Epigenetic regulation of Mash1 expression

Mash1 is a proneural gene important for specifying the neural fate. The Mash1 locus undergoes specific epigenetic changes in ES cells following neural induction. These include the loss of repressive H3K27 trimethylation and acquisition of H3K9 acetylation at the promoter, switch to an early replication timing and repositioning of the locus away from the nuclear periphery. Here I examine the relationship between nuclear localization and gene expression during neural differentiation and the role of the neuronal repressor REST in silencing Mash1 expression in ES cells. Following neural induction of ES cells, I observed that relocation of the Mash1 locus occurs from day 4-6 whereas overt expression begins at day 6. Mash1 expression was unaffected by REST removal in ES cells as well as the locus localization at the nuclear periphery. In contrast bona fide REST target genes were upregulated in REST -/- cells. Interestingly, among REST targets, loci that were more derepressed upon REST removal showed an interior location (Sthatmin, Synaptophysin), while those more resistant to REST withdrawal, showed a peripheral location (BDNF, Calbidin, Complexin). To ask whether the insulator protein CTCF together with the cohesin complex might be involved in regulating Mash1 in ES cells, I performed ChIP analysis of CTCF and cohesin binding across the Mash1 locus in ES cells and used RNAi to deplete CTCF and cohesin expression. A slight increase in the transcription of Mash1 was seen in cells upon Rad21 knock down, although it was not possible to exclude this was a consequence of delayed cell cycle progression. Finally ES cell lines that carried a Mash1 transgene were created as a tool to look at whether activation of Mash1 can affect the epigenetic properties of neighbouring genes

Characterization of Nucleolus-Associated Domains in Mouse Embryonic Stem Cells

In eukaryotic interphase cells, heterochromatin mostly localizes either at the nucleolar periphery or at the nuclear lamina. Genome localization studies are crucial due to evidence that spatial organization of the genome affects gene function. Nucleolus-associated domains (NADs) are mainly heterochromatic regions that have been mapped only in a handful of mouse and human somatic cells, and in plants. The extent to which changes in NAD localization occur during cellular differentiation remains unknown. In this thesis, we characterize a map of genome-wide NADs in F121-9 mouse embryonic stem cells (mESCs). We identified NADs by deep sequencing chromatin associated with biochemically purified nucleoli and using NADfinder software to call NAD peaks. F121-9 NADs are mostly comprised of genomic regions with inactive or lowly transcribed genes and overlap extensively with lamina-associated domains (LADs) and regions with late replication timing. Similar to somatic mouse embryonic fibroblasts (MEFs), where NADs have been previously characterized by our laboratory, F121-9 mESCs display abundant “Type I” NADs. This subset of NADs frequently associates with nuclear lamina and nucleolar periphery and resembles constitutive heterochromatin. Compared to MEFs, F121-9 mESCs have fewer “Type II” NADs; this subset of NADs is frequently found at the nucleolar periphery but not at the nuclear lamina. mESC NADs are also less enriched in H3K27me3 modified regions compared to MEF NADs. This suggests that Polycomb complex-mediated facultative vii heterochromatin expansion is part of NAD maturation during cellular differentiation. Comparison of MEF and mESC NADs also revealed enrichment of developmentally regulated genes in NADs specific to these cell types. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. Our F121-9 mESC NAD studies identified distinct features of stem cell NADs and will facilitate future studies of genome organization changes during mammalian development

Local rewiring of genome-nuclear lamina interactions by transcription

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning toward the nuclear interior. How this process works and how it impacts flanking chromosomal regions are poorly understood. We addressed these questions by systematic activation or inactivation of individual genes, followed by detailed genome-wide analysis of NL interactions, replication timing, and transcription patterns. Gene activation inside LADs typically causes NL detachment of the entire transcription unit, but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. Inactivation of active genes can lead to increased NL contacts. These extensive datasets are a resource for the analysis of LAD rewiring by transcription and reveal a remarkable flexibility of interphase chromosomes

Nuclear Envelope, Nuclear Lamina, and Inherited Disease

The nuclear envelope is composed of the nuclear membranes, nuclear lamina, and nuclear pore complexes. In recent years, mutations in nuclear-envelope proteins have been shown to cause a surprisingly wide array of inherited diseases. While the mutant proteins are generally expressed in most or all differentiated somatic cells, many mutations cause fairly tissue-specific disorders. Perhaps the most dramatic case is that of mutations in A-type lamins, intermediate filament proteins associated with the inner nuclear membrane. Different mutations in the same lamin proteins have been shown to cause striated muscle diseases, partial lipodystrophy syndromes, a peripheral neuropathy, and disorders with features of severe premature aging. In this review, we summarize fundamental aspects of nuclear envelope structure and function, the inherited diseases caused by mutations in lamins and other nuclear envelope proteins, and possible pathogenic mechanisms

Epigenetic Regulation of Progenitor Cell Commitment by Hdac3

Tissue-specific progenitor cells emerge during development to expand and differentiate into the multiple cell lineages that populate the embryo. Appropriate differentiation of these precursor cells requires coordinated expression of numerous lineage-specific genes and repression of alternative fate programs. Epigenetic regulators are enzymes capable of activating or silencing large genomic domains by altering histone modifications, DNA methylation status and chromatin organization. Although differentiating progenitor cells undergo epigenetic changes and epigenetic factors are required for appropriate cell behavior, the precise mechanism of how these proteins influence cell fate remains unclear. In this dissertation, I examine the role of histone deacetylase 3 in control of neural crest and cardiac progenitor cell commitment. Using Cre-mediated genetic deletion, I generated tissue-specific mouse models to study the function of Hdac3 in both neural crest and cardiac cells. These studies revealed a critical role for Hdac3 in maintaining neural crest proliferation and cell survival through regulation of a core network of factors required for craniofacial development. In cardiac progenitors, Hdac3 maintains appropriate differentiation into the cardiomyocyte, smooth muscle and endothelial cell lineages that make up the developed heart. Hdac3 represses a cardiomyocyte-specific gene program and prevents precocious differentiation of progenitors into the myocyte lineage. Surprisingly, this protein does not require deacetylase activity to repress myocyte commitment, and instead serves as a tether to retain myocyte-specific genomic loci at the nuclear periphery. This novel mechanism of gene repression and lineage specification highlights the role that nuclear architecture plays in controlling transcriptional activity and progenitor cell behavior

Chromosome organization in 4D: insights from C. elegans development

Eukaryotic genome organization is ordered and multilayered, from the nucleosome to chromosomal scales. These layers are not static during development, but are remodeled over time and between tissues. Thus, animal model studies with high spatiotemporal resolution are necessary to understand the various forms and functions of genome organization in vivo. In C. elegans, sequencing- and imaging-based advances have provided insight on how histone modifications, regulatory elements, and large-scale chromosome conformations are established and changed. Recent observations include unexpected physiological roles for topologically associating domains, different roles for the nuclear lamina at different chromatin scales, cell-type-specific enhancer and promoter regulatory grammars, and prevalent compartment variability in early development. Here, we summarize these and other recent findings in C. elegans, and suggest future avenues of research to enrich our in vivo knowledge of the forms and functions of nuclear organization