86 research outputs found
Structure and expression of the human serum amyloid A gene family
Serum amyloid A (SAA) proteins comprise a family of phylogenetically conserved major acute phase reactants. To delineate the structure of the human SAA genes extensive Southern analysis was carried out using SAA probes. Multiple hybridizing fragments were detected with a range of restriction enzymes. Restriction fragment length polymorphisms (RPLPs) were identified for Bg1I, HindIII, NcoI and PstI which were inherited in a Mendelian fashion for the former two enzymes. Three contiguous clones isolated from a human λEMBL3 genomic library spanned 30kb, and contained a single SAA gene predicted to encode apoSAA1β. SAA1β was approximately 4kb long with 4 exons and showed >90% sequence homology to the previously characterized SAA2β in exon, intron and 5'-controlling regions. A fourth λ clone encoded an allele of SAA2β, namely SAA2α with HindIII, NcoI and PstI polymorphic sites. A third novel SAA gene (SAA4) was isolated from a cos202 cosmid library and mapped 10kb downstream of SAA2. These studies demonstrated that the human SAA gene family comprises four discrete loci.
The effects of inflammatory mediators on gene expression were studied following transient transfection of SAACAT reporter genes into HepG2 cells. Similar responses were obtained for SAA1 and SAA2 constructs following treatment with IL1β, IL6 and TNFα. Whereas IL1 acts via the NFκB site in the SAA promoter, IL6 was shown to require both an intact NFκB site and a putative IL6 responsive element (IL6RE) upstream for full induction. In vitro bandshift and interference footprinting studies demonstrated binding of novel nuclear factors from IL6 treated HepG2 cells to both the NFκB region and an NFIL6-like recognition sequence at the IL6RE. IL1 and IL6 activated SAACAT synergistically. Functional studies indicated that this synergism was mediated through the NFκB site and in part through the putative IL6RE
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WWOX: a tumor suppressor gene mutated in multiple cancers
The present invention provides the isolation and cloning of WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3–24.1, a region frequently affected in several cancers. This gene encodes a tumor suppressor with apoptotic functions. The invention provides WWOX nucleic acid- and polypeptide-based cancer therapies. The invention also provides methods for cancer detection, diagnosis and prognosis involving WWOX nucleic acids and polypeptides.Board of Regents, University of Texas Syste
Modern Tools for Genetic Engineering
Site-specific endonucleases create double-strand breaks within the genome and can be targeted to literally any genetic mutation. Together with a repair template, a correction of the defective locus becomes possible. This book offers insight into the modern tools of genome editing, their hurdles and their huge potential. A new era of in vivo genetic engineering has begun
Temporal dynamics of gene expression in the lung in a baboon model of E. coli sepsis
BACKGROUND: Bacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons. RESULTS: Using human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs. CONCLUSION: The analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy
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Ubiquitin E3 ligase mediated regulation of HMG-CoA Reductase
Loss-of-function genetic screens are a powerful approach to identify the genes involved in biological processes. For nearly a century, forward genetic screens in model organisms have provided enormous insight into many cellular processes. However, the difficulty in generating and recovering bi-allelic mutations in diploid cells severely hindered the performance of forward genetic screens in mammalian cells. The development of a retroviral gene-trap vector to mutagenise the human near-haploid KBM7 cell line transformed forward genetic screens in human cells. The re-purposing of the microbial CRISPR/Cas9 system now offers an effective method to generate gene knockouts in diploid cells. Here, I performed a head-to-head comparison of retroviral gene-trap mutagenesis screens and genome-wide CRISPR knockout screens in KBM7 cells. The two screening approaches were equally effective at identifying genes required for the endoplasmic reticulum (ER)-associated degradation of MHC class I molecules.
The ER-resident enzyme HMG-CoA reductase (HMGCR) catalyses the rate-limiting step in the cholesterol biosynthesis pathway and is targeted therapeutically by statins. To maintain cholesterol homeostasis, the expression of HMGCR is tightly regulated by sterols transcriptionally and post-translationally. Sterols induce the association of HMGCR with Insig proteins, which recruit E3 ubiquitin ligase complexes to mediate degradation of HMGCR by the ubiquitin proteasome system. However, the identity of the E3 ligase(s) responsible for HMGCR ubiquitination is controversial. Here, I use a series of genome-wide CRISPR knockout screens using a fluorescently-tagged HMGCR exogenous reporter and an endogenous HMGCR knock-in as an unbiased approach to identify the E3 ligases and any additional components required for HMGCR degradation. The CRISPR screens identified a role for the poorly characterised ERAD E3 ligase RNF145. I found RNF145 to be functionally redundant with gp78, an E3 ligase previously implicated in HMGCR degradation, and the loss of both E3 ligases was required to significantly inhibit the sterol-induced degradation and ubiquitination of HMGCR. A focused E3 ligase CRISPR screen revealed that the combined loss of gp78, RNF145 and Hrd1 was required to completely block the sterol-induced degradation of HMGCR. I present a model to account for this apparent complexity.Wellcome Trus
The role of DNA modifications in pluripotency and differentiation
DNA methylation plays a crucial role in the epigenetic control of gene expression during
mammalian development and differentiation. Whereas the de novo DNA methyltransferases
(Dnmts), Dnmt3a and Dnmt3b, establish DNA methylation patterns during development;
Dnmt1 stably maintains DNA methylation patterns during replication. DNA methylation
patterns change dynamically during development and lineage specification, yet very little is
known about how DNA methylation affects gene expression profiles upon differentiation.
Therefore, we determined genome-wide expression profiles during differentiation of severely
hypomethylated embryonic stem cells (ESCs) lacking either the maintenance enzyme Dnmt1
(dnmt1-/- ESCs) or all three major Dnmts (dnmt1-/-; dnmt3a-/-, dnmt3b-/- or TKO ESCs),
resulting in complete loss of DNA methylation, and assayed their potential to transit in and
out of the ESC state. Our results clearly demonstrate that upon initial differentiation to
embryoid bodies (EBs), wild type, dnmt1-/- and TKO cells are able to activate differentiation
processes. However, transcription profiles of dnmt1-/- and TKO EBs progressively diverge
with prolonged EB culture, with dnmt1-/- EBs being more similar to wild type EBs, indicating a
higher differentiation potential of dnmt1-/- EBs compared to TKO EBs. Remarkably though,
after dissociation of late EBs and further cultivation under pluripotency promoting conditions,
both dnmt1-/- and TKO but not wild type cells rapidly revert to expression profiles typical of
undifferentiated ESCs. Thus, while DNA methylation is dispensable for the initial activation of
differentiation programs, it seems to be crucial for permanently restricting the developmental
fate during differentiation.
Based on the essential role of Uhrf1 in maintenance DNA methylation, we investigated the
structurally highly similar second member of the Uhrf protein family, Uhrf2, whose function in
maintenance methylation or other biological processes is completely unknown. Expression
analysis of uhrf1 and uhrf2 in various cell lines and tissues revealed a time- and
developmental switch in transcript levels of both genes with uhrf1 being highly expressed in
undifferentiated, proliferating cells and uhrf2 being predominately expressed in differentiated,
non-dividing cells. These opposite expression patterns together with no detectable effect on
DNA methylation levels upon knock down of uhrf2 suggests that Uhrf2 is rather involved in
maintaining DNA methylation patterns in differentiated cells and points to non-redundant
functions of both proteins.
The discovery of the “6th base” of the genome, 5-hydroxymethylcytosine (5hmC), resulting
from the oxidation of 5mC by the family of Tet dioxygenases (Tet1-3), once again ignited the
debate about how DNA methylation marks can be modified and removed. To gain insights
into the biological function of this newly identified modification, we developed a sensitive
enzymatic assay for quantification of global 5hmC levels in genomic DNA. Similar to 5mC
levels, we found that also 5hmC levels dynamically change during differentiation of ESCs to
EBs, which correlates with the differential expression of tet1-3. Furthermore, we
characterized a novel endonuclease, PvuRts1I that selectively cleaves 5hmC containing
DNA and show first data on its application as a tool to map and analyze 5hmC patterns in
mammalian genomes.
Finally, we investigated designer transcription activator-like effector (dTALEs) proteins
targeting the oct4 locus. Our results show that the epigenetic state of the target locus
interferes with the ability of dTALEs to activate transcriptionally silent genes, which however
can be overcome using dTALEs in combination with low doses of epigenetic inhibitors.
In conclusion, this work gives further insights into the biological roles of methylation mark
writers (Dnmts), readers (Uhrfs) and modifiers (Tets) and advances our understanding on the
function of DNA methylation in the epigenetic control of gene expression during development
and cellular differentiation
Molecular interactions between childhood acute lymphoblastic leukaemia cells and the bone marrow microenvironment
Acute lymphoblastic leukaemia (ALL) is the most common cause from death of disease in children. Whilst cure rates over the last 30 years have drastically improved, the children that do go on and relapse have a very poor prognosis. Additionally, the ones that do survive can have significant long term side effects from existing treatments. Understanding the molecular mechanisms of the relationship between leukaemia and its microenvironment is essential for the identification of novel targets for treatment and/or the manipulation of existing treatments.
The role that vascular endothelial growth factor (VEGF), an integral component of both neovascularisation and normal haematopoiesis, plays in the progression and invasiveness of solid tumours is well established. However, its function in haematological malignancies has been a more recent and thus less considered observation. Human leukaemia cells secrete VEGF, which may act in a paracrine manner with the bone marrow microenvironment to promote the survival and proliferation of leukaemia cells. In addition to VEGF being produced by leukaemias, it also increases vascularity in the bone marrow and lymph nodes of patients.
Our previous work has established a panel of 10 childhood acute lymphoblastic leukaemia xenografts from patient biopsies in NOD/SCID mice. Several of these secrete VEGF, and express the FMS-like tyrosine kinase-3 (FLT-3). FLT 3, a receptor tyrosine kinase (RTK), and its ligand, play an essential role in regulating normal haematopoiesis. This thesis builds on the previous work by examining the relationship between VEGF and FLT 3, two widely, yet independently studied molecules in leukaemia, with the aberrant expression of either having adverse outcomes for patients.
The results show that the high expression and activation of FLT 3, significantly increases the secretion VEGF. To assess whether VEGF secretion is triggered by FLT-3 signalling, we measured VEGF in the absence and presence of a class III receptor tyrosine kinase (RTK) inhibitor (SU11657), humanised anti-FLT 3 blocking antibodies as well as decreasing the receptors with siRNA. All of these manipulations were able to decrease the secretion of VEGF in leukaemia cells. To further investigate this relationship, we examined the phosphorylation status of FLT-3 and the downstream signalling pathway. Our results indicate that FLT 3 signalling may be an important factor in the induction of VEGF secretion in a sub-type of leukaemia cells and in turn, VEGF secretion can be attenuated by an FLT-3 specific inhibitor. Two separate microarray studies were also used to assess simultaneous gene expressions between the leukaemia and bone marrow microenvironment, and to examine the effects of FL on ALL xenograft cells. The results of the microarray studies confirm the previously observed results regarding the manipulation of the microenvironment by the leukaemic cells.
Inhibition of the FLT-3/VEGF pathway may disrupt paracrine signalling between leukaemia cells and the bone marrow microenvironment, and future studies into how this disruption may influence leukaemia cell responses to conventional chemotherapy are warranted
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