12.6: Regulating all the Genes on a Chromosome at Once

Consider again the different levels of chromatin structure, as seen in Figure 12.20. (below). Transcription factors bind specific DNA sequences by detecting them through the grooves (mainly the major groove) in the double helix. The drawing reminds us, however, that unlike the nearly naked DNA of bacteria, eukaryotic (nuclear) DNA is coated with proteins that, in aggregate, are greater in mass than the DNA that they cover. The protein-DNA complex of the genome is, of course, chromatin. Again, as a reminder, DNA coated with histone proteins forms the 9 nm diameter beads-on-a-string structure in which the beads are the nucleosomes. The association of specific nonhistone proteins with the nucleosome “necklace” causes it to fold over on itself to form the 30 nm solenoid.

Figure 12.20: Chromatin organization: different levels of chromatin structure result from differential association of DNA with histone and non-histone chromosomal proteins.

As we saw earlier, it is possible to selectively extract chromatin. Take a second look at the results of typical extractions of chromatin from isolated nuclei (Figure 12.21).

Figure 12.21: High-salt chromatin extraction from nuclei, or high-salt treatment of 10 nm filaments, yields 30 nm solenoid structures, essentially coils of 10 nm filaments.

Further accretion of nonhistone proteins leads to more folding and the formation of the euchromatin and heterochromatin seen in nondividing cell nuclei. In dividing cells, the chromatin further condenses to form the chromosomes that separate during either mitosis or meiosis.

Recall that biochemical analysis of the 10 nm filament extract revealed that the DNA wraps around histone-protein octamers—that is, the nucleosomes or beads in this beads-on-a-string structure. Histone proteins are highly conserved in eukaryotic evolution; they are not found in prokaryotes. They are also very basic (having many lysine and arginine residues) and therefore very positively charged. This explains why they can arrange themselves uniformly along DNA, binding to the negatively charged phosphodiester backbone of DNA in the double helix.

Since the DNA in euchromatin is less tightly packed than it is in heterochromatin, perhaps active genes are to be found in euchromatin and not in heterochromatin. Experiments in which total nuclear chromatin extracts were isolated and treated with the enzyme deoxyribonuclease (DNase) revealed that the DNA in active genes was degraded more rapidly than nontranscribed DNA. More detail on these experiments can be found at the following two links.

228 Question: Is Euchromatic DNA Transcribed?

229 Experiment and Answer: Euchromatin is Transcribed

The results of such experiments are consistent with the suggestion that active genes are more accessible to DNase because they are in less-coiled or less-condensed chromatin. DNA in more-condensed chromatin is surrounded by more proteins; thus, it is less accessible to and protected from DNase attack. When packed up in chromosomes during mitosis or meiosis, all genes are largely inactive.

Regulating gene transcription must occur in non-dividing cells or during the interphase of cells, times when it is easier to silence or activate genes by chromatin remodeling (i.e., changing the shape of chromatin). Changing chromatin conformation involves chemical modification of chromatin proteins and DNA. For example, chromatin can be modified by histone acetylation, de-acetylation, methylation and phosphorylation reactions, catalyzed by histone acetyltransferases (HAT enzymes), de-acetylases, methyl transferases and kinases, respectively.

Acetylation of lysines near the amino end of histones H2B and H4 tends to unwind nucleosomes and open the underlying DNA for transcription. De-acetylation promotes condensation of the chromatin in the affected regions of DNA. Likewise, the methylation of lysines or arginines (basic amino acids that characterize histones!) in H3 and H4 typically opens DNA for transcription, while demethylation has the opposite effect. These chemical modifications affect recruitment of other proteins that alter chromatin conformation and ultimately activate or block transcription. This reversible acetylation and its effect on chromatin are illustrated in Figure 12.22 (below).

Figure 12.22: The chemical modification of histones e.g., acetylation catalyzed by HAT enzymes (Histone Acetyl-Transferases) shown here, can change chromatin conformation, opening or closing it to transcription.

CHALLENGE

How do you imagine enzymes that acetylate, methylate, de-acetylate and de-methylate recognize which histones to attack?

Epigenetic alterations can also account for changes in broader features of chromatin structure. For example, topologically associated domains (TADs) are megabase regions of DNA that associate with each other. The boundary DNA between TADs is methylated and a CTCF protein bound to those regions is thought to insulate the TADs from one another, minimizing interactions between TADs. CTCF is ubiquitous in the nucleus (on chromatin at least) and thought generally to mediate chromatin interactions at a distance. Disruption of this insulation is seen in cancer cells and can result from mutation (e.g., of CTCF) or epigenetic alteration. In fact, hypermethylation of the boundary DNA between TADs is associated with many cancers. For more details, check out Epigenetic Cancer Chromatin Signatures (P. Pinoli et al., 2020).