Cell-type-specific long-range looping interactions identify distant regulatory elements of the CFTR gene

Identification of regulatory elements and their target genes is complicated by the fact that regulatory elements can act over large genomic distances. Identification of long-range acting elements is particularly important in the case of disease genes as mutations in these elements can result in human disease. It is becoming increasingly clear that long-range control of gene expression is facilitated by chromatin looping interactions. These interactions can be detected by chromosome conformation capture (3C). Here, we employed 3C as a discovery tool for identification of long-range regulatory elements that control the cystic fibrosis transmembrane conductance regulator gene, CFTR. We identified four elements in a 460-kb region around the locus that loop specifically to the CFTR promoter exclusively in CFTR expressing cells. The elements are located 20 and 80 kb upstream; and 109 and 203 kb downstream of the CFTR promoter. These elements contain DNase I hypersensitive sites and histone modification patterns characteristic of enhancers. The elements also interact with each other and the latter two activate the CFTR promoter synergistically in reporter assays. Our results reveal novel long-range acting elements that control expression of CFTR and suggest that 3C-based approaches can be used for discovery of novel regulatory elements

Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease

The worldwide burden of sickle cell disease is enormous, with over 200,000 infants born with the disease each year in Africa alone. Induction of fetal hemoglobin is a validated strategy to improve symptoms and complications of this disease. The development of targeted therapies has been limited by the absence of discrete druggable targets. We developed a unique bead-based strategy for the identification of inducers of fetal hemoglobin transcripts in primary human erythroid cells. A small-molecule screen of bioactive compounds identified remarkable class-associated activity among histone deacetylase (HDAC) inhibitors. Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease

Phenotypic Consequences of Copy Number Variation: Insights from Smith-Magenis and Potocki-Lupski Syndrome Mouse Models

The characterization of mice with different number of copies of the same genomic segment shows that structural changes influence the phenotypic outcome independently of gene dosage

Honey as a Source of Natural Antioxidants

146 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2002.Dietary antioxidants might prevent oxidative damage to tissues and therefore protect against chronic diseases. Previous research demonstrated that honeys from various floral sources exhibit significant antioxidant activity, rendering honey effective as a food antioxidant. The objective of the research presented in this dissertation was to extend the knowledge of the protective antioxidant effects of honey in food systems and in in vitro assays into determining the potential of honey as a dietary source of antioxidants. In vitro antioxidant activity of honey as measured by the oxygen radical absorbance capacity (ORAC) assay, was found to be similar to the antioxidant activity of many fruits and vegetables on a fresh weight basis. The ORAC values of honeys from various floral sources were significantly correlated with the in vitro inhibitory effect of honeys on copper-catalyzed serum lipoprotein oxidation. Wide variation in antioxidant activity was observed among honeys from different floral sources, with the darker colored honeys having higher antioxidant capacity. Specific honey components responsible for the observed antioxidant capacity were identified and quantified, including phenolics, proteins, gluconic acid, enzymes, hydroxymethylfuraldehyde and ascorbic acid. A linear correlation was observed between ORAC activity of the investigated honeys and their phenolic and protein content. In addition, the first evidence for an ex vivo antioxidant effect of honey was established in a human intervention study. Consumption of water with buckwheat honey significantly increased deproteinized serum antioxidant capacity relative to water consumption. A hypercholesterolemic rabbit model was used to further elucidate the in vivo effect of soy and buckwheat honey consumption on atherosclerosis. Feeding the rabbits soy or buckwheat honey in addition to cholesterol did not significantly change their serum lipid profiles, serum antioxidant capacity or atherosclerotic plaque formation as compared to feeding the rabbits cholesterol alone. These studies are the first to examine the biological potential of honey as a dietary antioxidant. Now it becomes crucial to study factors such as bioavailability and tissue distribution of honey phenolics and other components to fully understand its biological activity in vivo before incorporation of honey as a source of dietary antioxidants into the human food supply can be supported.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

The active FMR1 promoter is associated with a large domain of altered chromatin conformation with embedded local histone modifications

We have analyzed the effects of gene activation on chromatin conformation throughout an ≈170-kb region comprising the human fragile X locus, which includes a single expressed gene, FMR1 (fragile X mental retardation 1). We have applied three approaches: (i) chromosome conformation capture, which assesses relative interaction frequencies of chromatin segments; (ii) an extension of this approach that identifies domains whose conformation differs from the average, which we developed and named chromosome conformation profiling; and (iii) ChIP analysis of histone modifications. We find that, in normal cells where FMR1 is active, the FMR1 promoter is at the center of a large (≈50 kb) domain of reduced intersegment interactions. In contrast, in fragile X cells where FMR1 is inactive, chromatin conformation is uniform across the entire region. We also find that histone modifications that are characteristic of active genes occur tightly localized around the FMR1 promoter in normal cells and are absent in fragile X cells. Therefore, the expression-correlated change in conformation affects a significantly larger domain than that marked by histone modifications. Domain-wide changes in interaction probability could reflect increased chromatin expansion and may also be related to an altered spatial disposition that results in increased intermingling with unrelated loci. The described approaches are widely applicable to the study of conformational changes of any locus of interest

Sample preparation for small RNA massive parallel sequencing

High-throughput sequencing has allowed for a comprehensive small RNA (sRNA) expression analysis of numerous tissues in a diverse set of organisms. The computational analysis of the millions of generated sequencing reads has led to the discovery of novel miRNAs and other sRNA species, and resulted in a better understanding of the roles these sRNAs play in development and disease. This chapter describes the generation of sRNA deep-sequencing libraries for the Illumina massively parallel sequencing platform by using a cloning method that anneals specific RNA sequences to the 5′-and 3′-ends of the sRNA molecules

Mapping chromatin interactions by chromosome conformation capture

Chromosome conformation capture (3C) is one of the only techniques that allows for analysis of an intermediate level of chromosome structure ranging from a few to hundreds of kilobases, a level most relevant for gene regulation. The 3C technique is used to detect physical interactions between sequence elements that are located on the same or on different chromosomes. For instance, physical interactions between distant enhancers and target genes can be measured. The 3C assay uses formaldehyde cross-linking to trap connections between chromatin segments that can, after a number of manipulations, be detected by PCR. This unit describes detailed protocols for performing 3C with yeast Saccharomyces cerevisiae and mammalian cells