1,595 research outputs found
The molecular basis of variable phenotypic severity among common missense mutations causing Rett syndrome
Get PDFRett syndrome is caused by mutations in the X-linked MECP2 gene, which encodes a chromosomal protein that binds to methylated DNA. Mouse models mirror the human disorder and therefore allow investigation of phenotypes at a molecular level. We describe an Mecp2 allelic series representing the three most common missense Rett syndrome (RTT) mutations, including first reports of Mecp2[R133C] and Mecp2[T158M] knock-in mice, in addition to Mecp2[R306C] mutant mice. Together these three alleles comprise ∼25% of all RTT mutations in humans, but they vary significantly in average severity. This spectrum is mimicked in the mouse models; R133C being least severe, T158M most severe and R306C of intermediate severity. Both R133C and T158M mutations cause compound phenotypes at the molecular level, combining compromised DNA binding with reduced stability, the destabilizing effect of T158M being more severe. Our findings contradict the hypothesis that the R133C mutation exclusively abolishes binding to hydroxymethylated DNA, as interactions with DNA containing methyl-CG, methyl-CA and hydroxymethyl-CA are all reduced in vivo. We find that MeCP2[T158M] is significantly less stable than MeCP2[R133C], which may account for the divergent clinical impact of the mutations. Overall, this allelic series recapitulates human RTT severity, reveals compound molecular aetiologies and provides a valuable resource in the search for personalized therapeutic interventions.</p
Book Review: Bird Migration: A General Survey
No full textBook Review: Bird Migration: A General Survey (2nd edn)Book Author: P. Berthold.Oxford Ornithology Series, Oxford University Press, Oxford. 2001.Pp. 253. Price £27.50. ISBN 0 19 850786 0 (hardback); 019 850787 9 (paperback
Global analysis of the methyl-CpG binding protein MeCP2
Get PDFMeCP2 was initially identified as an abundant protein in the brain, with an affinity for
methylated DNA in vitro. Interestingly, both deficiency and excess of the protein leads to
severe neurological problems, such as Rett syndrome, which is the result of mutations in the
MECP2 gene. Subsequent transfection experiments showed that MeCP2 can recruit corepressor
complexes and inhibit gene expression in vivo. MeCP2 was therefore thought to
repress specific gene targets and the aetiology of Rett syndrome was proposed to result from
aberrant gene expression in the MeCP2-deficient brain. Although gene expression is
perturbed in the Mecp2-null mouse brain, few specific targets have been verified and
alternative hypotheses for MeCP2 function have been put forward. Previous binding studies
have also failed to clearly identify MeCP2 targets. To shed light on these matters, a novel
technique was generated to isolate neuronal and glial nuclei and established that the amount
of MeCP2 is unexpectedly high in neurons, with an abundance approaching that of the
histone octamer. Chromatin immunoprecipitation experiments on mature mouse brain
showed widespread binding of MeCP2, consistent with its high abundance, tracking the
methyl-CpG density of the genome. MeCP2 deficiency results in global changes in neuronal
chromatin structure, including elevated histone acetylation and a doubling of histone H1.
The mutant brain also shows elevated transcription of repetitive elements, which are
distributed throughout the mouse genome. Based on this data, we propose that MeCP2 binds
genome wide and suppresses spurious transcription through binding in a DNA methylation
dependent manner
Role of CpG island methylation and MBD2 in immune cell gene regulation
Get PDFThe phenomenon of cell type-specific DNA methylation has received much attention
in recent years and a number of DNA methylation differences have been described
between cells of the immune system. Of particular interest when studying DNA
methylation are CpG islands (CGIs) which are distinct from the rest of the genome
due to their elevated CpG content, generally unmethylated state and promoter
association. In the instances when they become methylated this is associated with
gene repression although it is unclear the extent to which differential methylation
corresponds to differential gene expression. I have used an immune system model to
assess the role of CGI methylation and the role of the methylation reader MBD2 in
regulation of gene expression.
A relatively small number of DNA methylation differences were seen between
immune cell types with the most developmentally related cells showing the fewest
methylation differences. Interestingly, the vast majority of CGI-associated cellspecific
methylation occurred at intragenic CGIs located, not at transcription start
sites, but in the gene body. Increased intragenic CGI methylation tended to associate
with gene repression, although the precise reason for this remains unclear. Most
differentially methylated CGIs were depleted for the active chromatin mark
H3K4me3 regardless of their methylation state but some of these were associated
with the silencing mark H3K27me3 when unmethylated. These findings suggest that
intragenic CGIs are a distinct class of genomic element particularly susceptible to
cell type-specific methylation. I also looked at the effect of removing the methyl-
CpG binding domain protein MBD2 from immune system cells. Immune cells from
Mbd2-/- mice showed a number of previously uncharacterised phenotypes as well as
a number of differences in gene expression compared to wild-type animals. Most of
these genes increased their expression in the absence of MBD2 consistent with
MBD2’s role as a transcriptional repressor and Mbd2-/- Th1 cells showed increases
in histone H3 acetylation compared to wild-type Th1 cells. This work provides an
insight into the role played by cell-specific CGI methylation and MBD2 in regulating
gene expression
Electrochemical-control of abrasive polishing and machining rates, U.S. Patent 6,171,467
No full textAn apparatus and method is disclosed; both of which use electrochemistry to selectively grow and remove hard oxide coatings on metals, and capacitive double layers on non-metals and semiconductors in order to predict and control the rate of surface abrasion during planarization of the surface of such materials
Book design: Adrian Bradshaw – The Door Opened: 1980s China
No full textAdrian Bradshaw: Book author
Phil Cleaver: Book design, branding, exhibition design and poster design
Art editor on content and sequencing of photography.
Designed in two languages Chinese and English in a way which combines them both together. Design reflects Chinese culture in a modern way with a western whist.
Embossed slip case with metal badge on cover of book
Defining the protein complement of CpG islands
Get PDFIn higher eukaryotes, the DNA base Cytosine can exist in a variety of modified forms when in the
dinucleotide CpG. Although a methylated form tends to dominate within the genome, approximately
1% of all CpG dinucleotides are found unmodified at high densities spanning around 1Kb and tend to
co-localise to the 5’ ends of around 60% of annotated gene promoters. These unique DNA sequences
are known as CpG islands (CGIs) and their role within the genome to date is largely unknown.
Methylation of CGIs in cancers however has been linked to silencing of associated genes implying a
role in gene regulation. Furthermore these sites are also interesting as they remain specifically nonmodified
within a genome rich in methylated CpG.
We set out to better understand the roles for CGIs through the characterisation of any specific CGI
binding proteins. Digestion of nuclei with methyl sensitive restriction enzymes facilitates the
purification of CGI fragments. Subsequent immunohistochemistry on the CGI chromatin fragments
along with ChIP-PCR over several CGIs revealed an enrichment of the “active” histone modifications
including H3K4me3, a depletion of the “silencing” marks such as H3K27me3, as well as a group of
CGI specific binding factors. These latter proteins contained a domain previously shown to bind to
non-methylated CpG dinucleotides (the CXXC domain) and as such were ideal candidates for CGI
specific factors, in particular a protein called Cfp1. Genome wide sequencing revealed a striking
correlation between Cfp1 and H3K4me3 which were both seen at around 80% of islands.
Furthermore, the presence of Cfp1/H3K4me3 at islands tended to have a negative correlation with the
presence of chromatin rich in the silencing histone modification H3K27me3. Closer investigation of
the Cfp1 protein reveals it to be a true non-methyl CGI binding factor in vivo and shRNA reduction of
Cfp1 levels to around 10% of wild type resulted in a precipitous drop in H3K4me3 levels over CGIs
without a dramatic reduction in global H3K4me3 levels. As Cfp1 has been shown to be part of the
Set1 histone H3K4 methyltransferase complex responsible for this modification, this CXXC protein
may be attracting this histone modifying complex and as such represents a method whereby the
underlying DNA sequence (CpG) can drive the overlying epigenetic state. This study may go some
way to understanding the functional significance of CGIs within the genome
Molecular biology - Methylation talk between histones and DNA
Get PDFThe addition of methyl groups to DNA or histones is a way to directly or indirectly silence gene expression. Although the two events are conceivably connected, they have always been studied separately. In his Perspective, Bird explores the exciting notion (supported by data published elsewhere) that the two events are irrevocably linked, that is, DNA methylation depends on histone methylatio
Establishment of DNA methylation patterns during mouse development
Get PDFMethylation is the only known modification of DNA and in animals it mainly
occurs at cytosines in a CpG context. The pattern of DNA methylation varies among
organisms; some invertebrates are totally devoid of it, while others have densely
methylated regions embedded in an otherwise unmethylated genome. The genome of
mammals on the other hand, is very rich in DNA methylation with the exception of
regions with high CpG frequency, known as CpG islands, that are often found devoid
of methylation. Little is known about the factors that determine the genome-wide
pattern of DNA methylation. Moreover, although there appears to be a specific
developmental program for the establishment of methylation in specific genomic
regions, the molecular events that lead to methylation establishment remain
unknown. The establishment of methylation in the regulatory region of the murine
Oct4 gene as well as the occurrence and establishment of methylation in mouse CpG
islands are investigated in this study.
The promoter of Oct4, which encodes an important developmental regulator,
is known to gain methylation as the gene becomes silenced during early
development. An in vitro model of murine early development has been used to
recapitulate the events that lead to the gene’s silencing. In accordance to other
reports, detailed methylation analysis of the gene’s entire upstream region and
expression analysis showed that DNA methylation establishment follows the gene’s
downregulation. Moreover, establishment of methylation at the Oct4 locus seems to
start from the gene’s proximal enhancer and then spread towards the distal enhancer
and the promoter. Although the initial establishment of methylation in the distal enhancer was not impaired in G9a -/- cells, methylation in these cells was unable to
spread and accumulate. These findings demonstrate that the promoter of the gene is
not the primary target for methylation as previously assumed and give rise to two
possible mechanisms for DNA methylation establishment at this gene; one
possibility is that methylation is actively targeted to the proximal enhancer, while the
other is that the promoter and the distal enhancer are resistant to methylation,
perhaps because of transcription factors bound to them. Moreover, the finding that
G9a is not necessary for DNA methylation establishment but appears to have a role
in methylation spreading, together with observations on the kinetics of the
downregulation and the timing of methylation establishment, allowed the formation
of a possible model for the role of DNA methylation in this gene’s downregulation.
According to this model, DNA methylation acts to accelerate the gene’s
downregulation ensuring its coordinated repression in the developing organism.
For the study of methylation in CpG islands, first a novel algorithm was
applied for the identification of CpG islands in the mouse genome. Approximately
21,000 CpG islands were identified in the mouse genome, half of which localised at
the 5’ of genes, while the majority of the remaining was equally distributed in
intragenic and intergenic regions. Only a very small proportion of the CpG islands
localised at the 3’ of genes. When the gene ontology terms related with the CpG
island-associated genes where interrogated, two main gene functions emerged as
being preferentially associated with CpG islands, development and cell maintenance.
Then, an affinity purification method, together with microarray hybridisation was
applied for the identification of methylated CpG islands from mouse brain.
Approximately 18% of all CpG islands were methylated in brain, with the big
majority localised at 5’ and intragenic regions. When the gene ontology of the
methylated CpG island-associated genes was analysed, developmental but not
housekeeping genes were overrepresented in the methylated fraction. In order to
further investigate the relationship of CpG islands with developmental genes, the
same methodology was applied for the identification of CpG islands that become
methylated after the in vitro induction of differentiation of ES cells. Although this
approach failed to produce genome-wide data, it enforced the idea of a
developmental program for CpG island methylation
Two PERMANOVAs (one per survey period) of bird assemblages at bird baths between bioregions and urban versus rural areas.
No full textTwo PERMANOVAs (one per survey period) of bird assemblages at bird baths between bioregions and urban versus rural areas.</p
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