehrlich ascites tumour (EAT) cell line.
Trypsinization was found to increase the number of X-ray induced
chromosomal anaphase abnormalities in EAT cells.
To investigate the possibility that the basis of the trypsin
effect lies in its action at the DNA level, further experiments were
performed to monitor DNA damage and repair using the DNA unwinding and
neutral elution techniques. No difference was seen in the unwinding
kinetics or in the DNA unwinding dose-effect curves for induction of DNA
single strand breakage (ssb); when using neutral elution however, treatment of cells with trypsin or buffer alone increased the incidence
of X-ray induced double strand breaks (dsb) at higher doses.
Trypsinized EAT cells were found to repair ssb after 12 Gy less
rapidly than those treated with buffer (EDTA) indicating an inhibitory
effect of trypsin on repair. A progressive decrease in repair capacity
with increase in time of trypsin treatment was seen.
The dsb repair kinetics as measured by the DNA unwinding technique
after 50 Gy showed that either trypsin of buffer (EDTA) alone reduced
the dsb repair rate, no difference between their repair kinetics being
evident (this was also seen with neutral elution repair after 40 Gy).
This indicates that the EDTA/buffer solution in which the trypsin is
dissolved may also be contributing to the trypsin effect.
A new nucleoid assay was developed and used to investigate the
effect of prolonged trypsinization and electroporation on nucleoid
morphology.
The results confirm that routine trypsinization of cells enhances
X-ray induced cell killing in some cell lines. It is postulated that
this may occur by reducing the repair capacity of the cells rather than
by increasing the amount of damage initially caused
Responsiveness of genes to long-range transcriptional regulation
Developmental genes are highly regulated at the level of transcription and exhibit complex spatial and temporal expression patterns. Key developmental loci are frequently spanned by clusters of conserved non-coding elements (CNEs), referred to as genomic regulatory blocks (GRBs), that have been subject to extreme levels of purifying selection during metazoan evolution. CNEs have been shown to function as long-range enhancers, activating transcription of their developmental target genes over vast genomic distances and bypassing more proximally located unresponsive genes (bystanders). Despite their role in the establishment of cell identity during development, many of these long-range regulatory landscapes remain poorly characterised.
In this thesis, I develop a computational method for the genome-wide identification of regulatory enhancer-promoter associations in human and mouse, based on co-variation of enhancer and promoter transcriptional activity across a comprehensive set of tissues and cell types, in combination with chromatin contact data. Using this method, I demonstrate that previously predicted GRB target genes are amongst the genes with the highest level of enhancer responsiveness in the genome, and are frequently associated with extremely long-range
enhancers. Remarkably, the activity of some previously predicted bystanders is also weakly but significantly associated with enhancer activity, challenging the notion that the promoters of bystanders are unresponsive to enhancers. Next, I systematically annotate human genes with elevated enhancer responsiveness and identify more than 600 putative target genes, associated with the regulation of a wide range of developmental processes, from pattern specification to axonogenesis, as well as with disease. The analysis performed in this thesis has facilitated the identification of hundreds of previously uncharacterised enhancer-responsive genes and their long-range regulatory landscapes, allowing the study of their unique properties.Open Acces
Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging
Both multi-cell biochemical assays and single cell fluorescence measurements
have revealed that the elongation rate of Polymerase II (PolII) in eukaryotes
varies largely across different cell types and genes. However, there is not yet
a consensus whether intrinsic factors such as the position, local mobility or
the engagement by an active molecular mechanism of a genetic locus could be the
determinants of the observed heterogeneity. Employing high-speed 3D
fluorescence nanoimaging we resolve here at the single cell level multiple,
distinct regions of mRNA synthesis within a labeled transgene array. By
employing phasor analysis, a fluorescence fluctuation spectroscopy technique,
we demonstrate that these regions are active transcription sites that release
mRNA molecules in the nucleoplasm, and we extract the local PolII elongation
rate. While we detect a range of 10-100 bp/s for PolII elongation from cell to
cell, we are now also able to measure up to a four-fold variation in the
average elongation between identical copies of the same gene measured
simultaneously within the same cell. Furthermore, we are able to visualize
changes of PolII elongation as a function of time. We observe a correlation
between the average elongation rate measured in a locus and its local mobility.
Finally, by cross-correlating the transcriptional activity with the nm-sized
movements of the active loci, we provide evidence of an active molecular
mechanism determining displacements of the transcription sites concomitant to
increases in transcriptional activity. Together these observations demonstrate
that local factors, such as chromatin local mobility and the microenvironment
of the transcription site, are an important source of transcription kinetics
variability.Comment: 56 pages, 5 main figures and 10 supplementary figure
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