An insulator embedded in the chicken α-globin locus regulates chromatin domain configuration and differential gene expression

Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5′ non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF

CTCF: Comprehending The Complex Functions of an 11 zinc finger transcription factor

In a multi-cellular organism, every somatic cell nucleus broadly contains the same sequence of DNA, yet clearly most cells are very different to each other. Specific sets of genes encoding proteins become activated whereas others are repressed. Within the genome, independently regulated genes are often found in close proximity to other genes that have different patterns of expression. How specific gene loci are organised in nuclear space is only recently emerging. CTCF is a protein that has been strongly implicated in mediating many distinct processes of gene regulation, including transcription, chromatin structure, and the structural organisation of gene loci. The aim of this thesis was to investigate the function of the CTCF protein in vivo, in particular the role of CTCF in regulating cellular proliferation, differentiation and the organisation of gene loci within the nucleus. The introduction aims to give an overview of the information required to understand the foundations of studies presented and discussed in this thesis. The transcription or activation of genes occurs in the cell nucleus and requires specific modifications of chromatin. Chapter 1 describes the formation of chromatin and key factors that modify this structure. How transcription is initiated, and influenced by cis-regulatory elements is also discussed. Since the initial characterisation as a transcription factor, many structural and regulatory functions have been attributed to CTCF, as detailed in chapter 2, which imply CTCF is a key regulator of development and cell viability. The haematopoietic system is used in this thesis as a model for investigating the function of CTCF in two distinct lineages. In chapter 3 the development of erythrocytes and T-lymphocytes is introduced. Chapters 4 and 5 describe the experiments used to address questions regarding CTCF function at the b-globin locus and during T-cell differentiation respectively. Published data strongly demonstrate the clustering of cis-regulat

Identification and functional validation of genomic boundaries in mammals

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de lectura: 30-06-2014Eukaryotic genomes are divided into expression domains, which contain DNA coding sequences together with all the regulatory elements needed for their correct spatio-­‐ temporal expression pattern. Genomic boundaries, also known as insulators, flank these domains preventing undesirable crosstalk between the regulatory elements of neighboring domains. They employ various mechanisms and thus, are functionally rather than structurally defined. For this reason, in an attempt to find boundaries in a genome-­‐ wide unbiased fashion in mammals, we focused on identifying those loci where the presence of boundary function would be required to satisfy a biological need. For example, we hypothesized that adjacent genes with opposite expression patterns would need to be separated by boundaries to maintain the independency of their different expression domains. Also, boundaries could be found partitioning the chromatin into inactive heterochromatic and active euchromatic domains, impeding the deleterious effects the spread of the former would have on the latter. Finally, boundaries could also bracket clusters of co-­‐expressed genes to ensure their co-­‐regulation and co-­‐expression. Different algorithms, based on the analysis of gene expression data, were developed in order to explore these scenarios. The resulting evolutionarily conserved non-­‐coding putative insulator sequences were functionally validated using a number of assays. Their enhancer-­‐ blocking properties were evaluated in vitro in human cells in culture, and then in vivo by using transgenic zebrafish. Additionally, one of the most powerful elements was further tested for its ability to protect from chromosomal position effects in transgenic mice. The description and characterization of new genomic boundaries would shed some light into the way mammalian genomes are organized, as well as expand the repertoire of genetic tools that can be incorporated in heterologous constructs to improve the gene transfer technologies by preventing chromosomal position effects.Los genomas de eucariotas están divididos en dominios de expresión, que se definen como aquellas porciones del genoma que contienen uno o varios genes y todos los elementos reguladores necesarios para que que se expresen de acuerdo con un patrón espacio-­‐temporal concreto. Los aisladores genómicos, también llamados insulators, flanquean estos dominios y los protegen de la influencia no deseada de los elementos reguladores contenidos en los dominios vecinos. Existen diversos mechanismos de aislamiento, por lo que los insulators no se definen por una secuencia de ADN concreta, sino porque comparten una misma función. Así, para encontrar aisladores en el genoma de mamíferos de una forma no sesgada, nos propusimos identificar aquellas posiciones del genoma donde se requiere la presencia de función aisladora para satisfacer un problema biológico. Por ejemplo, genes adyacentes con perfiles de expresión completamente distintos deberían estar separados por aisladores que mantuviesen dominios de expresión independientes. Asimismo, cabe esperar la presencia de aisladores entre dominios silentes de heterocromatina y dominios activos de eucromatina. Aquí, impedirían los efectos perjudiciales que el avance de los primeros tendrían sobre los segundos. Finalmente, también podrían encontrarse aisladores flanqueando grupos de genes co-­‐expresados para asegurar su co-­‐regulación y, por tanto, co-­‐expresión. Basándonos en estos escenarios, se desarrollaron diversos algoritmos que usaban datos de expresión génica para predecir la presencia de aisladores. Como resultado de estos algoritmos, se obtuvo una serie de secuencias conservadas evolutivamente y no codificantes que se validaron funcionalmente empleando varios tests. La capacidad de bloqueo de enhancers se evaluó mediante ensayos in vitro en células humanas en cultivo primero, y luego in vivo mediante el uso de peces cebra transgénicos. Además, se analizó la capacidad de uno de los elementos más potentes para proteger de efectos de posición cromosomales en ratones transgénicos. La descripción y caracterización de nuevos aisladores genómicos no sólo sirve para entender mejor cómo se organizan los genomas de mamíferos. También es útil para ampliar el abanico de herramientas disponibles que se pueden usar en construcciones heterólogas para bloquear los efectos de posición cromosomales que se dan comúnmente en experimentos de transferencia genética

Fingers in action! Chromatin Organization and Transcriptional Regulation by CTCF and CTCFL

__Abstract__ Chromatin is hierarchically folded and wrapped in order to compact DNA. It is accessible to specific proteins to allow regulation of various cellular processes. Although chromatin is organized into higher-order structures it is highly dynamic and it can influence genome configuration and transcription via interactions with various subnuclear compartments. CTCF is the most important factor involved in chromatin structure regulation, in particular the spatial organization of higher-order chromatin configurations. CTCF-like (CTCFL) is a testis specific paralogue of CTCF, whose function has been characterized to a lesser extent. The aim of this thesis is to obtain more insight in the biological roles of CTCF and CTCFL. A general introduction to the field of nuclear organization and transcription regulation is provided in chapter 1. This chapter also provides an overview of the process of spermatogenesis during which both CTCF and CTCFL are thought to perform important functions. Finally this chapter also summarizes already known aspects of CTCF, CTCFL and their functional interaction partners. The role of CTCF and CTCFL in the regulation of ribosomal repeat DNA is the main focus of chapter 2. CTCF and CTCFL interact with the key regulator of RNA polymerase I, UBF. Furthermore, CTCF regulates the spacer promoter by recruiting RNA polymerase I, H2A.Z and UBF to rDNA. In chapter 3 the focus shifts towards the study of CTCF binding motifs and the binding of CTCF zinc fingers to DNA. Using a genome-wide binding analysis on CTCF zinc finger mutants we propose a model for DNA binding by CTCF. Chapter 4 and 5 examine the functional relationship between CTCF and CTCFL extensively in mouse embryonic stem cells and testis by examining genome-wide binding and transcription profiles. Finally, chapter 6 provides a general discussion elaborating on the findings in this thesis. Furthermore, the findings are positioned in perspective with curren

Partners in Long Distance Interactions

The genome of higher eukaryotes consists of DNA, which in case of the human genome measures 2m in length and is divided over 46 chromosomes. These long DNA molecules are packed in a nucleus that measures about 10μm in diameter. In order to fit the complete DNA into such a small volume, DNA is folded and compacted by proteins in a structure called chromatin. During mitosis, is even further compacted into condensed chromosomes (Kornberg, 1974). All the information needed for the formation and proper function of an organism is stored in these structures and it is reasonable to expect that this overcrowded situation is organized in a very specific manner, with controlled three-dimensional contacts within the nucleus. The need for controlled chromatin contacts is also suggested by the fact that gene regulation is a tightly regulated process. Different levels of control must be involved in regulating proper spatio-temporal expression of genes throughout the process of cellular differentiation. These processes are coordinated by interactions of an “army” of general, cell-type and stage specific proteins that bind to chromatin and DNA. Several techniques allow the identification and study of chromatin regions that interact with each other. These include functional genetic analysis, microscopic analysis after DNA or RNA fluorescent in situ hybridization (FISH) in combination with 3D microscopy, as well as biochemical methods, such as chromosome conformation capture (3C) and the more sophisticated variation thereof (4C). The combination of these methods reveals a network of contacts in the nucleus. These interactions are mediated by insulators and other regulatory sequences, including enhancers and promoters, which mediate/promote certain functional three-dimensional interactions while preventing other enhancer-promoter contacts. In this chapter, I will introduce several factors: Ldb1, Delangin, Cohesin and CTCF which have important role long-range interactions

Regulatory architecture of the Fgf8 locus and tissue specific control of gene expression

During vertebrate development, precise spatio-temporal expression of genes is necessary for growth and tissue differentiation in the embryo. These genes are usually controlled by multiple elements scattered hundreds of kilobases up- and downstream of a target gene, sometimes embedded in introns of functionally non-related genes. This intricate distribution along the chromosome raises the question on the importance of the regulatory architecture for correct gene expression. This is additionally emphasized by several genetic disorders where no mutations in coding regions were found. Instead, it seems they are associated with the disruption of the normal structure of chromosomal domains. Furthermore, distribution of genes and their regulatory elements is mostly conserved across distant species, suggesting they are organized following a specific architecture. To address the role of structural organization of genes and their regulatory elements in achieving proper gene expression, we studied TLX1-FGF8 interval mapped to human chromosome 10q24. This 600kb gene-rich region harbors seven functionally and phylogenetically unrelated genes, representing a “normal” genomic situation. Gene order of the whole region is extremely conserved in tetrapods and to some extent in teleosts and beyond. In addition, human condition split hand-foot malformation type 3 (SHFM3), characterized by the loss of central digits on hand and feet, is caused by 0.5Mb tandem duplication within this locus. FGF8 is coding for a signaling molecule involved in developmental processes, including limb development. Although FGF8 is not within the duplicated interval, the early termination of its expression in the apical ectodermal ridge (AER) contributes to the phenotype. Despite the earlier mapping attempts conducted in mice and fish, full scope of Fgf8 regulatory elements is not determined. Combining mouse transgenesis and chromosomal engineering, I narrowed down the region critical for Fgf8 expression spanning 200kb downstream of the gene. Within it, I characterized individual regulatory elements. Many of them guided the expression of LacZ reporter gene in overlapping domains, suggesting functional redundancy. Also, when tested individually, they express much more regulatory potential than is eventually utilized by Fgf8. Additional experiments using artificial chromosomes (BACs) with inserted LacZ revealed filtering of this potential when elements are in their natural genomic environment. Fine-tuning of regulatory potential can be achieved either by negative elements or the structure of the locus itself. Close proximity of Fgf8 enhancers and promoters of other genes in the region raised the question on how do regulatory elements discriminate between their target and promoters of genes nearby. A series of chromosomal rearrangements reallocating different promoters into Fgf8 regulatory region showed that Fgf8 enhancers are intrinsically capable to activate heterologous promoters and that enhancer-promoter specificity is not exclusively guided by the sequence of the promoter. Rather, the relative position of the two plays a significant role in achieving proper target gene activation. Based on the results of our study, we propose a novel concept of gene regulation: a holo-enhancer. Within a holo-enhancer, vast regulatory potential of multiple enhancers is filtered by their relative position towards the target gene and the activity of potential negative regulators. Also, individual enhancers are able to activate heterologous promoters. However, this intrinsic promiscuity is refined by their position-dependent activity. In a complex genomic environment like the one of Fgf8, gene regulation is not composed of simple binary promoter-enhancer interactions, but is embedded in the structure of the region itself. Once a holo-enhancer is divided into individual elements, their full potential is revealed and perturbations of the region show the potential of enhancers to act on other promoters. This novel concept emphasizes the holistic nature of the interactions of genes and their regulatory elements in achieving gene and tissue specificity, with the overall organization of the locus being a key aspect in this process. These observations led us to suggest the mechanism leading to SHFM3. Duplication breakpoints disrupt the holo-enhancer, reallocating part of the enhancers and releasing them from potential negative elements needed to refine their activities. In addition, a new position brings them to the appropriate distance to heterologous promoters. Their intrinsic promiscuity and broad regulatory potential allows activation of other genes in the region, potentially leading to their up-regulation. Moreover, complex interactions within this region could also explain ancient linkage between functionally unrelated genes (like Fgf8 and Fbxw4), which is most probably due to structural constraints of the regulatory scaffold upon which genes are transcribed

Transcriptional regulation of the stem cell leukaemia gene (SCL/TAL1) via chromatin looping

The bHLH protein TAL1 (SCL) is a critical regulator of vertebrate hematopoiesis and is misregulated in T-cell acute lymphoblastic leukemia (T-ALL). This thesis studied chromatin looping interactions at the TAL1 locus – defining the first structural model which accounts for a number of phenomena associated with TAL1, its flanking genes and its relationship with its functional paralogue LYL1. The chromosome conformation capture (3C) and its high-throughput variant 4C-array technologies have been applied to characterise the chromatin interactions. Intriguing chromatin organisations have been identified at the TAL1 and LYL1 loci, which are closely associated with transcriptional regulation, chromosomal abnormality and regulatory remodelling through evolution. Firstly, in TAL1 expressing cells, the locus adopts a “cruciform” configuration – forming an active chromatin hub which brings together the TAL1 promoters, its stem cell and erythroid enhancers, and two CTCF/Rad21-bound insulators. Secondly, loss of a GATA1-containing complex bound by the TAL1 erythroid enhancer and its promoter is sufficient to disrupt the formation of the hub and the entire cruciform structure and results in decreased TAL1 expression. Thirdly, it demonstrates that genes flanking TAL1 are also dependent on this hub and that TAL1 promoters interact directly with intron 1 of the neighbouring STIL gene. This TAL1/STIL interaction also provides a structural link between the DNA sequences which mediate micro-deletions in 25% of cases of T-ALL. Finally, it demonstrates that a GATA1-dependent chromatin looping mechanism also exists at the LYL1 locus which is strikingly similar to that mediating contact between the TAL1 promoter and its erythroid enhancer. Conservation of core chromatin looping at the TAL1 and LYL1 loci may account for some aspects of their functional relationships. It also suggests that looping mechanisms at both loci could also facilitate cis-regulatory maintenance and/or remodelling during vertebrate evolution

CTCF: A Crucial Regulator of Gene Expression in Lymphocytes

In vertebrates, the immune system is responsible for the protection against pathogens such as viruses, bacteria, fungi or parasites. This remarkably effective defense system depends upon the white blood cells or leukocytes, which mediate both innate and adaptive immune responses. Innate immunity provides an immediate but non-specific front line of host defense against many pathogens and involves granulocytes (neutrophils, eosinophils and basophils), mast cells, macrophages and natural killer (NK) cells. Germ lineencoded surface receptors to common pathogens constituents on innate effector cells trigger the elimination of pathogens by phagocytosis and the release of inflammatory mediators, such as cytokines and chemokines. These receptors are referred to as pattern recognition receptors (PRRs), which recognize a limited set of molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). Acquired or adaptive immunity is characterized by gradual, though highly specific and effective immune responses against pathogens. Three major cell types are involved in adaptive immunity: B-lymphocytes, T-lymphocytes and antigen presenting cells (APCs), the most potent of which are dendritic cells (DCs). DCs act as messengers between the innate and adaptive immune system. Their main function is to take up, process and present pathogen constituents (also called antigens) with major histocompatibility complex (MHC) proteins to T lymphocytes. B- and T-lymphocytes are considered to be the central players of the adaptive immune system. Their unique and virtually limitless capacity to specifically recognize antigens relies on the generation of a wide repertoire of antigen receptors – B-cell receptor (BCR) in B-lymphocytes and T-cell receptor (TCR) in T-lymphocytes –

Differential chromatin topology and transcription factor enhancer binding regulate spatiotemporal gene expression in limb development

Many developmental genes are located in gene-poor genomic regions and are activated by long-range enhancers located up to 1Mb away. Modification and reorganisation of chromatin structure is pivotal to such long-range gene regulation. A prerequisite for enhancer activity is the binding of transcription factors and co-factors with the interplay between activating and repressive factors determining tissue, spatial and temporal specificity. Spatiotemporal control of sonic hedgehog (Shh) and the 5′ Hoxd genes (especially Hoxd13) is crucial for vertebrate limb anterior-posterior (A-P) axis and autopod patterning. Shh tissue specificity is controlled by multiple enhancers throughout an adjacent gene desert. The ~0.8Mb-distant limb enhancer (ZRS) bypasses nearby genes to activate only Shh. In contrast, limb-specific HoxD expression is regulated by multiple enhancers, with the ~200kb-distant global control region (GCR) regulatory element the most characterised. In this thesis I investigated the mechanisms of ZRS and GCR regulation of Shh and Hoxd13 respectively. The model system used was immortalised cell lines derived from the anterior and posterior distal forelimb buds of E10.5 and E11.5 mouse embryos. Cell line data were confirmed in dissected limb tissue. Increased expression of the 5′ Hoxd genes, particularly Hoxd13, correlated with the loss of the repressive, polycomb catalysed, histone modification H3K27me3 and decompaction of chromatin structure over the HoxD locus at the distal posterior forelimb bud at stage E10.5. Moreover, I show that the GCR spatially co-localises with the 5′ HoxD locus at the distal posterior region of E10.5-11 embryos. These data are consistent with the formation of a chromatin loop between Hoxd13 and the GCR at the time and place of distal limb bud development when the GCR is required to initiate 5′ Hoxd gene expression. This is the first example of A-P differences in chromatin compaction and local folding in the limb. Point mutations within the ZRS cause ectopic (anterior) Shh expression, which results in preaxial polydactyly (PPD). The ZRS contains multiple canonical ETS transcription factor binding motifs, and point mutations in two families with PPD results in the formation of additional ETS binding sites. The point mutations cause the loss or reduction of ETV4/5 transcription factor binding at a non-canonical ETS binding site and enable additional binding instead of ETS1. I show that ETV4/5, ETS1 and another ETS protein GABPα all bind to the ZRS. This work has revealed the differential effect on Shh expression of two groups of ETS factors mediated through the ZRS. The binding of ETS1/ GABPα determines the posterior Shh expression domain while ETV4/5 restricts anterior Shh expression. Two point mutations alter the ETS-binding profile, creating an additional ETS1/ GABPα site that is sufficient to drive ectopic Shh expression. DNA FISH on E11.5 forelimb and floorplate tissue sections revealed that the Shh-ZRS genomic locus is in a compact chromatin conformation in both Shhexpressing and non-expressing cells. However, I show that the ZRS co-localises with Shh to a significantly greater extent in the distal posterior limb bud and the floorplate compared with cells where Shh is not expressed. This thesis presents novel research into long-range gene regulation during limb development, elucidating the role of chromatin re-organisation and how spatial-specific enhancer activity is determined by opposing sets of binding factors