Rebuilding a superenhancer to investigate the additive and synergistic effects of its individual components

Superenhancers are clusters of putative enhancers, densely occupied by histone 3 lysine 27 acetylation, transcription factors, and coactivators such as the mediator complex. There is a tendency for superenhancers to regulate key cell-type-specific genes, and their dysregulation is often associated with disease. It remains unclear whether superenhancer constituent elements work independently or collaboratively and whether they have distinct roles in activating expression of their cognate genes. During my DPhil, I address these two questions by studying a model mammalian superenhancer at the mouse α-globin locus, active exclusively in erythroid cells. I sought to determine how the five constituents (R1, R2, R3, Rm, R4) of the mouse αglobin superenhancer exert control over α-globin gene expression during erythropoiesis. Firstly, I tested the sufficiency of the previously described strongest αglobin superenhancer constituent (R2) to independently activate α-globin expression; this entailed characterising, in detail, a mouse model in which the other four α-globin superenhancer elements have been removed from the native locus. Surprisingly, I show that this strong enhancer is incapable of driving the expected level of expression, independently. Secondly, following synthetic biology and genome editing techniques in mouse Embryonic Stem Cells (mESCs), I rebuilt the native α-globin superenhancer in all informative combinations, starting from an enhancer-less baseline in which all five constituents have been removed. Examination of molecular phenotypes of erythroid cells derived from the engineered mESCs revealed a complex relationship between inequivalent constituents which cooperate in additive, synergistic, and redundant fashions. I also uncovered a novel class of regulatory element within the αglobin superenhancer, which I named facilitators. Unlike canonical enhancers (R1, R2), facilitators (R3, Rm, R4) have no intrinsic enhancer activity. However, they are necessary to potentiate canonical enhancers, in order to attain optimal levels of target gene expression. Lastly, by comparing the impact of the three facilitators on gene expression, I discovered a functional hierarchy that seems to be position-dependent. Ultimately, I have rigorously dissected the α-globin superenhancer and described a new class of regulatory element, which I named facilitators, that were previously mistaken for weak enhancers. Furthermore, I unravelled a necessary mode of cooperation manifested between two types of superenhancer constituent elements, canonical enhancers and facilitators, highlighting an emergent property of a superenhancer

An analysis of at-home demand for ice cream in the United States

Ice cream has been manufactured commercially in the United States since the middle of the 19th century. Ice cream and frozen dessert products comprise an important and relatively stable component of the United States dairy industry. As with many other dairy products, ice cream is differentiated in several dimensions. A censored translog demand system model was employed to analyze purchases of 3 ice cream product categories. The objective of this study was to determine the effect that changes in retail prices and consumer income have on at-home ice cream consumption. The analysis was based on Nielsen 2005 home scan retail data and used marital status, age, race, education, female employment status, and location in the estimations of aggregate demand elasticities. Results revealed that price and consumer income were the main determinants of demand for ice cream products. Calculated own-price elasticities indicated relatively elastic responses by consumers for all categories except for compensated bulk ice cream. All expenditure elasticities were inelastic except for bulk ice cream, and most of the ice cream categories were substitutes. Ongoing efforts to examine consumer demand for these products will assist milk producers, dairy processors and manufacturers, and dairy marketers as they face changing consumer responses to food and diet issues.Nielsen home scan retail data; dairy demand; elasticity; ice cream

Understanding fundamental principles of enhancer biology at a model locus: analysing the structure and function of an enhancer cluster at the α-globin locus

Despite ever-increasing accumulation of genomic data, the fundamental question of how individual genes are switched on during development, lineage-specification and differentiation is not fully answered. It is widely accepted that this involves the interaction between at least three fundamental regulatory elements: enhancers, promoters and insulators. Enhancers contain transcription factor binding sites which are bound by transcription factors (TFs) and co-factors expressed during cell fate decisions and maintain imposed patterns of activation, at least in part, via their epigenetic modification. This information is transferred from enhancers to their cognate promoters often by coming into close physical proximity to form a 'transcriptional hub' containing a high concentration of TFs and co-factors. The mechanisms underlying these stages of transcriptional activation are not fully explained. This review focuses on how enhancers and promoters are activated during differentiation and how multiple enhancers work together to regulate gene expression. We illustrate the currently understood principles of how mammalian enhancers work and how they may be perturbed in enhanceropathies using expression of the α-globin gene cluster during erythropoiesis, as a model

An analysis of at-home demand for ice cream in the United States

Ice cream has been manufactured commercially in the United States since the middle of the 19th century. Ice cream and frozen dessert products comprise an important and relatively stable component of the United States dairy industry. As with many other dairy products, ice cream is differentiated in several dimensions. A censored translog demand system model was employed to analyze purchases of 3 ice cream product categories. The objective of this study was to determine the effect that changes in retail prices and consumer income have on at-home ice cream consumption. The analysis was based on Nielsen 2005 home scan retail data and used marital status, age, race, education, female employment status, and location in the estimations of aggregate demand elasticities. Results revealed that price and consumer income were the main determinants of demand for ice cream products. Calculated own-price elasticities indicated relatively elastic responses by consumers for all categories except for compensated bulk ice cream. All expenditure elasticities were inelastic except for bulk ice cream, and most of the ice cream categories were substitutes. Ongoing efforts to examine consumer demand for these products will assist milk producers, dairy processors and manufacturers, and dairy marketers as they face changing consumer responses to food and diet issues

Scalable in vitro production of defined mouse erythroblasts

Mouse embryonic stem cells (mESCs) can be manipulated in vitro to recapitulate the process of erythropoiesis, during which multipotent cells undergo lineage specification, differentiation and maturation to produce erythroid cells. Although useful for identifying specific progenitors and precursors, this system has not been fully exploited as a source of cells to analyse erythropoiesis. Here, we establish a protocol in which characterised erythroblasts can be isolated in a scalable manner from differentiated embryoid bodies (EBs). Using transcriptional and epigenetic analysis, we demonstrate that this system faithfully recapitulates normal primitive erythropoiesis and fully reproduces the effects of natural and engineered mutations seen in primary cells obtained from mouse models. We anticipate this system to be of great value in reducing the time and costs of generating and maintaining mouse lines in a number of research scenarios

An Analysis of Demand Elasticities for Fluid Milk Products in the U.S.

This study examines retail fluid milk products purchase data from Nielsen 2005 home scan data. The demand for seven categories of fluid milk products were estimated: whole milk, whole flavored milk, reduced fat milk, flavored reduced fat milk, buttermilk, canned milk and all other fluid milk products. Analyses of the purchases of seven fluid milk categories based on the Nielsen 2005 home scan retail data are used to determine the roles marital status, age, race, education, female employment status and location play in the empirical estimations of aggregate demand elasticities. To derive the demand elasticities, a censored translog demand system is used. The results reveal that price and income are the main determinants of demand for fluid milk products with a few minor determinants. All own-price elasticities are greater than unity for all fluid milk categories except for the compensated reduced fat milk. All expenditure elasticities are inelastic except for reduced fat milk and most of the fluid milk categories are substitutes

A Cross-Sectional Analysis of U.S. Yogurt Demand

Among processed and manufactured dairy products marketed in the United States, yogurt has one of the shortest histories and sharpest growth trends. To examine the socioeconomic factors associated with U.S. at-home yogurt demand, a demand system is used to analyze three yogurt products. Findings suggest that own-prices have negative effects and total expenditure has a positive effect on U.S. yogurt demand. Demographic factors do not have a large impact

Unexpectedly High Levels of Inverted Re-Insertions Using Paired sgRNAs for Genomic Deletions

Use of dual sgRNAs is a common CRISPR/Cas9-based strategy for the creation of genetic deletions. The ease of screening combined with a rather high rate of success makes this approach a reliable genome engineering procedure. Recently, a number of studies using CRISPR/Cas9 have revealed unwanted large-scale rearrangements, duplications, inversions or larger-than-expected deletions. Strict quality control measures are required to validate the model system, and this crucially depends on knowing which potential experimental outcomes to expect. Using the dual sgRNA deletion approach, our team discovered high levels of excision, inversion and re-insertion at the site of targeting. We detected those at a variety of genomic loci and in several immortalized cell lines, demonstrating that inverted re-insertions are a common by-product with an overall frequency between 3% and 20%. Our findings imply an inherent danger in the misinterpretation of screening data when using only a single PCR screening. While amplification of the region of interest might classify clones as wild type (WT) based on amplicon size, secondary analyses can discover heterozygous (HET) clones among presumptive WTs, and events deemed as HET clones could potentially be full KO. As such, screening for inverted re-insertions helps in decreasing the number of clones required to obtain a full KO. With this technical note, we want to raise awareness of this phenomenon and suggest implementing a standard secondary PCR while screening for deletions