Bachelorarbeit, 2018
40 Seiten, Note: 2.5
ABSTRACT
ACKNOWLEDGEMENTS
DEDICATION
TABLE OF CONTENT
LIST OF FIGURES
CHAPTER ONE
INTRODUCTION
CHAPTER TWO
LITERATURE REVIEW
2.1 Prdm gene family
2.2 Cis -regulatory architecture of the genome
2.3 Transcriptional regulation by enhancers
2.3.1 Transcriptional enhancers
2.3.2 Deregulation of enhancer function in human disease
2.4 Chromatin signatures in different enhancer states
2.4.1 Interaction between transcription factors and chromatin
2.5 Genome wide identification of enhancer element
CHAPTER THREE
MATERIALS AND METHODS
3.1 Search for cis -regulatory element(s) in the vicinity of Prdm15 by comparative genomics
3.2 Delineation of candidate brain enhancers of Prdm15 using brain-specific enhancer chromatin modification signatures
CHAPTER FOUR
RESULTS
CHAPTER FIVE
DISCUSSION
6.1 Conclusions
6.2 Recommendations
REFERENCES
The interaction between transcriptional factors and cis -regulatory DNA element has given rise to the diversity in the expression of eukaryotic gene. Prdm15 gene is known to be expressed in the brain throughout development of mouse and zebrafish however; there are no known enhancer(s) for this gene in the brain. The aim of this study was to identify brain specific enhancer(s) for the gene Prdm15 during brain development. The VISTA enhancer browser and Encyclopedia of DNA Elements (ENCODE) data were used in this study to determine our candidate brain enhancer. The VISTA enhancer browser was used to quest for cis -regulatory element(s) in the environs of Prdm15 by comparative genomics and only one candidate enhancer we designated Prdm15 control element 1(PCE1) was identified downstream of the gene. PCE1 was found to be conserved in organisms such as chicken, zebrafish, rhesus, chimp, dog and cow. To ascertain if PCE1 is a brain enhancer, we delineated PCE1 using brain-specific enhancer chromatin modification signatures H3K4me1 and H3K27ac in the ENCODE data and they showed enrichment for PCE1. In conclusion, our data set revealed that only one brain candidate enhancer (PCE1) exist for Prdm15 and that this brain enhancer for Prdm15 will help in identifying important upstream specific transcription factors of PrdmI5 during brain development.
First and foremost, I thank Dr. Samuel Abassah-Oppong for his helpful advice and valuable comments on the manuscript. I also thank the lectures of the Biochemistry department for their helpful and informative discussions during my studies.
It has been my great delight to work with the many gifted scientists in the Biochemistry department laboratory. I thank Mr. Eric Ofori, Mr. Baffour Kyei-Asante, Mr. Gilbert K. Wiabo-Asabil and Miss Vivian Geraldo for their excellent technical assistance during my undergraduate studies. I also thank the teaching assistant of the department for their invaluable advice in particular Sampson Avornor for giving me a good initiation into the study of cis -regulatory elements of Prdm15 during brain development.
Finally, I am grateful to my family and friends for their love and support throughout the past four years. My parents have been a huge source of support during my undergraduate career, and I am so grateful that they were always there for me, both to help me through tough times, and to celebrate happy moments.
I dedicate this work to my family for their support during my undergraduate studies.
Figure 1 Expression of Prdm15 in some tissues
Figure 2 The effect of deletion and mutation of limb enhancer of sonic hedgehog (Shh)
Figure 3 DNA elements that affect transcription by RNA polymerase II
Figure 4 Non-coding sequence conservation around Prdm15
Figure 5 Enhancer predictive chromatin marks around Prdm15
Figure 6 Enhancer predictive chromatin marks around Prdm15
Figure 7 Enhancer reporter assay
Figure 8 Chromatin immunoprecipitation technique
Multicellular eukaryotes are made of diverse cell types, each with specialized functions and properties critical for the distinctive development of the organism. Despite this specificity, with few exceptions, these cells have the same genome. Strangely, eukaryotes exhibit incredible multifariousity in cellular function as a result of differences in gene expression between cell types. Gene expression however, is principally regulated by a series of precise interaction between transcriptional factors and the cis -regulatory DNA element like enhancers (Zhang et al., 2013). These interactions occur both at the core promoter and at distal regulatory regions which are looped into close physical proximity with the core promoter. Enhancer regions are typically utilized in a cell-type-specific manner and help to drive the distinct gene expressions that define the diverse cell identities (Yu et al., 2007).
In the past, developmental diseases were associated with mutations in coding sequences of DNA, but studies have now display the fact that alteration in non-coding sequences of DNA can alter the transcription of a gene and therefore may also lead to diseases; example the thalassemia disease (Smith and Shilatifard, 2014). This phenomenon can be due to the fact that the non-coding regions of DNA contain regulatory sequences that act as repressors, enhancers and silencers to regulate gene expression. This connote that to understand the mechanism that regulates gene expression, it is vital to identify and define the network of cis- acting regulatory DNA element which can be viewed as the regulatory code wired within the genome (Yu et al., 2007).
According to Banerji et al., (1981), transcriptional enhancers are regulatory sequence that can modulate gene expression independently of their position in that they can be located upstream, downstream or within the gene they regulate. They are found in both eukaryotes and prokaryotes, usually 50-1500 base pair in length and found bound to activators to increase the likelihood that transcription will occur (Pennacchio et al., 2013). In eukaryotic cells, the structure of the chromatin is folded in a way such that enhancers may be far from the gene on the same chromosome but spatially close to the promoter due to DNA looping allowing enhancer-gene interaction with the general transcription factors and RNA polymerase II (Maston et al,. 2006).
The Prdm family are characterized by an N-terminal positive regulatory (PR) domain that is related to the suppressor of variegation 3-9, enhancer of zeste and trithorax (SET) methyltransferase domain and multiple zinc fingers that mediate sequence specific DNA binding and interaction between proteins (Hohenauer and Moore, 2012). Some members of this family known to be involved in growth and development of cells include Prdm1, Prdm8, Prdm15 and Prdm16 (Sun et al., 2008). Prdm factors acts directly as histone methyltransferase or recruit a suite of histone modifying enzymes to target promoters. By this, they function in many developmental contexts to drive and keep up cell state transition and to modify the activity of developmental signaling pathways (Hohenauer and Moore, 2012). Prdm15 gene is known to be expressed in the brain during development of mouse and zebra fish (Sun et al., 2008); however, there are no known enhancers for this gene in the brain. Considering that enhancers are crucial for the control of gene expression, we hypothesized that Prdm15 gene is regulated by one or more enhancers .
Although, studies have shown that non-coding variation is a factor for diseases the mechanism by which this variation contributes to diseases remains obscure. This work therefore seeks to identify candidate brain enhancers for Prdm15 gene. Identifying brain enhancers for Prdm15 will help to identify important upstream transcription factors partners of PrdmI5 during brain development. In this work, the methods that will be used to identify candidate enhancer(s) for Prdm15 gene are; (i) comparative genomics for chicken, zebrafish, rhesus, chimp, dog, cow and mice sequences using the VISTA enhancer browser and (ii) enhancers specific discriminative chromatin signatures profiling with Encyclopedia of DNA Elements (ENCODE) data.
The Prdm family members which are vital in the proliferation and differentiation of cells control gene expression through modification of the chromatin state at target gene promoters. Hohenauer and Moore (2012) reported that Prdm members are characterized by the presence of an N-terminal positive regulatory (PR) domain found at various levels of conservation across species. The products of Prdm act as direct histone methyltransferases or recruit a suite of additional histone-modifying enzymes to specific target promoters. They act simultaneously at multiple sites across the genome, and they exhibit context-dependent activity. These features create the capacity to drive and keep up cell state transitions, and to modify the transcriptional output of developmental signaling pathways. In addition, Prdm proteins modulate crucial cellular process including regulation of brown fat differentiation and hematopoiesis (Fog et al., 2011). Experimental evidence has shown that the Prdm proteins act as transcriptional regulators either through intrinsic chromatin modifying activity or recruitment of chromatin modifiers in a cell and promoter specific context to regulate cellular proliferation and differentiation (Banerji, 1981).
According to Bernstein (2012) Prdm4, 8, 12 and 13 are involved in the development of the nervous system notwithstanding, the expression fashion of Prdm proteins on mouse and zebrafish development is very dynamic as demonstrated by expression of multiple Prdms in the nervous system (Kinameri et al., 2008). Sun et al., (2008) reported that the functional relevance of this expression pattern has only been investigated for few Prdms including zebra fish Prdm1, which is vital for the development of the neural crest and sensory neurons. In addition, Hernandez-Lagunas et al., (2005); Komai et al., (2009) cited that the expression of Prdm8 and Prdm10 in the mouse embryo nervous system has been characterized in detail. Prdm16 is also expressed in neural stem or progenitor cells and regulates their survival by controlling transcription of genes involved in oxidative stress (Chuikov et al., 2010). Like any other gene, deregulation of Prdm causes severe human disease like cancer and autism (Fog et al., 2011).
In a work by Sun et al., (2008), they reported that Prdm15 is expressed in cranial ganglia neurons as well as in muscle pioneer cells providing important information on the involvement of the gene in neural development. Although Prdm15 gene is known to be expressed in the brain during development of mouse and zebra fish however, there are no known enhancers for this gene in the brain. Therefore considering that enhancers are crucial for the control of gene expression the gene may be regulated by one or more enhancers.
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Figure 1 Expression of Prdm15 in some tissues
a and b shows the side view of Zebrafish with expression of Prdm15 in its muscle pioneer cells whiles a’ and b’ is a dorsal view of the same species showing the expression of Prdm15 in the cranial ganglia neurons both at 18 and 24 hour post fertilization (hpf). Figure obtained from Sun et al., (2008).
Gene expression is regulated through the interacted action of promoter proximal element and many cis -regulatory element DNA element (enhancers, insulators and silencers) that are located at greater distance from the transcription start site (Lenhard et al.,2012; Levine,2010). These elements are located far upstream or downstream or inside gene and even inside adjacent gene (Wijgerde et al., 1995). Hence it is not always obvious which element control which gene and the identification of essential distal element has to be accomplished by experimental means using genetic manipulation. Also, each of these regulatory modules performs a specific function in a specific cell type or at a particular stage in development (Spitz et al., 2003). Many key developmental cis -regulatory element like enhancers act not only on their biologically relevant target gene but also on unrelated neighboring genes (Zuniga, 2004). According to Ruf (2011), among these elements enhancers and their associated transcriptional factors have a leading role in the initiation of gene expression.
As stated earlier on, the cycle of transcription onrushes through several stages each of which is subject to regulation. It begins by the recruitment of factors to the core promoter, including RNA polymerase II, general transcription factors and other cofactors (Hoke, 2014). This regulation of gene is influenced by both the core promoter region of a gene and distal enhancer regions. The core promoter is the region immediately flanking the transcription start site of a gene while enhancer elements can be locked many mega bases away from the gene that they control. Both core promoter and enhancers contain binding sites for sequence specific transcriptional factors that recruit transcriptional cofactors and the transcription machinery (Hoke, 2014). Although enhancers may be far from the gene they regulate, the interaction between promoter-enhancer bound proteins help mediate looping between the core promoter and enhancer, bringing the regions into close physical proximity. Also, transcriptional factors at enhancer and promoter influence transcription through the recruitment of both activating and repressive cofactor that can influence several stages of the transcriptional cycle.
Although transcription relies critically on core promoters, enhancers can greatly increase the level of transcription from target gene and through binding of cell type specific transcriptional factors to influence the timing and spatial distribution of gene expression within a cell. According to Bernstein et al., (2012) there may be over a million enhancer regions in the human genome hence investigating how these enhancer regions mediate cell type specific gene expression programs is critical to understanding human development as well as disease.
The first enhancer element identified was 72 base pair sequence from the SV40 viral genome. This viral sequence was competent in inducing a two hundred folds increase in the expression of a reporter gene in human Hela cells (Banerji et al., 1981). This enhancer region retained its function when placed at diverse distances and orientations to the reporter gene and it was able to enhance expression to a similar degree whether located proximal to the promoter or several kilobases upstream or downstream of the gene (Banerji et al., 1983). Subsequently, enhancer elements were identified in metazoan genome. One of the first enhancer described was a sequence located in the immunoglobulin heavy chain locus that acts as a transcriptional enhancer in reporter assays. The enhancer activity of the immunoglobulin heavy chain locus enhancer activity was found to be cell type specific (Davidson et al., 1986). In another example, enhancer regions were found to control expression of the β-globin gene in a cell type and developmental stage specific manner in chicken and later on in human cells (Antoniou et al., 1988).
Transcriptional enhancers region play vital role in driving the cell type specific gene expression programs that underlie the diversity in cell function within the human body. Due to the conservation of transcriptional enhancers, their mutation may lead to disease (Hoke, 2014) and as such mutation of the proteins that regulates enhancer function may also lead to diseases like Kabuki syndrome (Smith and Shilatifard, 2014). Early work in decoding the cause of genetic disease uncovered several examples in which mutations in regulatory regions rather than coding sequence was associated with disease phenotypes (Kleinjan & Lettice, 2008). For instance thalassemia (a blood disorder resulting from imbalance levels of the oxygen carrying factors α-globin and β-globin) can be caused by mutations in the protein coding regions of these gene or by alterations that affect regulatory regions. Point mutation affecting a regulatory region upstream of the sonic hedgehog (SHH) gene, vital regulator of brain-limb development were found to be associated with inherited preaxial polydactyl (Lettice et al., 2003). Mutations causing preaxial polydactyl occur in a limb-specific enhancer resulting in limb rather than brain defect (Lettice et al., 2003).
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