PLNT 3140 Introductory Cytogenetics - 2023

Chromatin

The eukaryotic genome has conflicting goals

1. Eukaryotic genomes are huge

Prokaryotes have small genomes, usually less than 10 ⁷ bp. They get along just fine carrying out replication, transcription and translation simultaneously.


Eukaryotic genomes are huge, ranging from  10 ⁷ bp to 10 ¹² bp.  As we saw in the exercise, the eukaryotic nucleus has to pack and unpack a tremendous quantity of chromatin once every cell cycle. That's a lot of spaghetti to keep organized! For this reason, eukaryotes need the elaborate mechanisms of mitosis and meiosis.

2. Two conflicting requirements of the cell:

a. On one hand - DNA needs to be accessible to transcriptional  and replicative enzymes, so that  gene expression and DNA replication can occur.
b. On the other hand - DNA needs to be packaged tightly so that it can be easily divided between two daughter cells during cell division.

The solution to this conflict is the eukaryotic chromosome. Chromatin is DNA complexed with proteins. The chromosome is a single DNA molecule complexed with proteins. It is organized to allow for a hierarchal packing scheme. 

Summary of Chromosome Structure

DNA coils around histone octamer particles to form nucleosomes

While there are multiple proteins that complex with DNA, histones are by far the predominant class of chromatin proteins. Histone proteins are responsible for helping DNA coil, as well as regulating transcriptionally "open" and "closed" genes. As they have an overall positive charge, they attract negatively-charged DNA. This charge attraction alone is sufficient for nucleosomes to form. There are five main histone proteins you should know:

Nucleosome structure

Treatment of nuclei with limiting amounts of micrococcal nuclease (an endonuclease) typically results in a "step-ladder" of bands, showing that DNA is cut into discrete fragments in multiples of ~200bp. If you digest with higher concentrations of nuclease, the periodicity changes to 146bp.

Interpretation: something in chromatin is protecting pieces of DNA, about 146 bp long, from being digested. Most of the chromosomal DNA is being protected.

MICROCOCCAL NUCLEASE DIGESTION OF CHROMATIN [Spiker et al. (1983) PNAS 80:815 Fig. 4] (Note: this figure compares 3 alternative methods for chromatin isolation) Fig. 4. Micrococcal nuclease digestion of wheat embryo nuclei and chromatin. Nuclei and chromatin substrates were adjusted to 1µg of DNA/ml and digested with micrococcal nuclease at 50 units/ml at 37°C. Time course of digestion experiments were carried out for all samples. The DNA fragments were then purified and separated electrophoretically on agarose gels (pH8.1). A HaeIII-digested phi-X174DNA. B chromatin isolated by the method of Simon and Becker. A 6min. digest is shown; this extent of digestion and quantitiy of DNA applied to the gel showas a nucleosome pattern better than that found under any other conditions although the faint nucleosome pattern is barely noticeable against the background of variable length fragments produced by micrococcal nuclease. C chromatin isolated by the modified method of Bonner et al.; a 10 min. digest is shown. D nuclei isolated as described in the text; a 2-min. digest is shown. E HaeIII-digested Lambda phage DNA.

The histone core particle is an octamer, and contains 2 copies each of H2A, H2B, H3, and H4. H1 is a linker histone that has three distinct regions: a basic (+), random coil at the N-terminus, a central globular region, and a highly conserved, highly basic C-terminus. DNA coils around histone octamers to form nucleosomes. Nucleosomes are the first stage of the coiling process, and contain the histone octamer and two turns of DNA. Figure from http://en.wikipedia.org/wiki/Nucleosome

The nucleosome structure includes the core histone octomer plus 2 turns of DNA. Two rotated views of the nucleosome are shown in ball-and-stick representation. The two strands of the DNA double helix are shown in light blue and cream color. The eight histone proteins in the nucleosome core are each colored differently. Wrapped around these particles is two-turns of a DNA helix. These are left-handed turns. They take up, not surprisingly, 146 bp. These figure were produced from PDB entry 2CV5, using the NCBI Cn3D viewer.

Complementarity between positive charges on the core protein and the DNA phosphodiester backbones. The (H3-H4)2 is white and the H2A-H2B dimers are blue. the C-alpha atoms of lysines and arginines on the cylindrical surface are indicated by red, and the atoms marking the positions of the positive end of helix dipoles are indicated by orange. Atoms of the DNA backbone are "undersized" so as to allow visualization of the protein surface. Nucleosomes can form spontaneously in-vitro The positive charges of the histone proteins and the negative charges of the DNA backbone are sufficient to drive nucleosome formation. In chromatin reconstitution experiments, histones H2a, H2b, H3 and H4 are added to DNA, resulting in nucleosome formation. Therefore, the process of nucleosome formation is solely due to charge. From: Moudreinakis, EN and Arents, G (1993) Structure of the Histone Octamer Core of the Nucleosome and Its Potential Interactions with DNA. in Cold Spring Harbor Symposia on Quantitative Biology Vol. LVIII DNA and Chromosomes.pp. 273-279. Cold Spring Harbor Press 1993. Fig. 6

Which is the correct model for chromatin fibers: 10 nm, or 30 nm?

NUCLEOSOMES: "BEADS ON A STRING" If you isolate nuclei (eg. Drosophila) and extract chromatin at low ionic strength (<100mM salt), you see "beads on a string" images in electron microscopy (a). These beads are nucleosomes (b). An interpretation of the structure is shown below: Two turns of DNA are coiled around each nucleosome, with a short region of naked DNA linking each nucleosome. Since the width of a nucleosome is about 10nm, these are called "10nm fibers". (In contrast, a DNA double helix is 2nm wide.) If you extract chromatin at high salt (200-300mM), or re-constitute chromatin by increasing Na+ or Mg++ conc, you get a 30nm fiber, or solenoid (c). The 30nm fiber is a coil of nucleosomes made up of 6 nucleosomes per turn.

Image displayed by hyperlink to https://media.springernature.com/full/springer-static/image/art%3A10.1038%2Fnrm1225/MediaObjects/41580_2003_Article_BFnrm1225_Fig3_HTML.jpg?as=webp

Alternative model: Irregular folding of 10-nm fibers. Maeshima, K., Imai, R., Tamura, S., & Nozaki, T. (2014). Chromatin as dynamic 10-nm fibers. Chromosoma, 123(3), 225–237. http://doi.org/10.1007/s00412-014-0460-2 Since the 30-nm fiber appears not to form at physiological salt conditions, one model proposes that 10-nm fibers associate in an unorganized fashion, governed by hydrophobic and hydrophilic interactions. The result is irregularly folded bundles of 10-nm fibers of chromatin. One advantage of this model is that it predicts that chromatin bundles will be less stable than the highly-organized 30-nm fibers would be. That means that bundles will be expected to open and close, to fold and unfold, giving transcriptional enzymes and DNA binding proteins better access to genes. Thus, transcription is easier to initiate on irregularly-folded chromatin, than it would be in 30-nm fibers.

Copyright © The Author(s) 2014 Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

Linker histones are important to coiling of nucleosomes. Histone 5 is also a linker histone, that is seen more often in transcriptionally active chromatin. Histone H1 tends to be seen more in inactive chromatin. The individual nucleosomes can be linked in three different ways, as per the diagram at right.

To facilitate tight compaction of DNA in sperm, histones are replaced by protamines

In mamalian spermatids, the nuclear volume is about 5% that of the somatic cell nucleus. To achieve this level of packaging, an arginine- and cysteine-rich class of proteins called protamines replace histones. As illustrated in the figure, a round of transcription occurs in haploid spermatids, producing copious quantities of protamines, which displace histones in mature spermatids. In mamalian spermatids, protamines do not directly displace histones. Mamalian spermatids first displace histones with basic "transition proteins", which are in turn displaced by protamines. From P. Sassone-Corsi (2001) Unique chromatin remodeling and transcriptional regulation in spermatogenesis. Science 296: 2176-2178.

For transcription to occur, the chromatin structure of a gene must be 'open', that is, accessible to the transcriptional machinery

In this discussion about histone proteins and nucleosomes, you may have been wondering: if DNA is complexed with these proteins, won't that make it hard to transcribe DNA? The enzymes of the eukaryotic transcriptional apparatus are adapted to the presence of chromatin proteins. This is the price eukaryotic cells pay for having such large genomes. You can't have a large genome without having the extra overhead of organizing it, which is done by the chromatin proteins. Like everything else in eukaryotic cells, gene expression and replication are very deliberate and carefully orchestrated processes. 

So, for gene expression to occur, the DNA can be complexed with proteins, but it has to be in a transcriptionally "open" position. How can we detect this "open" chromatin structure?

We can compare chromatin structure around the beta-globin gene of chick embryo erythroblasts (precursors to red blood cells), which do express globin genes, and an undifferentiated cell line MSB, which does not express globin genes. In erythroblasts, the DNA of the globin gene is preferentially sensitive to DNAseI digestion. This suggests that the structure of the chromatin is looser in cells expressing globin.

Note: These treatments are done using isolated nuclei, not naked DNA
 

Procedure:   Isolate nuclei              nuclei                                |   Divide into several tubes    v                                |----+----+---+---+--+--|                                v    v    v   v   v  v  v   DNAseI [µg/ml]               0  .01  .05  .1  .5 1 1.5                                        | Isolate DNA                                        v                                        | Cut with BamH1                                        v                                        | Electrophoresis                                        v                                        |  Blot                                        v                                         |Probe with globin gene                                        v

In the autoradiogram, we see that even at the highest DNAseI concentration, the 4.6kb fragment is relatively insensitive to digestion in nuclei from a cell line that does not express the globin gene. In the erythroblasts, however, specific degredation of this sequence can be seen to occur even at the lowest concentration. The interpretation of this is that the globin gene is in a more open chromatin configuration, which provides greater access to DNAseI. This phenomenon is referred to as general nuclease sensitivity, because the whole gene appears to be digested. However, more detailed studies of certain genes can detect specific sites within transcriptionally active genes that are hypersensitive to nuclease digestion.

From Lodish et al. Molecualr Cell Biology http://www.ncbi.nlm.nih.gov/books/bookres.fcgi/mcb/ch9f32b.gif

The Adh1 (alcohol dehydrogenase) gene in maize can be induced (ie. made to produce mRNA) by anaerobic conditions. This helps the plant detoxify the end products of anaerobic respiration. In cell culture, anaerobic conditions can be simulated by bubbling Ar into cell suspensions for 2hr. This system permits the comparison of chromatin structure of the Adh1 gene between anaerobic and aerobic conditions. Below is a restriction map of the region near the Adh1 gene in maize. (Start of transcription is +1). We can map hypersensitive sites by indirect end-labeling. Note that in the general sensitivity experiment, we used a probe that overlapped the entire gene in question. Since it is possible that many sites in a given gene would be digested simultaneously by DNAseI, the Bam fragment above was chopped into pieces too small to detect on an agarose gel. But with indirect end labeling, we use a short probe that lays outside the region we wish to examine, from +1 to +210.

M is a marker lane, with a mixture of DNAs generated by digesting this gene with either BamH1, Xba1, Pst1 or Alu1, and then mixing all of these DNAs together.Now, when the DNAse-ed DNA is digested with Hind3, the +1 to +210 probe will only detect fragments upstream from the Hind3 site. If we do a limiting digestion with DNAseI, in any given fragment, only one of several possible hypersensitive sites will be digested in a given molecule. Thus, we get a "ladder", in which each rung represents a different cut site. Note that there are two constitutive hypersensitive sites, even in uninduced (aerobic) nuclei, whereas in induced nuclei, we see 8 distinct hypersensitive sites.

from Anna-Lisa Paul, Vimla Vasil, Indra K. Vasil, and Robert J. Ferl. Constitutive and anaerobically induced DNase-I-hypersensitive sites in the 5′ region of the maize Adh1 gene. PNAS 84:799-803

Transcriptionally active DNA remains associated with histones, although nucleosome configuration may be different in active vs. inactive chromatin

So, we can identify regions that are in a more transcriptionally "open" position. But what does that "open" configuration look like with the histones? We know from electron micrograph data that the 10nm beads on a string configuration is present during transcription.

What's happening here? Let's start with two hypotheses.

• H(0): Transcription cannot occur when DNA is complexed into nucleosomes
• H(1): Transcription can occur when DNA is complexed into nucleosomes

As a means of detecting whether or not DNA is bound up with histones, crosslinking mediated by psoralen was used to covalently crosslink any DNA that was accessible when cells were treated with UV. The spacer DNA between nucleosomes is more accessible to crosslinking, resulting covalent bonds between the two strands. When chromatin is isolated for Electron Microscopy, the proteins are lost, and only the DNA remains on the grid. The crosslinked spacer regions remain as doublestranded helix. The DNA that was complexed with histones becomes single-stranded, giving appearance of bubbles on the circular chromosome. Thus, each bubble tells us which DNA was complexed into nucleosomes. Since we see bubbles on the entire SV40 genome, and we also see an RNA transcript coming off of the circle, with nucleosomes on either side of the transcription site, we can conclude that nucleosome structure remains intact even during transcription.

Nucleosomes seem to protect little bubbles of DNA in transcribing genes from treatment with DNA crosslinking reagents (eg. 6-methyl psoralen). The bubbles can be shown to be roughly 200bp apart. Only the inter-nucleosome DNA is crosslinked. The long "tail" on the circle is the growing mRNA transcript.

Nucleosome structure "opens up" during transcription, in a process called "chromatin remodeling"

So how does the DNA become accessible to RNA polymerase while staying complexed with histone proteins? The answer is a process called chromatin remodeling.

Chromatin remodeling: insights and intrigue from single-molecule studies Bradley R Cairns. Nature Structural & Molecular Biology 14, 989 - 996 (2007) Published online: 5 November 2007 doi:10.1038/nsmb1333 The precise details of chromatin remodeling are still under investigation. However, the figure at right illustrates one model for chromatin remodeling. In (d), D and Tr domains of the ocatmer "walk" along the double helix by rotating around the Hinge domain. Thus, while the DNA is more accessible to the solvent, and hence, more sensitive to nucleases, the nucleosome never completely dissociates from the DNA helix. Image displayed by hypertext link to https://media.springernature.com/lw685/springer-static/image/art%3A10.1038%2Fnsmb1333/MediaObjects/41594_2007_Article_BFnsmb1333_Fig3_HTML.gif?as=webp