ß-globin gene regulation and chromatin structure

Oxygen transport in the blood is mediated by highly specialized red cells. The majority of the proteins of a red cell comprises the oxygen carrier protein hemoglobin, which is a hetero-tctrameric protein that consists of two a and two p globin chains. In humans different u- and p-like globin chains are expressed during development resulting in several different hemoglobin tetramers. The expression of different globin chains serves to facilitate the oxygen uptake by the emblYo, since embryonic hemoglobin has a higher affinity for oxygen. The genes that code for the u- and p-like globin chains reside in loci located on separate chromosomes. Many blood disorders, like u- and p-thalassemias and sickle cell anemia are the consequence of deletions or mutations of sequences in these loci and they initiated extensive research into the molecular basis of these diseases. Especially the p globin locus has served as a model system to study the regulation of multi gene loci. The five functional globin genes, 5'-s_Gy_Ay-o-P-3', are differentially expressed during development. Proper expression of these genes requires the presence of a region located 5' upstream of the s globin gene. This locus control region (LCR) contains 5 small regions that bind several trans-acting factors in an erythroid environment. The aim of this thesis is to study the role of the LCR in the regulation of the ~ globin genes ill vivo. Chapter 1 reviews the CUlTent knowledge in the regnlation of eukaryotic transcription and chromatin. Chapter 2 gives a broad introduction covering two decades of shldies conceming the regulation of the human and murine p globin genes and serves as an outline for the results that wiII be discussed in the chapters 3 to 6. Chapter 3 describes the role of EKLF in the y to p globin switching process. Chapter 4 and 5 describe experiments that show that the human and the murine LCR can only activate one globin gene at a given moment. Chapter 6 describes the characterization of intergenic transcription in the human p globin locus. Finally in chapter 7 the implications of the results presented in this thesis will be discussed

Xist-mediated chromatin changes that establish silencing of an entire X chromosome in mammals

X chromosome inactivation (XCI) ensures an equal gene dosage between the sexes in placental mammals. Xist, a modular multi-domain X-encoded long non-coding RNA coats the X chromosome in cis during XCI. Xist recruits chromatin remodelers and repressor complexes ensuring silencing of the inactive X (Xi). Here, we review the recent work focused on the role of Xist functional repeats and interacting RNA-binding factors in the establishment of the silent state. Xist orchestrates recruitment of remodelers and repressors that first facilitate removal of the active chromatin landscape and subsequently direct the transition into a repressive heterochromatic environment. Some of these factors affect silencing on a chromosome-wide scale, while others display gene-specific silencing defects. The temporal order of recruitment shows each silencing step is party dependent on one another. After the Xi is established, many of the factors are dispensable, and a different repertoire of proteins ensure the silenced Xi is maintained and propagated

FGF treatment of host embryos injected with ES cells increases rates of chimaerism

In spite of the emergence of genome editing tools, ES cell mediated transgenesis remains the most controllable way of creating genetically modified animals. Although tetraploid (4N) complementation of 4N host embryos and ES cells, is the only method guaranteeing that offspring are entirely ES cell derived, this technique is challenging, not always successful and difficult to implement in some laboratory settings. The current study shows that pretreatment of host blastocysts with FGF4 prior to ES cell injection can provide an alternative method for the generation of animals displaying high rates of chimaerism. Chimaerism assessment in E11 fetuses and born pups shows that a large percentage of resulting conceptuses show a high ES cell contribution from implantation onwards and that developing pups do not necessitate c-section for delivery

Buccal swab as a reliable predictor for X inactivation ratio in inaccessible tissues

Background As a result of the epigenetic phenomenon of X chromosome inactivation (XCI) every woman is a mosaic of cells with either an inactive paternal X chromosome or an inactive maternal X chromosome. The ratio between inactive paternal and maternal X chromosomes is different for every female individual, and can influence an X-encoded trait or disease. A multitude of X linked conditions is known, and for many of them it is recognised that the phenotype in affected female carriers of the causative mutation is modulated by the XCI ratio. To predict disease severity an XCI ratio is usually determined in peripheral blood samples. However, the correlation between XCI ratios in peripheral blood and disease affected tissues, that are often inaccessible, is poorly understood. Here, we tested several tissues obtained from autopsies of 12 female individuals for patch size and XCI ratio. Methods XCI ratios were analysed using methylsensitive PCR-based assays for the AR, PCSK1N and SLITRK4 loci. XCI patch size was analysed by testing the XCI ratio of tissue samples with decreasing size. Results XCI patch size was analysed for liver, muscle, ovary and brain samples and was found too small to confound testing for XCI ratio in these tissues. XCI ratios were determined in the easily accessible tissues, blood, buccal epithelium and hair follicle, and compared with ratios in several inaccessible tissues. Conclusions Buccal epithelium is preferable over peripheral blood for predicting XCI ratios of inaccessible tissues. Ovary is the only inaccessible tissue showing a poor correlation to blood and buccal epithelium, but has a good correlation to hair follicle instead

Xist and Tsix Transcription Dynamics Is Regulated by the X-to-Autosome Ratio and Semistable Transcriptional States

In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist. Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-to-autosome ratio, directed by regulators of XCI, and can be modulated by tissue culture conditions