36 research outputs found
γ-Globin Reactivation
β-thalassemia and sickle cell disease are major human genetic health problem in many
parts of the world. Available treatments are not satisfactory as none of them exhibit the
optimal combination of safety, efficiency and convenience of use that would make them
applicable to most hemoglobinopathy patients, especially to those who lack access to modern
medical facilities. The observation that induction of γ-globin gene expression ameliorates the
disease phenotype led to the proposal to induce γ-globin gene expression for the treatment of
these diseases. Screening of a small molecule libraries (186000 compounds) has been done
but new reagents with a higher HbF inducing effect and reduced cytotoxicity than those
already known (butyrates, HU) were not found. Despite all the attempts made during the last
30 years, it is still not known how the γ-globin genes are switched off after birth, e.g. whether
it is absence of activating factors or presence of suppressing factors or combination of both
that leads to the down regulation of the γ-globin genes in the adult. There is only very limited
information about the factors that are bound to the γ-globin promoters in vivo, certainly in its
repressed state. It is likely that factors other than those described in the literature (at the
start of this work) are essential for the suppression process. We therefore wanted to develop
new strategies that specifically target fetal gene activation without cytotoxicity, widespread
epigenetic alterations or difficult to manage side effects through the identification of the
relevant transcription factors acting at the promoter.
To this end we designed a strategy to isolate and identify protein factors bound in vivo
to the suppressed human γ-globin gene promoter and to study their effect on γ-globin
regulation (chapter 3). This targeted, in vivo single gene promoter chromatin purification has
been carried out for the first time and is a completely novel strategy. To optimize such a
purification protocol, different parameters have been tested including the use of various
purification tags, reagents, buffers, etcetera (presented in chapter2).
An alternative approach would be to identify target molecules within pathways that are
involved in γ-globin gene expression. These pathways can be studied in the context of high
and low HbF expressing β-thalassemia and sickle cell disease patients (chapter 4). There are
many reports describing the induction of different HbF levels in response to hydroxyurea
treatments in patients. We therefore wanted to understand the mechanism by which HU
induces γ-globin in patients using expression analysis between so-called ‘responder’ and ‘nonresponder’
patients, i.e. between those showing high γ-globin production versus low γ-globin
production. The results of this study are presented in chapter4.
Finally in chapter 5 we present a novel set of genetic markers that is associated with
increased γ-gobin expression in β-thalassemia patients followed by a discussion of future
prospects in Chapter 6
Hydroxyurea responsiveness in β-thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity
β-thalassemia is caused by mutations in the β-globin locus resulting in loss of, or reduced, hemoglobin A (adult hemoglobin, HbA, α2β2) production. Hydroxyurea treatment increases fetal γ-globin (fetal hemoglobin, HbF, α2γ2) expression in postnatal life substituting for the missing adult β-globin and is, therefore, an attractive therapeutic approach. Patients treated with hydroxyurea fall into three categories: i) 'responders' who increase hemoglobin to therapeutic levels; (ii) 'moderate-responders' who increase hemoglobin levels but still need transfusions at longer intervals; and (iii) 'non-responders' who do not reach adequate hemoglobin levels and remain transfusion-dependent. The mechanisms underlying these differential responses remain largely unclear. We generated RNA expression profiles from erythroblast progenitors of 8 responder and 8 non-responder β-thalassemia patients. These profiles revealed that hydroxyurea treatment induced differential expression of many genes in cells from non-responders while it h
Chemical, functional, and structural properties of spent coffee grounds and coffee silverskin
Spent coffee grounds (SCG) and coffee silverskin (CS) represent a great pollution hazard if discharged into the environment. Taking this fact into account, the purpose of this study was to evaluate the chemical composition, functional properties, and structural characteristics of these agro-industrial residues in order to identify the characteristics that allow their reutilization in industrial processes. According to the results, SCG and CS are both of lignocellulosic nature. Sugars polymerized to their cellulose and hemicellulose fractions correspond to 51.5 and 40.45 % w/w, respectively; however, the hemicellulose sugars and their composition significantly differ from one residue to another. SCG and CS particles differ in terms of morphology and crystallinity, but both materials have very low porosity and similar melting point. In terms of functional properties, SCG and CS present good water and oil holding capacities, emulsion activity and stability, and antioxidant potential, being therefore great candidates for use on food and pharmaceutical fields.The authors acknowledge the financial support of the Science and Technology Foundation of Portugal (FCT) through the grant SFRH/BD/80948/2011 and the Strategic Project PEst-OE/EQB/LA0023/2013. The authors also thank the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes", REF. NORTE-07-0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDER. Thanks are also given to Prof. Jose J.M. Orfao, from the Department of Chemical Engineering, Universidade do Porto (Portugal), for his assistance with the porosity analyses
Genetic Drivers of Epigenetic and Transcriptional Variation in Human Immune Cells
Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14 monocytes, CD16 neutrophils, and naive CD4 T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of -genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.This work was predominantly funded by the EU FP7 High Impact Project BLUEPRINT (HEALTH-F5-2011-282510) and the Canadian Institutes of Health Research (CIHR EP1-120608). The research leading to these results has received funding from the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement no 282510 (BLUEPRINT), the European Molecular Biology Laboratory, the Max Planck society, the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’, SEV-2012-0208 and Spanish National Bioinformatics Institute (INB-ISCIII) PT13/0001/0021 co-funded by FEDER "“Una Manera de hacer Europa”. D.G. is supported by a “la Caixa”-Severo Ochoa pre-doctoral fellowship, M.F. was supported by the BHF Cambridge Centre of Excellence [RE/13/6/30180], K.D. is funded as a HSST trainee by NHS Health Education England, S.E. is supported by a fellowship from La Caixa, V.P. is supported by a FEBS long-term fellowship and N.S.'s research is supported by the Wellcome Trust (Grant Codes WT098051 and WT091310), the EU FP7 (EPIGENESYS Grant Code 257082 and BLUEPRINT Grant Code HEALTH-F5-2011-282510) and the NIHR BRC. The Blood and Transplant Unit (BTRU) in Donor Health and Genomics is part of and funded by the National Institute for Health Research (NIHR) and is a partnership between the University of Cambridge and NHS Blood and Transplant (NHSBT) in collaboration with the University of Oxford and the Wellcome Trust Sanger Institute. The T-cell data was produced by the McGill Epigenomics Mapping Centre (EMC McGill). It is funded under the Canadian Epigenetics, Environment, and Health Research Consortium (CEEHRC) by the Canadian Institutes of Health Research and by Genome Quebec (CIHR EP1-120608), with additional support from Genome Canada and FRSQ. T.P. holds a Canada Research Chair
Behaviour of Dietary Fibre Supplements During Bread Dough Development Evaluated Using Novel Farinograph Curve Analysis
Hydroxyurea responsiveness in -thalassemic patients is determined by the stress response adaptation of erythroid progenitors and their differentiation propensity
Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation
Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de) sumoylation in the control of transcriptional activity. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.017194, 1263-1273, 2012
Novel insights in the genomic organization and hotspots of recombination in the human KIR locus through analysis of intergenic regions
The Killer Immunoglobulin-like Receptor (KIR) proteins constitute a family of highly homologous surface receptors involved in the regulation of the innate cytotoxicity of natural killer (NK) cells. Within the human genome, 17 KIR genes are present, many of which show large variation across the population owing to the high number of allelic variants and copy number variation (CNV). KIR genotyping and CNV determination were used to map the KIR locus in a large cohort of >400 Caucasian individuals. Gene order and structure was determined by sequence-specific polymerase chain reaction of the intergenic regions. In this way, we could show that KIR3DL1 and KIR2DS4 gene variants are linked and that--contrary to current views--the gene KIR2DS5 is only present in the telomeric half of the KIR locus. Our study revealed novel insights in the highly organized distribution of KIR genes. Novel recombination hotspots were identified that contribute to the diversity of KIR gene distribution in the Caucasian population. Next-generation sequencing of the KIR intergenic regions allowed for a detailed single-nucleotide polymorphism analysis, which demonstrated several gene-specific as well as haplotype-specific nucleotides for a more accurate genotyping of this notoriously complex gene cluster
Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation
Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de) sumoylation in the control of transcriptional activity. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.017194, 1263-1273, 2012