Chromatin Remodeling and Transcriptional Memory: A Dissertation

Transcriptional regulation of gene expression is critical for all unicellular and multicellular organisms. The ability to selectively induce or repress expression of only a few genes from the entire genome gives cells the ability to respond to changing environmental conditions, grow and proliferate. Multicellular organisms begin life as a single totipotent cell, which undergoes many cell divisions during embryonic and later postnatal development. During this process, the dividing cells of the embryo progressively lose their pluripotency and adopt restricted cell fates. Cell fate restriction leads different cell types to gain unique transcriptional profiles. This transcriptional profile or gene expression pattern not only defines the cell types and restricts the ways in which they can respond to signals, it also has to be faithfully re-established in the progeny of these fate-restricted cells when they divide. Different mechanisms have evolved in multicellular organisms to propagate transcriptional memory of cell identity. Most of mechanisms involve modifications of chromatin such as epigenetic modification of DNA or alterations of associated histones. In contrast to multicellular organisms which have considerable cellular diversity and a long lifespan for which cell fates and transcriptional memory needs to be maintained, single celled budding yeast, Sachharomyces cerevisiae have a life cycle of about 90 minutes in normal nutrient rich conditions. However, even budding yeast have tremendous potential to respond to changing environmental conditions like nutrient availability by inducing expression of various genes. We observed that members of the GAL gene cluster, which encodes genes induced in response to and for metabolizing the sugar galactose, showed heritable transcriptional memory of previous activation. This dissertation thesis describes the studies I have done for my graduate research to define this phenomenon of transcriptional memory at the yeast GALgenes and to determine the mechanism by which it can be formed and inherited. Chapter I gives an introduction to different mechanisms of establishing transcriptional memory in unicellular and multicellular organisms. Chromatin based mechanisms have been well studied in multicellular organisms but not observed in budding yeast. We compare chromatin based or nuclear inheritance with cytoplasmic inheritance that can be observed in yeast. Chapter II describes work done to define the phenomenon of transcriptional memory at GAL1 gene. We define this as a faster rate of induction of the GAL1 gene, compared to a naïve gene, after a brief period of repression. We show that this cellular memory persists through mitosis and can be passed on to the next generation. We also show that chromatin remodeling enzymes appear to be required for rapid reinduction, raising the question if yeast may also possess chromatin associated, nuclear mechanisms for cellular memory. Chapter III describes experiments that show that cellular memory observed at GAL1 is cytoplasmic in nature and also compares our work with similar examples observed recently by other groups. Finally, Chapter IV offers a perspective of the significance of such cellular memory mechanisms in budding yeast and outlines some potential further experiments to better understand the control of GAL1 induction kinetics

SWI/SNF is required for transcriptional memory at the yeast GAL gene cluster

Post-translational modification of nucleosomal histones has been suggested to contribute to epigenetic transcriptional memory. We describe a case of transcriptional memory in yeast where the rate of transcriptional induction of GAL1 is regulated by the prior expression state. This epigenetic state is inherited by daughter cells, but does not require the histone acetyltransferase, Gcn5p, the histone ubiquitinylating enzyme, Rad6p, or the histone methylases, Dot1p, Set1p, or Set2p. In contrast, we show that the ATP-dependent chromatin remodeling enzyme, SWI/SNF, is essential for transcriptional memory at GAL1. Genetic studies indicate that SWI/SNF controls transcriptional memory by antagonizing ISWI-like chromatin remodeling enzymes

Hybridization and immobilization of long ds-DNAs on polystyrene microspheres

Here, we develop a system where long double-stranded DNAs (ds-DNAs) are immobilized on the surface of a polystyrene (PS) microsphere. A simple synthetic strategy is adopted in order to achieve this goal in which a single DNA chain is anchored by one of its extremities to a latex (PS) microsphere. We chose hybridization as a unique method to attach long ds-DNA chains in solution with oligonucleotides grafted on modified aminated polystyrene microspheres. The DNAs chosen were of various sizes and sources: T7A1 DNA (4.48 kilobase pair (kbp)), a plasmid DNA; and DeltaDIIIT7 DNA (39.34 kbp), a mutant of bacteriophage T7 DNA. The temperature dependence of the kinetics of hybridization of T7A1 DNA (4.48 kbp) in solution with an appropriate oligonucleotide (20-mer sequence) grafted on modified aminated polystyrene microspheres yielded a value of activation energy of similar to5.3 kcal/mol, consistent with a non-diffusion-controlled mechanism

Polycomb Repressive Complex 1 Generates Discrete Compacted Domains that Change during Differentiation

Master regulatory genes require stable silencing by the polycomb group (PcG) to prevent misexpression during differentiation and development. Some PcG proteins covalently modify histones, which contributes to heritable repression. The role for other effects on chromatin structure is less understood. We characterized the organization of PcG target genes in ESCs and neural progenitors using 5C and super-resolution microscopy. The genomic loci of repressed PcG targets formed discrete, small (20-140 Kb) domains of tight interaction that corresponded to locations bound by canonical polycomb repressive complex 1 (PRC1). These domains changed during differentiation as PRC1 binding changed. Their formation depended upon the Polyhomeotic component of canonical PRC1 and occurred independently of PRC1-catalyzed ubiquitylation. PRC1 domains differ from topologically associating domains in size and boundary characteristics. These domains have the potential to play a key role in transmitting epigenetic silencing of PcG targets by linking PRC1 to formation of a repressive higher-order structure

Combined modulation of polycomb and trithorax genes rejuvenates β cell replication

Inadequate functional β cell mass underlies both type 1 and type 2 diabetes. β Cell growth and regeneration also decrease with age through mechanisms that are not fully understood. Age-dependent loss of enhancer of zeste homolog 2 (EZH2) prevents adult β cell replication through derepression of the gene encoding cyclin-dependent kinase inhibitor 2a (INK4a). We investigated whether replenishing EZH2 could reverse the age-dependent increase of Ink4a transcription. We generated an inducible pancreatic β cell–specific Ezh2 transgenic mouse model and showed that transgene expression of Ezh2 was sufficient to increase β cell replication and regeneration in young adult mice. In mice older than 8 months, induction of Ezh2 was unable to repress Ink4a. Older mice had an enrichment of a trithorax group (TrxG) protein complex at the Ink4a locus. Knockdown of TrxG complex components, in conjunction with expression of Ezh2, resulted in Ink4a repression and increased replication of β cells in aged mice. These results indicate that combined modulation of polycomb group proteins, such as EZH2, along with TrxG proteins to repress Ink4a can rejuvenate the replication capacity of aged β cells. This study provides potential therapeutic targets for expansion of adult β cell mass