Hb switching in chickens

We have taken advantage of the preferential digestion of active genes by DNAase I to investigate the chromosomal structure of embryonic and adult β-globin genes during erythropoiesis in chick embryos, and in particular to examine the question of hemoglobin switching during development. DNA in isolated red cell nuclei was mildly digested with DNAase I to about 10–15 kb, purified and restricted with a variety of restriction enzymes. The DNA was then separated on agarose gels, transferred to nitrocellulose filters and hybridized with an adult-specific β-globin cDNA clone or a genomic clone containing the genes coding for both an embryonic and an adult β-globin chain. Preferential sensitivity of the respective globin genes was monitored by the disappearance of specific restriction bands after DNAase I digestion of nuclei. In embryonic red cells, both adult and embryonic β-globin genes are very sensitive to DNAase I; however, in adult erythroid lines, the embryonic β-globin gene becomes relatively more resistant but the adult gene remains highly sensitive. Controls showed that all globin genes were resistant to DNAase I in brain nuclei and nuclei from lymphoid cells. Thus the switch from embryonic to adult globin expression is associated with an apparent change in the chromosome structure of the embryonic globin gene as reflected in the gene becoming less accessible to DNAase I in adult red cell nuclei. Our results also show that the chromosomal structure of both adult and embryonic genes is altered in embryonic red cell nuclei; thus the nonexpressed globin gene (that is, the adult gene in embryonic red cells) has already been “recognized” to some degree and marked by the erythroid compartment. The sensitivity of the adult globin gene in embryonic cells may represent a “pre-activation” state of the chromosome.     

Rapid reprogramming of globin gene expression in transient heterokaryons

Interspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell. Therefore, erythroid cells contain transacting regulatory factors that are capable of activating the expression of globin genes in a stage- and tissue-specific manner. These observations also indicate that globin genes are not irreversibly repressed in differentiated cells and that their expression can be rapidly reprogrammed in the presence of the appropriate regulatory factors.     

Interaction of HMG 14 and 17 with actively transcribed genes

The interaction of HMG 14 and 17 with actively transcribed genes was studied by monitoring the sensitivity of specific genes to DNAase I after reconstitution of HMG-depleted chromatin with HMG 14 and 17. Our experiments lead to the following conclusions: most actively transcribed genes become sensitized to DNAase I by HMG 14 and 17; either HMG 14 or HMG 17 can sensitize most genes to DNAase I; genes transcribed at different rates have about the same affinity for HMG 14 and 17; HMG 14 and 17 bind stoichiometrically to actively transcribed nucleosomes; and HMG 14 and 17 can restore DNAase I sensitivity to purified nucleosome core particles depleted of HMGs. This last observation suggests that during reconstitution, low levels of HMG 14 and 17 can associate with the active nucleosomes in the presence of a 10–20 fold excess of inactive nucleosomes. Consequently, we conclude that besides their association with HMGs, active nucleosomes also have at least one other unique feature that distinguishes them from bulk nucleosomes and insures proper HMG binding during reconstitution.     

DNAase I sensitivity of genes expressed during myogenesis.

Cultures of a rat myogenic cell line were used to examine the question of whether in proliferating precursor cells genes which are programmed to be expressed later in development, in the same cell lineage, differ in DNAase I sensitivity from genes which are never expressed in these cells. Nuclei isolated from proliferating mononucleated myoblasts, differentiated cultures containing multinucleaged fibers, and rat brain, were treated with DNAase I. The sensitivity of the genes coding for the muscle-specific alpha-actin, myosin light chain 2 and the nonmuscle beta-actin was measured by blot hybridization of nuclear DNA with the corresponding cloned cDNA and genomic DNA probes. The sensitivity of these genes was compared to that of a gene not expressed in the muscle tissue. The results showed that in the muscle precursor cells, the potentiality of tissue-specific genes to be expressed is not reflected in DNAase I sensitivity. The changes which render these genes preferentially sensitive to DNAase I take place during the transition to terminal differentiation. The results showed also that the region of DNAase I sensitivity of the alpha-actin gene in the differentiated cells ends between 40 to 700 bp 5' to the structural gene. No DNAase I hypersensitive site was detected 5' to the alpha-actin gene.     

Mapping of DNAase I sensitive regions on mitotic chromosomes

We have shown that in fixed mitotic chromosomes from female G. gerbillus cells the inactive X chromosome is distinctly less sensitive to DNAase I than the active X chromosome, as demonstrated by in situ nick translation. These results indicated that the specific chromatin conformation that renders potentially active genes sensitive to DNAase I is maintained in fixed mitotic chromosomes. We increased the sensitivity and accuracy of in situ nick translation using biotinylated dUTP and a specific detection and staining procedure instead of radioactive label and autoradiography and now show that in both human and CHO chromosomes, the DNAase I sensitive and insensitive chromosomal regions form a specific dark and light banding pattern. The DNAase I sensitive dark D-bands usually correspond to the light G-bands, but not all light G-bands are DNAase I sensitive. Identifiable regions of inactive constitutive heterochromatin are in a DNAase I insensitive conformation. Our methodology provides a new and important tool for studying the structural and functional organization of chromosomes.     

An altered DNA conformation detected by S1 nuclease occurs at specific regions in active chick globin chromatin

The single-stranded activity of S1-nuclease cleaves globin chromatin in red cell nuclei in specific regions. The cleavages are observed only in tissues in which the globin genes are active, and they "switch" to reflect the switching pattern of globin-gene expression in embryonic and adult red cells. The positions of the S1 cleavages in the beta- and alpha-globin chromatin correspond to the general region of known DNAase I-hypersensitive sites, but can be distinguished in detail. When DNA segments containing these regions are subcloned into pBR322 and the supercoiled molecules are treated with S1, similar sites are cleaved in the purified supercoiled (but not linear) recombinant plasmid DNA. However, the dominant S1 cutting sites are shifted in the plasmid vis-a-vis the chromatin. We believe that some aspect of DNA sequence is translated into an altered DNA structure in chromatin and that it is this altered structure that is recognized by s1 nuclease and possibly by certain chromosomal proteins. Several physical properties reflected in the S1 digestion of supercoiled plasmids suggest a mechanism for generating differences in daughter cells during development.     

The chromatin structure of specific genes: II. Disruption of chromatin structure during gene activity.

We have compared the chromatin structure in the active and inactive states at loci encoding the major heat shock protein in Drosophila. DNAase I and micrococcal nuclease were used as probes of higher order organization and nucleosomal integrity. Such integrity is gauged here by the characteristic pattern of discrete DNA fragments produced at specific chromosomal loci by nucleolytic cleavage. The specific fragment patterns are visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets, hybridization with 32P-labeled cloned DNA containing the heat shock genes and autoradiography. Using this criterion, a disruption in nucleosomal and possibly in higher order organization are observed as indicated by a relative loss or smearing of the characteristic discrete DNA fragment patterns from the heat shock loci in the active state. The fragment patterns are restored when cells are allowed to recover from heat shock and these loci return to the inactive state.     

Selective release of chromosomal proteins during limited DNAase 1 digestion of avian erythrocyte chromatin

Duck erythrocyte chromatin has been treated with DNAase 1 under conditions that are known to digest selectively the structural genes coding for globin mRNAs. This limited digestion releases specific sets of nonhistone chromosomal proteins that are not preferentially released during limited digestion with micrococcal nuclease, which does not selectively attack the globin sequences. Analysis of nucleosome monomer and multimer peaks separated on sucrose gradients after limited digestion with micrococcal nuclease shows that the proteins which are released by DNAase 1 digestion remain associated with the chromatin subunits and can be removed by extraction in 0.5 M NaCl. These proteins are tentatively identified as members of the high mobility group (HMG) proteins (originally described by Goodwin, Sanders and Johns, 1973) in terms of their extractability, electrophoretic characteristics and amino acid composition.     

Structural state of active and inactive genes during chromatin decondensation

Chromatin structure of globin and ovalbumin genes in chicken erythrocyte nuclei has been investigated by means of the "nuclease criterion" (described earlier). In intact nuclei (i.e. in the presence of 3 mM MgCl2) DNase I cleaves chromatin of both genes generating fragments multiple of a double-nucleosome repeat (2N-periodicity). However, in the case of the globin gene, apart from the 2N-periodicity, fragments were observed that are multiple of 100 b.p. and are characteristic for partially unfolded chromatin. This distinction in nuclease cleavage patterns correlates with a higher sensitivity of the globin gene as compared with the inactive ovalbumin gene. At 0.5-0.7 mM MgCl2 the transition from dinucleosomal fragmentation with DNase I and DNase II to fragmentation via a 100 b.p. interval occurs and the difference in digestibility of both genes is dramatically increased. If chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer DNase Il generates an usual nucleosomal repeat, and in this ionic conditions one may not observe any difference in nuclease sensitivity of the analyzed genes. The data allow to suggest that the high nuclease sensitivity of potentially active genes can be conditioned by more relaxed arrangement of nucleosomes in higher order chromatin structure.