Maintenance of a <em>Drosophila melanogaster</em> Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...

National Institutes of Health

We have performed a very extensive electron microscopy investigation of the chromatin structures extruded from partially denatured metaphase chromosomes from HeLa cells under a wide variety of conditions. Denatured chromosomes having fibres as the dominant structural element are obtained in the presence of buffers of very low concentration or after incubation with water. At slightly higher ionic concentrations, metaphase chromosomes become granulated. The most frequently observed granules have a diameter of about 35 nm and show the same structural characteristics as the compact cylindrical chromatin bodies previously found in our laboratory in studies performed using small chromatin fragments. Our results suggest that fibres are formed by the face-to-face association of 35-nm chromatin bodies. We have observed a very compact morphology of chromosomes in solutions containing intracellular concentrations of monovalent cations and the Mg2+ concentration found in metaphase. The most abundant structural elements observed in chromatin extruded from partially denatured compact metaphase chromosomes are multilayered plate-like structures. This is the first time that these planar structures have been reported. The observation of the irregular plates found in some preparations and of the small planar structures seen in aggregates of small chromatin fragments suggests that plates are formed by side-by-side association of compact chromatin bodies.

Structural elements of bulk chromatin within metaphase chromosomes.

We have performed a very extensive investigation of chromatin folding in different buffers over a wide range of ionic conditions similar to those found in eukaryotic cells. Our results show that in the presence of physiological concentrations of monovalent cations and/or low concentrations of divalent cations, small chicken erythrocyte chromatin fragments and chromatin from HeLa cells observed by transmission electron microscopy (TEM) show a compact folding, forming circular bodies of approximately 35 nm in diameter that were found previously in our laboratory in studies performed under very limited conditions. Since TEM images are obtained with dehydrated samples, we have performed atomic force microscopy (AFM) experiments to analyze chromatin structure in the presence of solutions containing different cation concentrations. The highly compact circular structures (in which individual nucleosomes are not visible as separated units) produced by small chromatin fragments in interphase ionic conditions observed by AFM are equivalent to the structures observed by TEM with chromatin samples prepared under the same ionic conditions. We have also carried out experiments of sedimentation and trypsin digestion of chromatin fragments; the results obtained confirm our AFM observations. Our results suggest that the compaction of bulk interphase chromatin in solution at room temperature is considerably higher than that generally considered in current literature. The dense chromatin folding observed in this study is consistent with the requirement of compact chromatin structures as starting elements for the building of metaphase chromosomes, but poses a difficult physical problem for gene expression during interphase.

Highly compact folding of chromatin induced by cellular cation concentrations. Evidence from atomic force microscopy studies in aqueous solution.

In a previous work we observed multilayered plate-like structures surrounding partially denatured HeLa chromosomes at metaphase ionic conditions. This unexpected finding has led us to carry out an extensive investigation of these structures. Our results show that plates can also be found in metaphase chromosomes from chicken lymphocytes. We have used atomic force microscopy (AFM) to image and investigate the mechanical properties of plates in aqueous solution. Plates are thin (approximately 6.5 nm each layer) but compact and resistant to penetration by the AFM tip: their Young's modulus is approximately 0.2 GPa and the stress required for surface penetration is approximately 0.03 GPa in the presence of Mg(2+) (5-20 mM). Low-ionic strength conditions produce emanation of chromatin fibers from the edges of uncrosslinked plates. These observations and AFM results obtained applying high forces indicate that the chromatin filament is tightly tethered inside the plates. Images of metal-shadowed plates and cryo-electron microscopy images of frozen-hydrated plates suggest that nucleosomes are tilted with respect to the plate surface to allow an interdigitation between the successive layers and a thickness reduction compatible with the observed plate height. The similarities between denatured plates from chicken chromosomes and aggregates of purified chromatin from chicken erythrocytes suggest that chromatin has intrinsic structural properties leading to plate formation. Scanning electron micrographs and images obtained with the 200-kV transmission microscope show that plates are the dominant component of compact chromatids. We propose that metaphase chromosomes are formed by many stacked plates perpendicular to the chromatid axis.

Dense chromatin plates in metaphase chromosomes.

While most transcription factors exit the chromatin during mitosis and the genome becomes silent, a subset of factors remains and "bookmarks" genes for rapid reactivation as cells progress through the cell cycle. However, it is unknown whether such bookmarking factors bind to chromatin similarly in mitosis and how different binding capacities among them relate to function. We compared a diverse set of transcription factors involved in liver differentiation and found markedly different extents of mitotic chromosome binding. Among them, the pioneer factor FoxA1 exhibits the greatest extent of mitotic chromosome binding. Genomically, ~15% of the FoxA1 interphase target sites are bound in mitosis, including at genes that are important for liver differentiation. Biophysical, genome mapping, and mutagenesis studies of FoxA1 reveals two different modes of binding to mitotic chromatin. Specific binding in mitosis occurs at sites that continue to be bound from interphase. Nonspecific binding in mitosis occurs across the chromosome due to the intrinsic chromatin affinity of FoxA1. Both specific and nonspecific binding contribute to timely reactivation of target genes post-mitosis. These studies reveal an unexpected diversity in the mechanisms by which transcription factors help retain cell identity during mitosis.

Juan Manuel Caravaca

Laboratory of Cellular and Developmental Biology,
National Institute of Diabetes,
Digestive and Kidney Diseases,
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes, Digestive and Kidney Diseases

National Institutes of Health

Maintenance of a Drosophila melanogaster Population Cage