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Globin Gene Expression Is Reprogrammed in Chimeras Generated by Injecting Adult Hematopoietic Stem Cells into Mouse Blastocysts

To elucidate whether the differentiation capacity of hematopoietic stem cells (HSCs) is influenced by specific microenvironments, adult mouse bone marrow-derived HSCs were injected into mouse blastocysts. Embryos developing from injected blastocysts contained donor-derived cells at various developmental stages, and progeny of the stem cells were detected in hematopoietic tissues. Thus, HSCs derived from an adult animal survive after injection into blastocysts and are able to participate in hematopoietic development. We further find that the erythroid progeny of transplanted adult HSCs express embryonic/fetal-type globin genes and, conversely, that embryonic and fetal progenitor cells transplanted into adult recipients transcribe the adult-type globin gene. Thus, the developmental potential of adult HSCs is evidently more plastic than previously thought, and the developmental stage of the hematopoietic microenvironment controls the developmental fate of transplanted progenitor cells.

The Chicken Lysozyme Locus as a Paradigm for the Complex Developmental Regulation of Eukaryotic Gene Loci

Gene loci of higher organisms have complex structural features. In some cases their coding regions occupy many, even hundreds, of kilobases of DNA. Additionally, the sequences that contain the information for the correct spatial and temporal regulation of a particular gene locus during development often exceed the extensions of the coding region by severalfold. The question of what type of information is encoded in these vast amounts of DNA has puzzled researchers from the beginning. It is now clear that eukaryotic genes are regulated by a number of different cis-regulatory elements distributed over large distances. A convenient way to assay the number and the distribution of cis-regulatory elements has been the mapping of DHS in chromatin. Such local chromatin perturbations are in most cases caused by the binding of transcription factors to their cognate DNA sequences. The pattern of DHS can undergo dramatic developmental changes, indicating a change in the activity of cis-regulatory elements. In addition, the analysis of protein-DNA interactions at a single-nucleotide resolution level in vivo has demonstrated that, depending on the developmental stage, different combinations of transcription factors can occupy the same cis-regulatory element (1, 2). These experiments indicate that the transcriptional activation of a gene locus is achieved by the cooperation of several different cisregulatory elements, which, in turn, assemble transcription factors in a sequential, developmentally controlled fashion. However, the assembly of active transcription factor complexes on natural genes does not occur on naked DNA but in a chromatin context, where nucleosome-DNA interactions have to be counteracted. Hence, the activation of a gene locus requires at least the following steps: the perturbation of chromatin structure by the binding of transcription factors on cis-regulatory elements, the developmentally controlled reorganization of transcription factor complexes, the assembly of the basal transcription machinery and its interaction with upstream regulatory elements, the onset of mRNA synthesis, and, in many cases, the maintenance of an active transcriptional state during multiple rounds of DNA synthesis. How can the molecular basis of locus activation be experimentally studied? While the basal activities of individual cisregulatory elements of particular gene loci can be analyzed by transient and stable transfection experiments, the molecular mechanism of activation of a gene locus from the transcriptionally silent state can only be studied in a developing system, preferentially in transgenic animals. The ideal model locus should be small, thus facilitating the manipulation of individual cis-regulatory elements within the context of an entire genomic locus, and it should be extensively characterized on the molecular level. In addition, to dissect the role of different cis-regulatory elements in the developmental control of gene locus activation, it should be possible to follow cell differentiation experimentally, thus enabling the linkage of a stage-specific chromatin structure with the transcriptional activity of the gene. Here, we summarize recent studies on the molecular basis of the transcriptional activation of the chicken lysozyme locus, which may serve as a paradigm for other developmentally regulated eukaryotic gene loci.

ROLE OF POSITIVE AND NEGATIVE CIS-REGULATORY ELEMENTS IN THE TRANSCRIPTIONAL ACTIVATION OF THE LYSOZYME LOCUS IN DEVELOPING MACROPHAGES OF TRANSGENIC MICE

Expression of the chicken lysozyme locus in macrophages is regulated by at least six different positive and negative cis-regulatory elements. Chromatin of the chicken lysozyme locus is gradually reorganized during macrophage differentiation, indicating that each cis-regulatory element is activated at a different developmental stage. Irrespective of their differential developmental activation, individual cis-regulatory regions are capable of driving transcription of the lysozyme gene in mature macrophages of transgenic mice. In order to examine the role of different cis-regulatory regions in lysozyme locus activation, we analyzed the time course of transcriptional up-regulation of deletion mutants of the lysozyme locus in a new in vitro differentiation system based on enriched primary macrophage precursor cells from the bone marrow of transgenic mice. We show that constructs carrying cis-regulatory elements which are structurally reorganized early in development are also transcriptionally active at an early stage. A construct in which the early enhancer has been deleted shows a delay in transcriptional activation. The presence or absence of a negative regulatory element has no influence on the time course of transcriptional activation of the lysozyme locus.

Prerequisites for tissue specific and position independent expression of a gene locus in transgenic mice.

Abstract The elucidation of general parameters influencing the transcriptional activation of gene loci at distinct stages of development is an essential prerequisite for a reproducibly successful gene transfer in both gene therapy protocols and biotechnology. Up to now research has focused mostly on the identification and characterization of individual cis-regulatory elements by transient transfection and in vitro assays. However, the most relevant assay system to test gene constructs designed for gene therapy protocols is the transgenic animal. In such an experimental system exogenous genes are usually integrated randomly in the chromatin. For gene constructs not fulfilling the requirements for correct gene locus activation this can lead to genomic position effects on gene expression. The consequences are highly variable expression levels and a disturbance of temporal and spatial expression patterns. Hence it is important to examine how cis-elements function in a chromatin context, and how they cooperate during the developmentally controlled activation of an entire gene locus. One among a few gene loci which are sufficiently characterized to enable such investigations is the chicken lysozyme locus. This review summarizes recent results aimed at identifying the necessary prerequisites for a reproducibly correct expression of the lysozyme locus in transgenic mice and the implications of our findings for gene transfer. The complete lysozyme locus is expressed independent of the chromosomal position and at a high level in macrophages of transgenic mice. Correct transgene regulation requires the cooperation of all cis-regulatory elements. Chromatin of the lysozyme locus in both the active and the inactive state is highly structured. Each cis-regulatory element on the chicken lysozyme locus is organized in its own unique chromatin environment, with nucleosomes specifically placed on specific sequences. The transcriptional activation of the lysozyme locus is accompanied by extensive rearrangements of its chromatin structure, which are disturbed when the transgenes are subjects to genomic position effects. Based on these results, we propose that a complete locus is resistant to genomic position effects, and that a distinct chromatin architecture of a gene locus is required for its correct activation.