Comparative analysis of chromatin landscape in regulatory regions of human housekeeping and tissue specific genes

BACKGROUND: Global regulatory mechanisms involving chromatin assembly and remodelling in the promoter regions of genes is implicated in eukaryotic transcription control especially for genes subjected to spatial and temporal regulation. The potential to utilise global regulatory mechanisms for controlling gene expression might depend upon the architecture of the chromatin in and around the gene. In-silico analysis can yield important insights into this aspect, facilitating comparison of two or more classes of genes comprising of a large number of genes within each group. RESULTS: In the present study, we carried out a comparative analysis of chromatin characteristics in terms of the scaffold/matrix attachment regions, nucleosome formation potential and the occurrence of repetitive sequences, in the upstream regulatory regions of housekeeping and tissue specific genes. Our data show that putative scaffold/matrix attachment regions are more abundant and nucleosome formation potential is higher in the 5' regions of tissue specific genes as compared to the housekeeping genes. CONCLUSION: The differences in the chromatin features between the two groups of genes indicate the involvement of chromatin organisation in the control of gene expression. The presence of global regulatory mechanisms mediated through chromatin organisation can decrease the burden of invoking gene specific regulators for maintenance of the active/silenced state of gene expression. This could partially explain the lower number of genes estimated in the human genome

Complexity and conservation of regulatory landscapes underlie evolutionary resilience of mammalian gene expression.

To gain insight into how mammalian gene expression is controlled by rapidly evolving regulatory elements, we jointly analysed promoter and enhancer activity with downstream transcription levels in liver samples from 15 species. Genes associated with complex regulatory landscapes generally exhibit high expression levels that remain evolutionarily stable. While the number of regulatory elements is the key driver of transcriptional output and resilience, regulatory conservation matters: elements active across mammals most effectively stabilize gene expression. In contrast, recently evolved enhancers typically contribute weakly, consistent with their high evolutionary plasticity. These effects are observed across the entire mammalian clade and are robust to potential confounders, such as the gene expression level. Using liver as a representative somatic tissue, our results illuminate how the evolutionary stability of gene expression is profoundly entwined with both the number and conservation of surrounding promoters and enhancers

DNA methylation in the marbled crayfish Procambarus virginalis

The all-female marbled crayfish Procambarus virginalis is a freshwater crayfish which is the only known obligatory parthenogen among the decapod crustaceans. Marbled crayfish are recent descendants of the sexually reproducing slough crayfish Procambarus fallax and have most likely emerged through a recent evolutionary macromutation event in P. fallax. Marbled crayfish reproduce by apomictic parthenogenesis, where oocytes do not undergo meiosis and all offspring are genetically identical clones of the mother. Nevertheless, marbled crayfish show a high degree of phenotypic variation and are a highly invasive species, where (through parthenogenesis) a single animal can establish a whole population. Moreover, they have been distributed via the pet trade and anthropogenic releases, and have formed stable populations in a variety of ecological habitats. Earlier this year, our group performed whole-genome sequencing for 11 marbled crayfish animals from different populations and countries, and found only four non-synonymous single nucleotide variances in coding regions. Since the marbled crayfish’s remarkable adaptability is not due to genetic variability, it is crucial to investigate epigenetic programming in this organism. I present here a comprehensive analysis of DNA methylation in marbled crayfish. Whole- genome bisulfite sequencing data was used to directly compare methylation patterns from multiple replicates in different tissues and from different marbled crayfish and Procambarus fallax animals. These methylation maps were integrated with RNA-seq and ATAC- seq data to comprehensively analyse the interplay between DNA methylation, chromatin accessibility, and gene expression. I found 18% of CpGs in marbled crayfish to be methylated. Repeats showed overall low methylation levels, with the exception of a single class of DNA transposons, which was ubiquitously methylated. DNA methylation was mainly targeted to the coding regions of housekeeping genes in marbled crayfish. In contrast to paradigmatic mammalian methylomes, I only observed very moderate methylation differences between tissues for both gene bodies and promoters. I did, however, identify a set of approximately 700 genes that showed a high variance in their methylation across tissues and animals. Gene body methylation was significantly inversely correlated with gene expression variability. Interestingly, the marbled crayfish shows overall lower methylation levels and higher gene expression variability than its parent species P. fallax. Since plasticity in gene expression can be a beneficial trait for adapting to new environments, this trait might contribute to the marbled crayfish’s adaptive and invasive success. The integrative analysis of DNA methylation, chromatin accessibility, and gene expression revealed that genes with highly methylated gene bodies were located in regions of poorly accessible chromatin and showed stable expression patterns. In contrast, lowly methylated genes were found in more accessible chromatin when stably expressed, and in more condensed chromatin when variably expressed. In this context, gene body methylation might function to stabilise gene expression in regions of limited chromatin accessibility. These findings broaden our knowledge of evolutionary conservation of DNA methylation patterns in invertebrates and provide novel insights on the interplay between gene body methylation, chromatin accessibility, and gene expression

Genome-wide gene expression surveys and a transcriptome map in chicken

The chicken (Gallus gallus) is an important model organism in genetics, developmental biology, immunology, evolutionary research, and agricultural science. The completeness of the draft chicken genome sequence provided new possibilities to study genomic changes during evolution by comparing the chicken genome to that of other species. The development of long oligonucleotide microarrays based on the genome sequence made it possible to survey genome-wide gene expression in chicken. This thesis describes two gene expression surveys across a range of healthy chicken tissues in both adult and embryonic stages. Specifically, we focus on the mechanisms of regulation of gene transcription and their evolution in the vertebrate genome. Chapter 1 provides a brief history of the chicken as a model organism in biological and genomics research. In particular a brief overview is presented about expression profiling experiments, followed by an introduction to gene transcription regulation in general. Finally, the aim and outline of this thesis is presented. An important aim of this thesis is to generate surveys of genome-wide gene expression data in chicken using microarrays. In chapter 2, we introduce microarray data normalization including background correction, within-array normalization and between-array normalization. Based on these results an analysis approach is recommended for the analysis of two-color microarray data as performed in the experiments described in this thesis. We also briefly explain the relevant methodology for the identification of differentially expressed genes and how to translate resulting gene lists into biological knowledge. Finally, specific issues related to updating microarray probe annotation in farm animals, is discussed. For the analysis of the microarray data in this thesis re-annotation of the probes on the chicken 20K oligoarray was done using the oligoRAP, analysis pipeline. The vast amount of data generated from a single transcriptomics study makes it impossible to extract meaningful biological knowledge by manually going through individual genes from a list with hundreds and thousands of differentially expressed genes. In chapter 3, we present a practical approach using a collection of R/Bioconductor packages to extract biological knowledge from a microarray experiment in farm animals. Furthermore, a locally adaptive statistical procedure (LAP) analysis approach is used to identify differentially expressed chromosomal regions in a microarray experiment. Chapter 4 presents a genome-wide gene expression survey across eight different tissues (brain, bursa of Fabricius, kidney, liver, lung, small intestine, spleen, and thymus from 10-week old chickens) in adult birds using a chicken 20K microarray. To a certain extent, most genes show some tissue-specific pattern of expression. Housekeeping and tissue-specific genes are identified based on gene expression patterns across the eight different tissues. The results show that housekeeping genes are more compact, i.e. are smaller, with shorter, coding sequence length, intron length, and smaller length of the intergenic regions. This observed compactness of housekeeping genes may be a result of selection on economy of transcription during evolution. Furthermore, a comparative analysis of gene expression among mouse, chicken, and frog showed that the expression patterns of orthologous genes are conserved during evolution between mammals, birds, and amphibians. The chicken embryo has been a very popular model for developmental biology. To study the overall gene expression pattern in whole chicken embryos at different developmental stages and/or embryonic tissues, a genome-wide gene expression survey across different developmental and embryonic stages was performed (chapter 5). The study included four different developmental stages (HH stage 3, 10, 15, 22) and eight different embryonic tissues (brain, bursa of Fabricius, heart, kidney, liver, lung, small intestine, and spleen from HH stage 36). We were able to identify several embryonic stage- and tissue-specific genes in our analysis. Genomic features of genes widely expressed under these 12 conditions suggest that widely expressed genes are more compact than tissue-specific genes, confirming the findings described in chapter 4. The analysis of the differentially expressed genes during the different developmental stages of whole embryo indicates a gradual change in gene expression during embryo development. A comparison of the gene expression profiles between the same organs, of adults and embryos reveals both striking similarities as well as differences. The overall goal of this thesis was to improve our understanding of the mechanisms of transcriptional regulation in the chicken. In chapter 6, a transcriptome map for all chicken chromosomes is presented based on the expression data described in chapter 4. The results reveal the presence of two distinct types of chromosomal regions characterized by clusters of highly or lowly expressed genes respectively. Furthermore, these regions show a high correlation with a number of genome characteristics, like gene density, gene length, intron length, and GC content. A comparative analysis between the chicken and human transcriptome maps suggests that the regions with clusters of highly expressed genes are relatively conserved between the two genomes. Our results revealed the presence of a higher order organization of the chicken genome that affects gene expression, confirming similar observations in other species. Finally, in chapter 7 I summarize the main findings and discuss some of the limitations of the analyses described in this thesis. I also discuss the different merits and shortcomings of studying gene expression using either microarrays or next-generation sequencing technology and propose directions for future research. The rapid developments in new-generation sequencing technology will facilitate better coverage and depth of the chicken genome. This will provide a better genome assembly and an improved genome annotation. The sequence-based approaches for studying gene expression will reduce noise levels compared to hybridization-based approaches. Overall, next-generation sequencing is already providing greatly enhance tools to further improve our understanding of the chicken transcriptome and its regulation. <br/