Marc R. Gartenberg - US grants

The long-range goal of this project is to understand parameters which determine spatial and functional organization of DNA in the nucleus. Specifically, the work will identify chromosomal DNA sites which are anchored to immobile objects in yeast Saccharomyces cerevisiae. DNA immobilization by specific DNA sequences may represent a fundamental unit of chromosome architecture; DNA anchors may segregate chromosomal DNA into independent structural domains for DNA packaging, regulated gene expression and positioning within the nucleus. DNA anchoring sequences and associated proteins will be characterized using a newly developed molecular biological technique. The approach employs site specific recombination in vivo to generate well-defined DNA rings which contain a single promoter and a DNA fragment to be tested. Transcription of anchored DNA rings ina yeast topoisomerase mutants leads to diagnostic changes in DNA topology, in accordance with the twin domain model of transcriptional supercoiling. models of DNA organization based on analyses of biochemically manipulated chromosomes, termed nuclear scaffold, argue that DNA is arranged into loops with specific DNA segments anchoring the bases of the loops to an organizational framework. The excision methodology will be used to investigate whether sequences which bind yeast scaffold preparations function as DNA anchors in vivo. Scaffold attachment regions (SARs) to be examined include chromosomal components such as telomeres, silencers, centromeres and origins of replication. DNA anchor formation. The cell- cycle dependent anchoring of centromeres and replication origins will be determined in synchronized cell cultures. A selection system will be developed to isolate sequences with DNA anchoring activity from libraries of the sequenced yeast Chromosome III. The DNA loop structure of specific chromosomal regions will be determined by mapping the DNA anchoring elements which reside within the regions. The influence of genomic DNA anchors on plasmid partitioning and gene expression will be examined.

The long-range goal of this project is to determine how large chromosomal domains are transcriptionally inactivated by packaging DNA into heterochromatin. This form of heritable repression inhibits some genes permanently and others in a developmentally regulated manner. Chromosomal translocations that move genes between transcriptionally- poised domains and heterochromatic domains lead to aberrant expression states and diseases. Leukemias and other cancer frequently arise from the inappropriate expression of translocated genes. Genom rearrangements that position a gene near heterochromatin result in an epigenetic pattern of expression: the gene is "off" in some cells and "on" in others and these expression states are maintained stably across many generations. This bimodal pattern of expression is attributed to the clonal propagation of heterochromatin structure that has spread along DNA to varying extents in different cells. In yeast Saccharomyces cerevisiae, genes near telomeres and the silent mating-type loci are repressed by a structure, termed silent chromatin, that bears remarkable similarity to heterochromatin. At the silent mating loci, repression requires cis-actin regulatory sequences, termed silencers, and a set of non-histone chromatin components known as the Sir proteins. To identify basic principles underling heterochromatin- like repression, a host of novel approaches will be used to investigate the structure, stability, and inheritance of silent chromatin in yeast. A primary strategy will involve the use of inducible site-specific recombination to form DNA rings of silent chromatin in vivo. Biochemically isolated rings will be used to analyze the structure and composition of silent chromatin and perform functional studies of transcriptional repression. To investigate silent chromatin stability, the persistence of transcriptional repression in rings uncoupled from chromosomal silencers will be analyzed in vivo. The heritable propagation of silent chromatin will be investigated by analyzing the establishment of the repressive structure on newly replicated templates. Factors that can function as boundaries of silent chromosomal domains will be identify by a simple genetic selection.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Large regions of eukaryotic chromosomes are transcriptionally repressed due to DNA packaging in heterochromatin, a structure that is heritably propagated from one mitotic cycle to the next. Other chromosomal regions are transcriptionally active for brief periods during early development only to be shut-off permanently by repressive structures related to heterochromatin. Work in the Gartenberg lab has focused on mechanisms of heterochromatic gene repression in budding yeast. The hallmark of our approach over the last decade has been the use of site-specific recombination to physically uncouple silenced loci from other chromosomal sequences and activities. This has led to discoveries regarding the role of cis-acting sequences, DNA replication and nuclear localization in silencing. It did not escape our attention that liberating pieces of heterochromatin might facilitate their purification. Previously we developed a differential centrifugation approach to biochemically enrich DNA circles formed by site-specific recombination in vivo. We showed that the isolated material retained characteristics of silenced chromatin. We have since engineered a tandem affinity purification reagent to further purify the rings. In this collaboration with the John Yates lab we aim to identify the protein constituents of yeast heterochromatin by mass spectrometry.

DESCRIPTION (provided by applicant): Cohesin is a multi-subunit protein complex that orchestrates proper segregation of chromosomes by mediating cohesion between sister chromatids. Defects in the cohesion pathway lead to certain developmental diseases, as well as chromosome segregation defects like those in cancer. Cohesin binds centromeres where it helps mount chromatids onto spindle microtubules. The complex also binds discrete sites in relation to the transcriptional landscape of the genome and recent work suggests that cohesin plays significant roles in transcriptional regulation. In the model eukaryote yeast Saccharomyces cerevisiae, cohesin binding is dynamic in euchromatic domains where genes are poised for transcription. The complex is also heavily enriched at heterochromatic domains where transcription is suppressed. These domains functionally resemble heterochromatin of higher eukaryotes and have been useful in deciphering principles of regional inactivation and epigenetic control. The Gartenberg laboratory has used the yeast system to define principles of chromosome architecture as they relate to chromosome function. Using site-specific recombination and other novel molecular genetic strategies, the laboratory has focused on how yeast heterochromatin intersects with the sister chromatid cohesion pathway. The laboratory recently discovered that Sir2, the evolutionarily conserved protein deacetylase responsible for yeast heterochromatin assembly, retains cohesin at heterochromatic loci. Aim 1 investigates the molecular basis of this event and the functional consequences of cohesin on heterochromatic silencing and genome stability. Other transcriptional regulators also appear to direct cohesin to unexpressed genes. In aim 2, the tools developed for the study heterochromatic cohesion will be used to determine how cohesin arrives at non-heterochromatic genes and how the complex is utilized when transcription is activated. One particular chromosomal domain under study contains heterochromatin juxtaposed to an active tRNA gene. Previously the Gartenberg laboratory found that the tRNA gene is required for heterochromatic cohesion. Now the laboratory has learned that the domain localizes with nuclear pores in a cohesin-dependent manner. In aim 3, the molecular basis for this new dimension in higher order chromosomal organization will be investigated. PUBLIC HEALTH RELEVANCE: Human diseases caused by mutations in the cohesin pathway (cohesinopathies) display a host of transcriptional and/or heterochromatin structure defects. To understand what goes wrong in these diseases, we need to first fully account for how cohesin normally functions at transcriptionally repressed and activated genes. Using budding yeast as a model system, we will investigate the behavior of cohesin on euchromatic genes and how the complex influences the silencing, structure and stability of heterochromatic loci.