The architecture of human chromosomes in interphase nuclei is still largely unknown. Microscopy studies have indicated that specific regions of chromosomes are located in close proximity to the nuclear lamina (NL). This has led to the idea that certain genomic elements may be attached to the NL, which may contribute to the spatial organization of chromosomes inside the nucleus. However, sequences in the human genome that interact with the NL in vivo have not been identified. Here we construct a high-resolution map of the interaction sites of the entire genome with NL components in human fibroblasts. This map shows that genome-lamina interactions occur through more than 1,300 sharply defined large domains 0.1-10 megabases in size. These lamina-associated domains (LADs) are typified by low gene-expression levels, indicating that LADs represent a repressive chromatin environment. The borders of LADs are demarcated by the insulator protein CTCF, by promoters that are oriented away from LADs, or by CpG islands, suggesting possible mechanisms of LAD confinement. Taken together, these results demonstrate that the human genome is divided into large, discrete domains that are units of chromosome organization within the nucleus.

Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions

Lipids are vital components of many biological processes and crucial in the pathogenesis of numerous common diseases, but the specific mechanisms coupling intracellular lipids to biological targets and signalling pathways are not well understood. This is particularly the case for cells burdened with high lipid storage, trafficking and signalling capacity such as adipocytes and macrophages. Here, we discuss the central role of lipid chaperones — the fatty acid-binding proteins (FABPs) — in lipid-mediated biological processes and systemic metabolic homeostasis through the regulation of diverse lipid signals, and highlight their therapeutic significance. Pharmacological agents that modify FABP function may provide tissue-specific or cell-type-specific control of lipid signalling pathways, inflammatory responses and metabolic regulation, potentially providing a new class of drugs for diseases such as obesity, diabetes and atherosclerosis.

/pdf/fatty-acid-binding-proteins-role-in-metabolic-diseases-and-u7ttdl83np.pdf

Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets.

https://www.cell.com/developmental-cell/pdf/S1534-5807(05)00421-1.pdf

Hyperdynamic Plasticity of Chromatin Proteins in Pluripotent Embryonic Stem Cells

Autophagy is a catabolic process involving self-digestion of cellular organelles during starvation as a means of cell survival; however, if it proceeds to completion, autophagy can lead to cell death. Autophagy is also a haploinsufficient tumor suppressor mechanism for mammary tumorigenesis, as the essential autophagy regulator beclin1 is monoallelically deleted in breast carcinomas. However, the mechanism by which autophagy suppresses breast cancer remains elusive. Here we show that allelic loss of beclin1 and defective autophagy sensitized mammary epithelial cells to metabolic stress and accelerated lumen formation in mammary acini. Autophagy defects also activated the DNA damage response in vitro and in mammary tumors in vivo, promoted gene amplification, and synergized with defective apoptosis to promote mammary tumorigenesis. Therefore, we propose that autophagy limits metabolic stress to protect the genome, and that defective autophagy increases DNA damage and genomic instability that ultimately facilitate breast cancer progression.

/pdf/autophagy-mitigates-metabolic-stress-and-genome-damage-in-4adtplmbaz.pdf

Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis

What makes a stem cell is still poorly understood. Recent studies have uncovered that chromatin might hold some of the keys to how embryonic stem cells maintain their pluripotency, their ability to self-renew and induce lineage specification. Embryonic stem (ES) cells are unique in that they are pluripotent and have the ability to self-renew. The molecular mechanisms that underlie these two fundamental properties are largely unknown. We discuss how unique properties of chromatin in ES cells contribute to the maintenance of pluripotency and the determination of differentiation properties.

Chromatin in pluripotent embryonic stem cells and differentiation

This disclosure provides a system for creating cloned cells and embryos that are genetically modified. Cells are treated to increase expression of telomerase and potentially extend replicative capacity. One or more genetic modifications is made to inactivate a gene or confer desirable features, growing and selecting the cells as needed. The modified nucleus can then be transferred to a suitable recipient cell, which can then be used to grow an embryo with the conferred attributes. This technology makes it possible to create embryos, animals and embryonic cell lines with multiple genetic modifications, including homozygously inactivated genes and gene substitutions.

Use of telomerase reverse transcriptase to create homozygous knockout animals

Co-integration and expression of bacterial and genomic transgenes in the pancreatic and intestinal tissues of transgenic mice.

For many years the lack of germline competent embryonic stem cell lines in livestock meant that the targeted modification of endogenous genes was not possible in these species. The demonstration that livestock could be cloned by nuclear transfer from cultured somatic cells has now provided an alternative route to accomplish gene targeting. This chapter describes protocols for culturing primary sheep fibroblasts, introducing and selecting targeted modifications into them and then using these modified cells in nuclear transfer experiments.

Genetic modification of sheep by nuclear transfer with gene-targeted somatic cells.

This invention provides a system for rapid determination of pharmacologic effects on target tissue types in cell populations cultured in vitro The cells contain a promoter-reporter construct that reflects a toxicologic or metabolic change caused by the agent being screened The promoter is taken from a gene known to be up- or down-regulated according to the metabolic state of the cell, and linked to a reporter gene that provides an external signal for monitoring promoter activity The promoter-reporter cells may be produced by placing these genetic alterations into a line of human embryonic stem cells, bulking up the cells to any extent desired, and then differentiating the cells into the desired tissue type This disclosure explains some of the powerful features of the promoter-reporter cells of this invention, and shows various ways the skilled reader can use the invention for pharmaceutical development and testing, or to monitor graft survival

Promoter-reporter cells for determining drug metabolism, drug interactions, and the effects of allotype variation

We have generated transgenic mice which express the gene encoding Escherichia coli nitro reductase (NTR) specifically in the luminal epithelial cells of the mammary gland and the glial cells of the brain. The enzyme activates an antitumour drug CB1954, to produce a cross-linking agent that kills all cells expressing the enzyme. We have shown that administration of the antitumour drug CB 1954 rapidly and selectively kills these cells. Original experiments demonstrated the ability to ablate the luminal cells in the mammary gland with no apparent bystander effect. Subsequently, astrocytes expressing nitroreductase under the targeting of the GFAP promoter were selectively ablated following administration of the prodrug CB 1954 produces a degeneration of granular neurones due to changes in glutamate levels. Recent experiments demonstrated inhibition of myc-dependent mammary tumours using the same enzyme (nitroreductase) — prodrug (CB 1954), combination. Owing to the ease of control of NTR-mediated cell ablation, we anticipate that this system will supersede herpes simplex virus type 1 thymidine kinase. There are Widespread potential applications for this approach in the dissection of complex cellular interactions during development and in the adult organism. The present transgenic models also have important applications for the study in vivo of novel prodrugs that can be selected for variable degrees of bystander effects. Such studies will have particular significance for those groups advocating the use of NTR as an appropriate enzyme for gene-directed enzyme prodrug therapy by providing models of a wide range of human disease for mechanistic and therapeutic experimentation. The results clearly demonstrate that the model has potential to study chemoprevention and fundamental questions on cell-cell interactions in cell biology.

A. John Clark

Papers

Use of telomerase reverse transcriptase to create homozygous knockout animals

Co-integration and expression of bacterial and genomic transgenes in the pancreatic and intestinal tissues of transgenic mice.

Genetic modification of sheep by nuclear transfer with gene-targeted somatic cells.

Promoter-reporter cells for determining drug metabolism, drug interactions, and the effects of allotype variation

Development of novel selective cell ablation in the mammary gland and brain to study cell-cell interactions and chemoprevention.