Sir2 is a histone deacetylase found in all eukaryotes. Sir3 and Sir4 associate with Rap1 and with deacetylated histone tails and with each other. Sir2 associates with Sir4, deacetylating the tails of neighboring histones and thus causing this repressing chromatin structure to “spread” along the chromosomes from the telomere. Below is a biochemical method for analyzing the extent of acetylation of histones in nucleosomes associated with a specific gene of interest:
|Isolation||Chromatin is isolated from cells that were treated with formaldehyde to cross-link proteins to DNA.|
|Shearing||Chromatin is mechanically sheared .|
|Labeling||This sheared chromatin is mixed with an antibody specific for acetylated (unrepressed) N-terminal histone tails.|
|Precipitation||Nucleosomes with their antibody-bound histone tails are immunoprecipitated.|
|Release||Immunoprecipitated DNA is released and examined by PCR, then run on a gel; bands indicate gene of interest was acetylated..|
|Conclusion||A good positive control -- just to verify your gene of interest is present -- is to run PCR on sheared chromatin without immunoprecipitation; your gene should undergo PCR regardless of its acetylation state. A good negative control is to immunoprecipitate with antibody from non-immunized animals, like a rabbit or mouse. Lastly, you can assay for active transcription of a gene by immunoprecipitating with an antibody for RNA Polymerase II.|
Modifying histones tails controls chromatin condensation. Each histone protein making up nucleosome core contains a flexible amino terminus of 11-37 rseidues extending from the fixed nucleosome structure. These termini are called histone tails. Each H2A also has a flexible C-terminal taial. The histone tails are required for chromatin to condense from the beads-on-a-string into the 30-nm fiber.Several positively charged lysine side chains in istone tails may interact with linker DNA, and tials of one nucleosome likely interact with neighboring nucleosomes. The histone tail lysines, especially those in H3 and H4, undergo reversible acetylation and deacetylation by enzymes that act on specific lysines in the N-termini. In the acetylated form, the positive charge of the lysine e-amino group is neutralized, therby eliminating interaction with a DNA phosphate group. The greater the acetylation of histone N-termini, the less likely chromatin is to form condensed 30-nm fibers and possibly higher-order folded structures. Histone tails can bind to other proteins associated with chromatin that influence chromatin structure and processes such as transcription and DNA replication.
The interaction of histone tails with these proteins can be regulaed by a variety of covalent modifications of histone tail amino acid side chains. These include acetylation of lysine e-amio groups, as mentioned earlier as wel as methylation of these groups a process preventing acetylatino, thus maintaining their positive charge. Arginine side chains can also be methylated. Serine and threonine side chains can be phosporylated, inducing a negative charge. Finally, a single 76-amino-acid ubiquitin molecule can be added to some lysines. Recall that addition of multiple linked ubiquitin molecules to a protein does not affect the ability of a histone but influencez chromatin structure. In summary, multiple types of covalent modifications of histone tails can influence chromatin structure by altering histone-DNA interactions and interactions between nucleosomes and by controlling interactions with additinoal proteins participating with transcriptional regulation.
There is a direct correlation between histone acetylation and susceptibility of chromatin DNA to digestion by nucleases. This can be demonstrated b digesting isolated nucei with DNase I. Following digestion, the DNA is completely separated from chromatin protein, digested to completin with restriction enzyme, and analyzed by Southern blotting. An ntact gene treateed with a restriction enzyme yields characteristic fragments. When a gene is first exposed to DNase, it is cleaved at random sites within boundaries of rseitrction enzyme cut sites. Consequently, any Southern blog bands normally seen will be lot This has shown that transcriptionall inactive Β-globin gene in nonerythroid cells (associated with unacetlated histones) is much more resistant to DNase I tan is the active, transcribed Β-globin gene in erythroaid recursor cells where associated with acetylated histones. Chromatin structure of nontranscribed DNA is more condensed, more protected from DNase digestion, than transcribed DNA. In condensed chromatin, the DNA is mostly inaccessible to DNase I because of close association with histones and other less abundnat chromatin proteins. In contrast, actively transcribed DNA is accessible to DNase I digestion because it is prsent in extended, beads-on-a-string chromatin form.
Yeast studies shown that specific hitone acetylases are required for full activation of transcription of a number of genes. Control of aetylation of histone N-termini in specific chromosomal regions is thought to contribute to gene control by regulating the strength of the interaction of histones with DNA and the folding of chromatin into condensed structures. Genes in condensed, folded regins of chroamating are inaccessible to RNA polymerase and other proteins required for transcription.
Chromatin decondensation appears to require two types of protein complexes each made of several polypeptide subunits:
|Histone acetylase complexes||These are often referred to as HATS for histone acetylases. Chromatin remodeling factors use energy from ATP hydrolysis to rearrange the packing of nucleosomes in higher order chromatin structures There are several different chromatin remodeling complexes in cells. Some of these bind to activation domains and decondense the associated chromatin. Some bind to repression domains and condense the associated chromatin.|
|Chromatin remodeling factors||These are often referred to as Swi/Snf factors because they were first identified as yeast mutants defective in mating type switching and in the ability to metabolize sucrose (sucrose non-fermenting).|
In higher eukaryotes, methylation at histone H3 lysine 9 (H3K9) resuts in the assembly of one kind of heterochromatin.
Another type of spreading repression mechanism that is crucial to development in all metazoans is the Polychome Complex repression mechanism. Polycomb repression is established early in embryogenesis by certain repressors that are expressed briefly. Initially these repressors associate with histone deacetylase complexes to repress their target genes. But after they stop being expressed, repression of the target genes is maintained by the Polycomb repression system, and is maintained through cell division during development.