β-globin LCR and intron elements cooperate and direct spatial reorganization for gene therapy

The Locus Control Region (LCR) requires intronic elements within b-globin transgenes to direct high level expression at all ectopic integration sites. However, these essential intronic elements cannot be transmitted through retrovirus vectors and their deletion may compromise the therapeutic potential for gene therapy. Here, we systematically regenerate functional bglobin intron 2 elements that rescue LCR activity directed by 5′HS3. Evaluation in transgenic mice demonstrates that an Oct-1 binding site and an enhancer in the intron cooperate to increase expression levels from LCR globin transgenes. Replacement of the intronic AT-rich region with the Igμ 3′MAR rescues LCR activity in single copy transgenic mice. Importantly, a combination of the Oct-1 site, Igm 39MAR and intronic enhancer in the BGT158 cassette directs more consistent levels of expression in transgenic mice. By introducing intron-modified transgenes into the same genomic integration site in erythroid cells, we show that BGT158 has the greatest transcriptional induction. 3D DNA FISH establishes that induction stimulates this small 5′HS3 containing transgene and the endogenous locus to spatially reorganize towards more central locations in erythroid nuclei. Electron Spectroscopic Imaging (ESI) of chromatin fibers demonstrates that ultrastructural heterochromatin is primarily perinuclear and does not reorganize. Finally, we transmit intron-modified globin transgenes through insulated self-inactivating (SIN) lentivirus vectors into erythroid cells. We show efficient transfer and robust mRNA and protein expression by the BGT158 vector, and virus titer improvements mediated by the modified intron 2 in the presence of an LCR cassette composed of 5′HS2-4. Our results have important implications for the mechanism of LCR activity at ectopic integration sites. The modified transgenes are the first to transfer intronic elements that potentiate LCR activity and are designed to facilitate correction of hemoglobinopathies using single copy vectors

Global Chromatin Architecture Reflects Pluripotency and Lineage Commitment in the Early Mouse Embryo

An open chromatin architecture devoid of compact chromatin is thought to be associated with pluripotency in embryonic stem cells. Establishing this distinct epigenetic state may also be required for somatic cell reprogramming. However, there has been little direct examination of global structural domains of chromatin during the founding and loss of pluripotency that occurs in preimplantation mouse development. Here, we used electron spectroscopic imaging to examine large-scale chromatin structural changes during the transition from one-cell to early postimplantation stage embryos. In one-cell embryos chromatin was extensively dispersed with no noticeable accumulation at the nuclear envelope. Major changes were observed from one-cell to two-cell stage embryos, where chromatin became confined to discrete blocks of compaction and with an increased concentration at the nuclear envelope. In eight-cell embryos and pluripotent epiblast cells, chromatin was primarily distributed as an extended meshwork of uncompacted fibres and was indistinguishable from chromatin organization in embryonic stem cells. In contrast, lineage-committed trophectoderm and primitive endoderm cells, and the stem cell lines derived from these tissues, displayed higher levels of chromatin compaction, suggesting an association between developmental potential and chromatin organisation. We examined this association in vivo and found that deletion of Oct4, a factor required for pluripotency, caused the formation of large blocks of compact chromatin in putative epiblast cells. Together, these studies show that an open chromatin architecture is established in the embryonic lineages during development and is sufficient to distinguish pluripotent cells from tissue-restricted progenitor cells

Visualizing the Structural Basis of Genome Silencing

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.Ph

Visualizing the Structural Basis of Genome Silencing

Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.Ph

Loss of pluripotency results in chromatin compaction in epiblast cells.

<p>Left panels show fluorescence microscopy of Oct4 immuno-labelled embryos; wildtype Oct4^+/+, heterozygous Oct4^−/+ (A), and null mutants Oct4^−/− (B). Panels in the second column show low magnification mass-sensitive image of the area indicated in fluorescence images. Merged phosphorus and nitrogen maps are shown in the right panels. Panels labelled 3 and 4 are from a separate null embryo (not shown). Epiblast cells in control embryos have a thin rim of chromatin on nuclear membrane (arrowheads in A 1,2), whereas Oct4^−/− mutant epiblast cells showed greater accumulation of chromatin at the nuclear periphery (arrowheads in B 1,2). Arrows in B indicate large blocks of compact chromatin, a structure rarely observed in control epiblast nuclei. C, Chromatin compaction was quantified by measuring the distribution of chromatin cluster size in Oct4 mutant and control embryos (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010531#s4" target="_blank">materials and methods</a>for details). Cells within Oct4^−/− embryos contained larger chromatin clusters than in control cells, suggesting that loss of Oct4 leads to increased chromatin compaction levels. Scale bar represents 500 nm in merged images.</p

Chromatin becomes highly dispersed into a mesh of 10 nm fibres between four- and eight-cell stage embryos, with high levels of RNPs in the intervening nucleoplasmic space.

<p>(A), Fluorescence image of a DAPI stained nucleus of a four-cell embryo. (B), low magnification mass image of cell from four-cell embryo (nuclear periphery highlighted with dotted line). Field from boxed region (B) showing phosphorus and nitrogen maps (C,D), nitrogen map after phosphorus subtraction merged with phosphorus map (E). Nuclear pores are indicated (arrowheads, E), nucleolar precursor body (NPB). Higher magnification phosphorus map (F) and merged with nitrogen (G) of a field from region shown in C–E. DCh, dispersed chromatin; CCh, compact chromatin blocks. H, higher magnification of a field from region in F,G. Fields delineated with dotted lines (F,H) contain RNP structures, characterized by phosphorus-rich granules, but of a lower phosphorus to nitrogen ratio than chromatin fibres. Arrowheads (H) indicate 10 nm chromatin fibres. (I), Fluorescence image of a DAPI stained nucleus of an eight-cell embryo. (J), low magnification mass image of a cell from eight-cell embryo (nuclear periphery highlighted with dotted line). K, field shown in J of the merge of phosphorus and nitrogen signals. L, high magnification of field in K. Arrows serve as fiduciaries between K, L and M, and also indicate two nuclear pores. Area indicated with dotted lines shows high concentration of RNPs in nucleoplasm (L). M, high magnification of field in L. Arrowheads indicate 10 nm chromatin fibres in compact chromatin region at nuclear envelope. Scale bars in A and I represents 5 µm. Scale bar in C represents 500 nm (C–E), 250 nm (F,G) and 125 nm (H). Scale bar in K represents 500 nm (K), 250 nm (L) and 125 nm (M).</p

Global chromatin architecture in <i>in vitro</i> stem cell populations is similar to their <i>in vivo</i> counterparts in the embryo.

<p>ESI images of stem cells are shown in the left column (embryonic stem cells (ES), epiblast stem cells (EpiSCs), trophectoderm stem cells (TS), and extra-embryonic endoderm (XEN). ESI of nuclei of embryo stages from which the stem cells were derived are shown in the right column (E3.5 epiblast, E5.5 epiblast, E5.5 extra-embryonic ectoderm, E3.5 primitive endoderm). Chromatin (yellow) and protein and RNPs (shades of blue) are determined from nitrogen and phosphorus maps. Scale bar represents 500 nm.</p

Major changes in nuclear and global chromatin structure occur in nuclei from the zygote to the E5.5 postimplantation stage embryo.

<p>Major changes in nuclear and global chromatin structure occur in nuclei from the zygote to the E5.5 postimplantation stage embryo.</p