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

Optimal use of tandem biotin and V5 tags in ChIP assays

Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes

Urban consumers\u2019 attitudes and willingness to pay for functional foods in Iran: A case of dietary sugar

Growing concerns for the incidence of incurable diseases and high costs of health care have attracted consumers to functional foods in the world. These foods are characterized with health improvement, lower risk of disease incidence and less health hazards. The present work examined consumers' attitude and willingness to pay for dietary sugar in Rasht city, Iran. The studied sample included 125 citizens of Rasht in spring and summer of 2016 whose size was determined by Mitchell and Carson approach. Results of contingent valuation method on the basis of one-and-one-half-bound choice model revealed that the descriptive variable of bid had negative, statistically significant impact on the acceptance of bid by participants. In addition, the descriptive variables of respondent's age, educational level, family size, monthly income of the family, record of diabetes in family, healthy purchase attitude, and attitude towards the benefits of dietary sugar had positive, significant influence on bid acceptance. Participants expressed their willingness to pay 35.59% extra for dietary sugar as compared to conventional sugar

EZH2-dependent chromatin looping controls INK4a and INK4b, but not ARF, during human progenitor cell differentiation and cellular senescence

<p>Abstract</p> <p>Background</p> <p>The <it>INK4b-ARF-INK4a </it>tumour suppressor locus controls the balance between progenitor cell renewal and cancer. In this study, we investigated how higher-order chromatin structure modulates differential expression of the human <it>INK4b-ARF-INK4a </it>locus during progenitor cell differentiation, cellular ageing and senescence of cancer cells.</p> <p>Results</p> <p>We found that <it>INK4b </it>and <it>INK4a</it>, but not <it>ARF</it>, are upregulated following the differentiation of haematopoietic progenitor cells, in ageing fibroblasts and in senescing malignant rhabdoid tumour cells. To investigate the underlying molecular mechanism we analysed binding of polycomb group (PcG) repressive complexes (PRCs) and the spatial organization of the <it>INK4b-ARF-INK4a </it>locus. In agreement with differential derepression, PcG protein binding across the locus is discontinuous. As we described earlier, PcG repressors bind the INK4a promoter, but not ARF. Here, we identified a second peak of PcG binding that is located ~3 kb upstream of the <it>INK4b </it>promoter. During progenitor cell differentiation and ageing, PcG silencer EZH2 attenuates, causing loss of PRC binding and transcriptional activation of <it>INK4b </it>and <it>INK4a</it>. The expression pattern of the locus is reflected by its organization in space. In the repressed state, the PRC-binding regions are in close proximity, while the intervening chromatin harbouring <it>ARF </it>loops out. Down regulation of EZH2 causes release of the ~35 kb repressive chromatin loop and induction of both <it>INK4a </it>and <it>INK4b</it>, whereas <it>ARF </it>expression remains unaltered.</p> <p>Conclusion</p> <p>PcG silencers bind and coordinately regulate <it>INK4b </it>and <it>INK4a</it>, but not <it>ARF</it>, during a variety of physiological processes. Developmentally regulated EZH2 levels are one of the factors that can determine the higher order chromatin structure and expression pattern of the <it>INK4b-ARF-INK4a </it>locus, coupling human progenitor cell differentiation to proliferation control. Our results revealed a chromatin looping mechanism of long-range control and argue against models involving homogeneous spreading of PcG silencers across the <it>INK4b-ARF-INK4a </it>locus.</p

Optimal use of tandem biotin and V5 tags in ChIP assays

Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes