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ChromatinComments

Chromatin

To pack DNA into the tiny nucleus (the DNA packing problem), DNA is tightly wound around special proteins to form a nucleoprotein complex called chromatin.

Chromatin proteins are predominantly histones, a family (H1, H2A, H2B, H3 and H4) of small proteins that are conserved in eukaryotes and contain many positively-charged basic amino acids that interact with negatively-charged DNA phosphate groups.

0.34 nm=Distance between base pairs in B-form DNA.
3 · 109 bp=Human Haploid Genome.
~1 m=Length of Haploid Genome.
3-10 · 10-6 m=Length of nucleus.
~2 m=Length of DNA per nucleus.
PrimaryTwo each of each histone (except H1) interlock to form a disc-shaped structure ~10 nm in diameter. In all eukaryotes, 147 bp of DNA wraps almost twice around the histone octamer to form the nucleosome. Nucleosome "beads" are each spaced by 15-55 bp (depending on species) "strings" of linker DNA. The basic and positively-charged histones bind tightly to DNA, protecting it from proteins. Careful nuclease treatment will digest linker DNA and release individual nucleosomes with their DNA still intact. However, linker DNA is somewhat protected by bound H1 and by inter-histone interactions. Newly replicated DNA in vivo assembles into nucleosomes shortly after the replication fork passes, nucleosomes do not spontaneously form in vitro at physiological salt concentration when histones are added to DNA. However, nuclear proteins have been characterized that bind histones and assemble them in vitro with DNA. These are thought to assemble new DNA into histones in vivo as well.
SecondaryNucleosomes (the 1° chromatin structural unit) will stack on top of each other at physiological conditions (~0.15 M KCl), and the stacks will then intertwine into an irregular spiral (solenoid arrangement) that is ~30 nm and contains ~6 nucleosomes per turn. H1, the 5th major histone, is bound to DNA on inside of solenoid with one H1 molecule associated with each nucleosome. The 30-nm solenoid is less uniform than a perfect solenoid; condensed chromatin may actually be quite dynamic, with regions occasionally partially unfolding and then refolding into a solenoid structure.
TertiaryAt special scaffold attachment regions (SARs), the 30 nm fibers attach to a flexible protein scaffold; the unattached regions form chromatin loops. DNA can be released from the protein scaffold by treatment with detergent. In addition to this general structure, thousands of low-abundance regulatory proteins associate with specific DNA sequences. During meiosis, chromatin further folds and compacts into visible metaphase chromosomes.

Condensed vs decondensed

Actively transcribed chromatin is in 10 nm form (beads-on-a-string). This can revert to 30 nm when genes are repressed. Highly inactive chromatin, such as that containing repetitive DNA, is still further condensed around the chromosome scaffold. Histones have unstructured tails (not seen in crystal structure) that are specifically modified (including acetylation, methylation, and phosphorylation) to mediate the regulated condensation and decondensation of the chromatin.

DNAse sensitivity is an effective way to determine if a gene is condensed or decondensed form. A condensed gene will be more resistant to DNAse than a gene that is not condensed. Afterward, decondensation of the entire genome and PCR analysis will reveal what has and has not been digested.

HeterochromatinHeterochromatin is made up of dense, tightly packed, portions of the chromosome that are mostly inactive and often contain repetitive simple sequence DNA. This appears very dark in the electron microscope.Heterochromatin is highly condensed but can be converted to euchromatin by transcriptional activators targeted to that chromosomal region.
EuchromatinGene rich regions of the chromosome are much less densely packed and make up what is called Euchromatin. The decondensation of chromatin upon transcriptional activation can also be observed through its sensitivity to DNAse. For example, globin genes expressed in erythrocytes (in these cells, DNAse sensitive) but not expressed in other cells (DNAse resistant).

Additional chromatin proteins

The total mass of histones associated with DNA in chromatin is about equal to that of the DNA. Interphase chromatin and metaphase chromsomes also contain small amounts of a complex set of other proteins.

For instance, a growing list of DNA-binding transcription factors have been identified associated with interphase chromatin. The structure and function of hese critical nonhistone proteins, which help regulate transcritpion, are examined in Chapter 11. Other low-abundance nonhistone proteins associated with chromatin regulate DNA replication during the eukaryotic cell cycle. A few other nonhistone DNA-binding proteins are present in much larger amounts than the transcription or replication factors. Some exhbit high mobility during electrophoretic separation and have thus been designated high-mobility group (HMG) proteins.

When genes encoding the most abundant HMG proteins are deleted from yeast cells, normal transcription is disturbed in most other genes examined. Some HMG proteins have been found to bind DNA cooperatively with transcription facors binding to specific DNA sequences to stabilize multiproteins complexes regulating transcription of a neighboring gene.

Chromatin scaffold

Nonhistone proteins provide a structural scaffold for long chromatin loops. Although histones are the predominant proteins in chromosomes, nonhistone proteins are also involved in organizing chromosome structure. Electron micrographs of histone-depleted metaphase chromosomes from HeLa cells reveal long loops of DNA anchored to a chromosome scaffold composed of nonhistone proteins. This scaffold is shaped like the metaphase chromosome and persists evn when DNA is digested by nucleases. Loops of 30-nm chromatin fiber a few megabases long associate with a flexible chromsome scaffold, yielding an extended form characteristic of chromosomes during interphase. Folding of scaffold produces highly condensed structure characteristic of metaphase chromosomes. But the geometry of scaffold folding in metaphase chromosomes has not yet been determined.

In situ hybridization with different fluorescent-labeled probes to DNA in human interphase cells support loop model shown. Some probe sequences mllion of base pairs apart in linear DNA appeared reproducibly very clse in interphase nuclei from different cells. hese are postulated to lie close to specific sequences in DNA called scaffold-associated regions (SARs) or matrix-attachment regions (MARs) bound to chromosome scaffold. SARs have been mapped by digesting histone-depleted chromsoemes with restriction enzymes and recovering fragment bound to scaffold proteins. In general, SARs are found between transcription units. Genes are located primarily within chromatin loops attached at bases to chromosome scaffold. Experiments with transgenic mice indicate that in some cases, SARs are required for transcription of neigboring genes.

Individual interphase chromosmes are less condensed than metaphase chromosomes and cannot be resolved by standard microscopy or electron microscopy. Nonetheless, it is associate with extended scaffolds and is further organized into specific domains. This can be demonstrated by in site hybridization of interphase nuclei with a large mixture of fluorescent-labeled probes specific for sequences along the length of a particular chromosome. Little overlap between chromosomes in interphase nuclei. Precicse positions are not reproducible between cells.