Divergence of Mammalian Higher Order Chromatin Structure Is Associated with Developmental Loci

Several recent studies have examined different aspects of mammalian higher order chromatin structure - replication timing, lamina association and Hi-C inter-locus interactions - and have suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and until now little has been known about the characteristics of any divergent loci present. Here, we generate a dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the vast majority of the detectably orthologous genome. This broad similarity is observed in spite of many loci possessing cell type specific structures. However, we also identify hundreds of regions (from 100 Kb to 2.7 Mb in size) showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. These regions show unusual shifts in human GC content, are unevenly distributed across both genomes, and are enriched in human subtelomeric regions. Divergent regions are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions cause divergent regulation. Particular divergent loci are strikingly enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in the evolution of mammalian developmental programmes. These data suggest that, though relatively rare in the mammalian genome, divergence in higher order chromatin structure has played important roles during evolution

Subtle changes in chromatin loop contact propensity are associated with differential gene regulation and expression.

While genetic variation at chromatin loops is relevant for human disease, the relationships between contact propensity (the probability that loci at loops physically interact), genetics, and gene regulation are unclear. We quantitatively interrogate these relationships by comparing Hi-C and molecular phenotype data across cell types and haplotypes. While chromatin loops consistently form across different cell types, they have subtle quantitative differences in contact frequency that are associated with larger changes in gene expression and H3K27ac. For the vast majority of loci with quantitative differences in contact frequency across haplotypes, the changes in magnitude are smaller than those across cell types; however, the proportional relationships between contact propensity, gene expression, and H3K27ac are consistent. These findings suggest that subtle changes in contact propensity have a biologically meaningful role in gene regulation and could be a mechanism by which regulatory genetic variants in loop anchors mediate effects on expression

Forces driving the three-dimensional folding of eukaryotic genomes

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it

Chromatin organizer CTCF in brain development and behaviour

Chromatin architecture is an important regulator of gene expression, which dictates development. Mutations in one copy of the CTCF chromatin organizer gene cause intellectual disability and autism. Polymorphisms in CTCF have also been associated with increased risk for schizophrenia, a condition that overlaps in biological etiology with autism and intellectual disability. In this thesis, we sought to understand the role of CTCF in neurodevelopment using brain-specific conditional knockout and heterozygote mouse models. Using the Ctcf-null animals, we identify a cell-autonomous role for CTCF in regulating cortical interneuron development in the medial ganglionic eminence (MGE) through the transcriptional control of Lhx6. In the absence of CTCF, MGE-derived cortical interneuron subtypes are inappropriately specified such that their cortical laminar position is altered and there is a reduction in the number of cells expressing PV and SST. These features are rescued with viral-mediated re-expression of Lhx6. In addition, there is a concomitant increase in the expression of Lhx8, which specifies ventral telencephalic cell types in the MGE, indicating CTCF is an important regulator of cell fate choice in the MGE. To model the human condition associated with CTCF mutation, we generated mice heterozygous for Ctcf deletion in the developing brain (CtcfNestinHet). These mice had spontaneous hyperactivity and impaired spatial learning on behavioural testing. In addition to these behaviours, male mice had decreased sociability, altered aggression, and decreased anxiety. Together, this constellation of behaviours is reminiscent of other mouse models of schizophrenia, autism and intellectual disability. In addition, structural MRI revealed that CtcfNestinHet mouse brains had decreased white matter volume, suggestive of hypoconnectivity, a feature commonly attributed to the pathophysiology of autism. There were also significant volume decreases in the cerebellar nuclei, and an increase in the anterior cerebellar lobe. These findings provide further evidence for the emerging role of the cerebellum in cognition and in neurodevelopmental disorders. In summary, this work addresses the consequence of reduced CTCF expression in the developing brain at cellular, structural and behaviour levels, and thus significantly furthers our understanding of chromatin architecture regulation in neurodevelopmental disease

Conservation and divergence in higher order chromatin structure

Aspects of higher order chromatin structure such as replication timing, lamina association and Hi-C inter-locus interactions have been recently studied in several human and mouse cell types and it has been suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and little is known about the characteristics of any divergent loci defined. Here we generate an orthologous dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between different mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the majority of the orthologous genome. This broad similarity is observed in spite of the substantial time since the species diverged, differences in experimental procedures among the datasets examined, and the presence of cell type specific structures at many loci. However, we also identify hundreds of regions showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. Divergent regions are enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in mammalian evolution. They are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions may underlie divergent regulation. Divergent regions show unusual shifts in compositional bias, sequence divergence and are unevenly distributed across both genomes. We investigate the mechanisms of divergence in higher order structure by examining the influence of sequence divergence and also many features of primary level chromatin, such as histone modification and DNA methylation patterns. Using multiple regression, we identify the dominant factors that appear to have shaped the physical structure of the mammalian genome. These data suggest that, though relatively rare, divergence in higher order chromatin structure has played important roles during evolution

Chromatin Structure and Differential Accessibility of Homologous Human Mitotic Metaphase Chromosomes

The human mitotic metaphase chromosome is a product of complex chromatin restructuring during interphase. Metaphase chromosomes exhibit considerable plasticity in condensation. This is evident as distinct regions of accessible and compact chromatin fiber or epigenetic differences in histone and non-histone proteins. Such differences in chromatin condensation have been extensively described along the length of individual mitotic chromosomes but have not been recognized between homologous loci during metaphase. This thesis characterizes localized differences in condensation of homologous metaphase chromosomes that are related to differences in accessibility (DA) of associated DNA probe targets. Reproducible DA was observed for ~10% of locus-specific, short (1.5-5 kb) single copy (SC) DNA probes used in fluorescence in situ hybridization. To investigate the physical and structural organization of chromatin at locus-specific sites, we developed correlated atomic force and fluorescence microscopy imaging. Comparison of centromeric DNA and protein distribution patterns in fixed homologous chromosomes indicated that CENP-B and α-satellite DNA were distributed distinctly from one another and relative to observed centromeric ridge topography. At non-centromeic locations, short DNA probes that did not exhibit DA showed greater accessibility to the accessible chromatin topography on both homologs. Localized differential accessibility between chromosome homologs in metaphase was non-random and reproducible but not unique to known imprinted regions or specific chromosomes. Second, non-random DA was shown to be heritable within a 2 generation family. Third, DNA probe volume and depth measurements of hybridized metaphase chromosomes showed internal differences in chromatin accessibility of homologous regions by super-resolution 3D-structured illumination microscopy. Finally, genomic regions with equivalent accessibility were enriched for epigenetic marks of open interphase chromatin to a greater extent than regions with DA, suggesting that observed structural differences in accessibility may arise during or preceding metaphase chromosome compaction. Inhibition of the topoisomerase IIα-DNA cleavage complex mitigated DA by decreasing DNA superhelicity and axial metaphase chromosome condensation. Inter-homolog probe intensity ratios, depth, and volume between chromosomes treated with a catalytic inhibitor of topoisomerase IIα, were equalized compared to untreated cells. These data altogether suggest that DA is a reflection of allelic differences in metaphase chromosome compaction, dictated by the catenation state of the chromosome

Bases genéticas de la evolución del cerebro humano: estudio evolutivo y funcional del gen NPAS3

Comprender el origen y la evolución del cerebro humano es uno de los desafíos más sobresalientes que enfrenta la ciencia. Los cambios genéticos que llevaron a la adquisición de las capacidades distintivas del cerebro humano están codificados en nuestro genoma. La disponibilidad de más de cincuenta genomas de vertebrados permite hoy reconstruir nuestra historia evolutiva. Nuestra hipótesis es que la adquisición de nuevos patrones de expresión de genes relacionados con el desarrollo y la función cerebral en el linaje humano, habría sido crítica para la evolución de las capacidades cognitivas excepcionales de nuestro cerebro. Haciendo uso de bases de datos públicas de secuencias no codificantes conservadas con evidencia de evolución acelerada en el linaje humano (denominados HAEs por human accelerated elements), se encontró que el factor de transcripción neuronal PAS domain-containing protein 3 (NPAS3) contiene 14 HAEs, el mayor número de HAEs para un solo gen en todo el genoma humano. Usando un ensayo de expresión en peces cebra transgénicos se demostró que 11 de los 14 HAEs son capaces de activar la expresión de la proteína reportera EGFP durante el desarrollo embrionario, particularmente en el sistema nervioso. Además, utilizando ratones transgénicos, se realizó un análisis comparativo estudiando los patrones de expresión de uno de los HAEs de NPAS3 y secuencias ortólogas de chimpancé y ratón. El enhancer humano muestra una extensión del patrón de expresión en el telencéfalo. Este cambio humano específico pudo haber contribuido con la evolución de alguna de las características de nuestro cerebro.Understanding the origin and evolution of the human brain is one of the greatest challenges that today science faces. The genetic changes that led to the acquisition of the distinctive capacities of the human brain are encoded in our genome. The availability of more than fifty vertebrate genomes allows unraveling our evolutionary history. Our hypothesis is that the acquisition of new expression patterns in the human lineage of genes involved with the development and functioning of the brain would have been critical for the evolution of our unique cognitive capacities. Using public databases of human accelerated conserved non coding sequences (HAEs or human accelerated elements), we found that the transcription factor neuronal PAS domain-containing protein 3 (NPAS3) contains 14 HAEs, the largest number detected for a human gene. Using an enhancer transcription assay in transgenic zebrafish we show that 11 out of the 14 HAEs activated the expression of the reporter gen EGFP during zebrafish development in the central nervous system. In addition, using transgenic mice we performed an expression pattern comparative analysis of human, chimpanzee and mouse ortholog sequences of a selected HAE. We found that the human enhancer shows an extended expression pattern in the forebrain. This human-specific change could have contributed to the evolution of some of the distinctive capacities of our brain.Fil:Kamm, Gretel Betiana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina