The upstream area of the chicken α-globin gene domain is transcribed in both directions in the same cells

AbstractIt was demonstrated previously that in erythroid chicken cells an extended upstream area of the α-globin gene domain is transcribed in both directions as a part of ggPRX gene and a part of a full domain transcript of the α-globin gene domain. Here, we show that both DNA chains of the above-mentioned region are transcribed in the same cells and that the corresponding transcripts coexist in nuclei. The data obtained suggest that cells possess a molecular mechanism which in some cases prevents the formation of dsRNA and subsequent destruction of both transcripts in spite of the presence of complementary RNA chains in the cell nucleus

TMEM8 – a non-globin gene entrapped in the globin web

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of α-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the α-globin cluster due to inversion of an ∼170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the α-globin genes. Surprisingly, TMEM8 is not simply recruited to the α-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs

Mapping of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure

We have developed an experimental procedure to analyze the spatial proximity of nuclear matrix-bound DNA fragments. This protocol, referred to as Matrix 3C (M3C), includes a high salt extraction of nuclei, the removal of distal parts of unfolded DNA loops using restriction enzyme treatment, ligation of the nuclear matrix-bound DNA fragments and a subsequent analysis of ligation frequencies. Using the M3C procedure, we have demonstrated that CpG islands of at least three housekeeping genes that surround the chicken α-globin gene domain are assembled into a complex (presumably, a transcription factory) that is stabilized by the nuclear matrix in both erythroid and non-erythroid cells. In erythroid cells, the regulatory elements of the α-globin genes are attracted to this complex to form a new assembly: an active chromatin hub that is linked to the pre-existing transcription factory. The erythroid-specific part of the assembly is removed by high salt extraction. Based on these observations, we propose that mixed transcription factories that mediate the transcription of both housekeeping and tissue-specific genes are composed of a permanent compartment containing integrated into the nuclear matrix promoters of housekeeping genes and a ‘guest’ compartment where promoters and regulatory elements of tissue-specific genes can be temporarily recruited

Visualization of individual DNA loops and a map of loop domains in the human dystrophin gene

The organization of the human dystrophin gene into loop domains has been studied using two different experimental approaches: excision of DNA loops mediated by nuclear matrix-bound topoisomerase II and in situ hybridization of different probes with histone-depleted nuclei (nuclear halos). Our objective was to examine if the DNA loops mapped by this biochemical approach coincide with loops visualized by microscopy. The results obtained using both approaches were in good agreement. Eight loops separated by attachment regions of different length were mapped in the upstream part (up to exon 54) of the gene by topoisomerase II-mediated excision. One of these loops was then directly visualized by in situ hybridization of the corresponding bacmid clone with nuclear halos. This is the first direct demonstration that a DNA domain mapped as a loop using a biochemical approach corresponds to a loop visible on cytological preparations. The validity of this result and of the whole map of loop domains was confirmed by in situ hybridization using probes derived from other attachment regions or loops mapped by topoisomerase II-mediated cleavage; these probes hybridized on the core or halo region, respectively, of nuclear halos. Our results demonstrate that a single transcription unit may be organized into several loops and that DNA loop attachment regions may be fairly long. Three out of four replication origins mapped in this gene co-localize with loop attachment regions, and the major deletion hot spot is harbored in an attachment region. These results strongly suggest that partitioning of genomic DNA into specific loops attached to a skeletal structure is a characteristic feature of eukaryotic chromosome organization in interphase

Verification of the <i>MLL</i> gene and chromosome 11 territory detection by a computer algorithm.

<p>Cells were treated with etoposide for 1.5 hour and cultivated under normal conditions for 1 h. In all cases, projections of the confocal sections are shown. A, B, E, F) Detection of the MLL signals. Original staining (red, A, E), detection of signals by the computer software (added green colour, resulting in yellow spots, B, F). C,D,G,H,I) Detection of a chromosome territory. Original staining (green, C, G), local brightness adjustment by the computer software (D,H), detection of the chromosomal territory by the computer software (I). Detected chromosome territories were automatically outlined in grey (I); during analysis border lines were not included in the territories. Chromosome territory rejected due to small size is indicated by an arrow (I).</p

The Broken <i>MLL</i> Gene Is Frequently Located Outside the Inherent Chromosome Territory in Human Lymphoid Cells Treated with DNA Topoisomerase II Poison Etoposide

<div><p>The mixed lineage leukaemia (MLL) gene is frequently rearranged in secondary leukaemias, in which it could fuse to a variety of different partners. Breakage in the MLL gene preferentially occurs within a ~8 kb region that possesses a strong DNA topoisomerase II cleavage site. It has been proposed that DNA topoisomerase II-mediated DNA cleavage within this and other regions triggers translocations that occur due to incorrect joining of broken DNA ends. To further clarify a possible mechanism for MLL rearrangements, we analysed the frequency of MLL cleavage in cells exposed to etoposide, a DNA topoisomerase II poison commonly used as an anticancer drug, and positioning of the broken 3’-end of the MLL gene in respect to inherent chromosomal territories. It was demonstrated that exposure of human Jurkat cells to etoposide resulted in frequent cleavage of MLL genes. Using MLL-specific break-apart probes we visualised cleaved MLL genes in ~17% of nuclei. Using confocal microscopy and 3D modelling, we demonstrated that in cells treated with etoposide and cultivated for 1 h under normal conditions, ~9% of the broken MLL alleles were present outside the chromosome 11 territory, whereas in both control cells and cells inspected immediately after etoposide treatment, virtually all MLL alleles were present within the chromosomal territory. The data are discussed in the framework of the “breakage first” model of juxtaposing translocation partners. We propose that in the course of repairing DNA topoisomerase II-mediated DNA lesions (removal of stalled DNA topoisomerase II complexes and non-homologous end joining), DNA ends acquire additional mobility, which allows the meeting and incorrect joining of translocation partners.</p> </div

Relative positions of the <i>MLL</i> gene and chromosome 11 territory in control cells and cells treated with etoposide.

<p>Confocal images of cells made using the 3D-FISH technique; blue colour – DNA stained with DAPI, green colour – territory of chromosome 11, red colour - genomic locus containing the 3’ fragment of the <i>MLL</i> gene. A) Untreated cells. B) Cells treated with etoposide for 1.5 hour C, D) Cells treated with etoposide for 1.5 hour and cultivated for 1 h under normal conditions. The 3' fragment of the <i>MLL</i> gene thatmoves out of chromosome 11 territory is marked with an arrowhead. The enlarged nucleus indicated by the white square is shown in section D.</p

DNA breaks within the <i>MLL</i> gene breakpoint cluster region upon etoposide treatment.

<p>A) A map of the <i>MLL</i> locus with the positions of regions visualised by the LSI MLL break-apart hybridization probe that covers approximately 350 kb upstream of the BCR (green) and approximately 190 kb downstream of the BCR (red). B) Images of untreated Jurkat cells, DNA stained with DAPI (blue), genomic locus containing the 5’ fragment of the <i>MLL</i> gene (green) and the 3’ fragment of the <i>MLL</i> gene (red). Arrowheads show the double coloured spots. C) Different cases of staining of the <i>MLL</i> gene by the LSI MLL break-apart hybridization probe in Jurkat cells treated with etoposide: upper row – a cell with one double coloured spot (arrowhead), representing a non-cleaved <i>MLL</i> allele, and two single-coloured spots, representing the upstream and downstream ends of a broken <i>MLL</i> allele (arrows); middle and the lower rows - cells with two double coloured spots (arrowheads) and one additional red or green spot, respectively (arrow). The percentage of nuclei representing each of the shown patterns and number of the analysed cells are indicated to the right of section C. D,E) Schemes demonstrating the possible ways to generate the staining patterns are shown in section C.</p