Epigenetics & chromatin: Interactions and processes

On 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the conference

Transcriptional Regulation of Multi-Gene Loci: Multi-Level Control.

Recent studies indicate that different levels of control operate within multigene loci. In addition to regulatory sequences immediately flanking the genes, there are also elements that act over long distances on more than one gene. Competition for these elements among genes can influence both the level and timing of gene expression during development

Fragmentation of bacteriophage S13 replicative from DNA by restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius.

The restriction enzymes Hind from Hemophilus influenzae and HaeIII from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and pyrimidine isostich analysis. The Hind and HaeIII fragments were ordered relative to each other by cross digestion and a physical map of the S13 genome constructed. Comparison of the Hind cleavage patterns of S13 RF DNA and X174 RF DNA showed the majority of the fragments from the two DNAs coincided with each other except for three of the thirteen S13 fragments and three of the thirteen X174 fragments. Comparison of the HaeIII patterns of the two DNAs revealed a lack of coincidence of one S13 fragment only and two X174 fragments. From the data obtained by the cleavage of the two DNAs by Hind and HaeIII, a correlation between the physical restriction enzyme cleavage maps and the genetic map of the two phages was made. The differences in cleavage of the two DNAs by the restriction enzymes have been explained by changes in two restriction enzyme sites in the AB region and one change of site in the F region of the genetic map of the two bacteriophages

The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimerism.

Mouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. However, the reasons why this contribution often appears erratic are poorly understood. We have tested the notion that the chromosome make-up may be important in contributing both to somatic cell chimaerism and to germ line transmission. We found that the percentage of chimaerism of ES cell-embryo chimaeras, the absolute number of chimaeras and the ratio of chimaeras to total pups born all correlate closely with the percentage of euploid metaphases in the ES cell clones injected into the murine blastocyst. The majority of the ES cell clones that we tested, which were obtained from different gene targeting knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline; in contrast, none of the ES cell clones with more than 50% of chromosomally abnormal metaphases transmitted to the germline. Euploid ES cell clones cultured in vitro for more than 20 passages rapidly became severely aneuploid, and again this correlated closely with the percentage of chimaerism and with the number of ES cell-embryo chimaeras obtained per number of blastocysts injected. At the same time, the ability of these clones to contribute to the germline was lost when the proportion of euploid cells dropped below 50%. This study suggests that aneuploidy, rather than 'loss of totipotency', in ES cells, is the major cause of failure in obtaining contributions to all tissues of the adult chimaera, including the germline. Because euploidy is predictive of germline transmission, karyotype analysis is crucial and time/cost saving in any gene-targeting experiment

DNaseI hypersensitive sites 1, 2 and 3 of the human β-globin dominant control region directs position-independent expression.

The human beta-globin dominant control region (DCR) which flanks the multigene beta-globin locus directs high level, site of integration independent, copy number dependent expression on a linked human beta-globin gene in transgenic mice and stably transfected mouse erythroleukemia (MEL) cells. We have assayed each of the individual DNaseI hypersensitive regions present in the full 15kb DCR for position independence and copy number dependence of a linked beta-globin gene in transgenic mice. The results show that at least three of the individual DNaseI hypersensitive site regions (sites 1, 2 and 3), though expressing at lower levels than the full DCR, are capable of position independent, copy number dependent expression. Site 2 alone directs the highest level of expression of the single site constructs, producing nearly 70% of the level of the full DCR. Sites 1 and 3 each provide 30% of the full activity. Deletion of either site 2 or 3 from the complete set significantly reduces the level of expression, but does not effect position independence or copy number dependence. This demonstrates that sites 2 and 3 are required for full expression and suggests that all the sites are required for the full expression of even a single gene from this multigene locus

Regulated expression of human A γ-, β-, and hybrid γ β-globin genes in transgenic mice: manipulation of the developmental expression patterns.

We have introduced the human fetal gamma- and adult beta-globin genes into the germ line of mice. Analysis of the resulting transgenic mice shows that the human gamma-globin gene is expressed like an embryonic mouse globin gene; the human beta-globin gene is expressed (as previously shown) like an adult mouse globin gene. These results imply that the regulatory signals for tissue- and developmental stage-specific expression of the globin genes have been conserved between man and mouse but that the timing of the signals has changed. Because the two genes are expressed differently, we introduced a hybrid gamma beta-globin gene construct. The combination of the regulatory sequences resulted in the expression of the hybrid gene at all stages in all the murine erythroid tissues

Forces driving the three-dimensional folding of eukaryotic genomes

The last decade has radically renewed our understanding of higher order chromatin folding in the eukaryotic nucleus. As a result, most current models are in support of a mostly hierarchical and relatively stable folding of chromosomes dividing chromosomal territories into A- (active) and B- (inactive) compartments, which are then further partitioned into topologically associating domains (TADs), each of which is made up from multiple loops stabilized mainly by the CTCF and cohesin chromatin-binding complexes. Nonetheless, the structure-to-function relationship of eukaryotic genomes is still not well understood. Here, we focus on recent work highlighting the biophysical and regulatory forces that contribute to the spatial organization of genomes, and we propose that the various conformations that chromatin assumes are not so much the result of a linear hierarchy, but rather of both converging and conflicting dynamic forces that act on it

DNA sequences required for regulated expression of β-globin genes in murine erythroleukaemia cells.

We introduced into MEL cells rabbit beta-globin gene deletion mutants and two sets of hybrid genes constructed from the inducible human beta-globin gene and noninducible human gamma-globin gene or the murine H-2Kbm1 class I MHC gene. S1 nuclease analysis of gene transcripts before and after MEL differentiation showed that induction of the rabbit beta-globin gene did not require more than 58 bp of DNA 5' to the transcription initiation site. Hybrid genes were constructed with human beta-globin DNA sequences from either 5' or 3' of the translation initiation site linked to the complementary parts of the gamma or H2Kbm1 genes. Both types of constructs were inducible during MEL differentiation. The relative rates of transcription of the 5' gamma-3' beta and 5'H2-3' beta hybrid genes show that induction of the hybrid gene transcripts results at least in part from transcriptional activation of the genes. We suggest that DNA sequences that regulate beta-globin gene transcription during MEL differentiation are located both 5' and 3' to the translation initiation site