Chromatin packaging

SIGNIFICANCE: The huge quantity of DNA present in each cell must be organized and highly condensed in order to fit into the discrete units of genetic material known as chromosomes. Gene expression can be regulated by the nature and extent of this DNA packaging in the chromosome, and errors in the packaging process can lead to genetic disease.

Chromosomes and Chromatin

Scientists have known for many years that an organism’s hereditary information is encrypted in molecules of DNA that are themselves organized into discrete hereditary units called genes and that these genes are organized into larger subcellular structures called chromosomes. James Watson and Francis Crick elucidated the basic chemical structure of the DNA molecule in 1953, and much has been learned since that time concerning its and expression. At the molecular level, DNA is composed of two parallel chains of building blocks called nucleotides, and these chains are coiled around a central axis to form the well-known “double helix.” Each on each chain attracts and pairs with a complementary nucleotide on the opposite chain, so a DNA molecule can be described as consisting of a certain number of these nucleotide base pairs. The entire human genome consists of more than six billion base pairs of DNA, which, if completely unraveled, would extend for more than 2 meters (6.5 feet). It is a remarkable feat of engineering that in each human cell this much DNA is condensed, compacted, and tightly packaged into chromosomes within a nucleus that is less than 10^-5 meters in diameter. What is even more astounding is the frequency and fidelity with which this DNA must be condensed and relaxed, packaged and unpackaged, for replication and expression in each individual cell at the appropriate time and place during both development and adult life. The essential processes of or (transcription) cannot occur unless the DNA is in a more open or relaxed configuration.

Chemical analysis of mammalian chromosomes reveals that they consist of DNA and two distinct classes of proteins, known as histone and nonhistone proteins. This nucleoprotein complex is called chromatin, and each chromosome consists of one linear, unbroken, double-stranded DNA molecule that is surrounded in predictable ways by these histone and nonhistone proteins. The are relatively small, basic proteins (having a net positive charge), and their function is to bind directly to the negatively charged DNA molecule in the chromosome. Five major varieties of histone proteins are found in chromosomes, and these are known as H1, H2A, H2B, H3, and H4. Chromatin contains about equal amounts of histones and DNA, and the amount and proportion of histone proteins are constant from cell to cell in all higher organisms. In fact, the histones as a class are among the most highly conserved of all known proteins. For example, for histone H3, which is a protein consisting of 135 “building blocks,” there is only a single amino acid difference in the protein found in sea urchins as compared with the one found in cattle. This is compelling evidence that histones play the same essential role in packaging in all higher organisms and that evolution has been quite intolerant of even minor sequence variations between vastly different species.

Nonhistones as a class of proteins are much more heterogeneous than the histones. They are usually acidic (carrying a net negative charge), so they will most readily attract and bind with the positively charged histones rather than the negatively charged DNA. Each cell has many different kinds of nonhistone proteins, some of which play a structural role in chromosome organization and some of which are more directly involved with the regulation of gene expression. Weight for weight, there is often as much nonhistone protein present in chromatin as histone protein and DNA combined.

Nucleosomes and Solenoids

The fundamental structural subunit of chromatin is an association of DNA and histone proteins called a “nucleosome.” First discovered in the 1970s by Ada and Donald Olins and Chris Woodcock, each consists of a core of eight histone proteins: two each of the histones H2A, H2B, H3, and H4. Around this histone octamer are wound 146 base pairs of DNA in one and three-quarter turns (approximately eighty base pairs per turn). The overall shape of each nucleosome is similar to that of a lemon or a football. Each nucleosome is separated from its adjacent neighbor by about fifty-five base pairs of “linker DNA,” so that in its most unraveled state they appear under the electron microscope to be like tiny beads on a string. Portions of each core histone protein protrude outside the wound DNA and interact with the DNA that links adjacent nucleosomes.

The next level of chromatin packaging involves a coiling and stacking of nucleosomes into a ribbonlike arrangement, which is twisted to form a chromatin fiber about 30 nanometers (nm) in diameter commonly called a “solenoid.” Formation of solenoid fibers requires the interaction of histone H1, which binds to the linker DNA between nucleosomes. Each turn of the chromatin fiber contains about 1,200 base pairs (six nucleosomes), and the DNA has now been compacted by about a factor of fifty. The coiled solenoid fiber is organized into large domains of 40,000 to 100,000 base pairs, and these domains are separated by attached that serve both to organize and to control their packaging and unpackaging.

Long DNA Loops and the Chromosome Scaffold

Physical studies using the techniques of X-ray crystallography and neutron diffraction have suggested that solenoid fibers may be further organized into giant supercoiled loops. The extent of this additional looping, coiling, and stacking of solenoid fibers varies, depending on the cell cycle. The most relaxed and extended chromosomes are found at interphase, the period of time between cell divisions. Interphase chromosomes typically have a diameter of about 300 nm. Chromosomes that are getting ready to divide (metaphase chromosomes) have the most highly condensed chromatin, and these structures may have a diameter of up to 700 nm. One major study on the structure of chromosomes has shown that a skeleton of nonhistone proteins in the shape of the metaphase chromosome remains even after all of the histone proteins and the DNA have been removed by enzymatic digestion. If the DNA is not digested, it remains in long loops (10 to 90 kilobase pairs) anchored to this nonhistone protein scaffolding.

In the purest preparations of metaphase chromosomes, only two scaffold proteins are found. One of these forms the latticework of the scaffold, while the other has been identified as toposiomerase II, an that is critical in DNA replication. This enzyme cleaves double-stranded DNA and then rapidly reseals the cut after some of the supercoiling has been relaxed, thus relieving torsional stress and preventing tangles in the DNA. Apparently this same enzyme activity is necessary for the coiling and looping of solenoid fibers along the chromosome scaffold that occurs during the transition between and metaphase chromosome structure. In the most highly condensed metaphase chromosomes, the DNA has been further compacted by an additional factor of one hundred.

Impact and Applications

Studies of chromatin packaging continue to reveal the details of the precise chromosomal architecture that results from the progressive coiling of the single DNA molecule into increasingly compact structures. Evidence suggests that the regulation of this coiling and packaging within the chromosome has a significant effect on the properties of the genes themselves. In fact, errors in DNA packaging can lead to inappropriate gene expression and developmental abnormalities. In humans, the blood disease thalassemia, several neuromuscular diseases, and even male sex determination can all be explained by the altered assembly of chromosomal structures.

Chromatin domains, composed of coiled solenoid fibers, may contain several genes, or the boundary of a domain can lie within a gene. These domains have the capacity to influence gene expression, and this property is mediated by specific DNA sequences known as locus control regions (LCRs). An LCR is like a powerful that activates transcription, thereby turning on gene expression. The existence of such sequences was first recognized from a study of patients with beta-thalassemia and a related condition known as hereditary persistence of fetal hemoglobin. In these disorders, there is an error in the expression of a cluster of genes, known as the beta-globin genes, that prevents the appearance of adult type hemoglobin. The beta-globin genes are linearly arrayed over a 50-kilobase-pair chromatin domain, and the LCR is found upstream from this cluster. Affected patients were found to have normal beta-globin genes, but there was a deletion of the upstream LCR that led to failure to activate the genes appropriately. Further investigation led to the conclusion that the variation in expression of these genes observed in different patients was caused by differences in the assembly of the genes into higher-order chromatin structures. In some cases, gene expression was repressed, while in others it was facilitated. Under normal circumstances, a nonhistone protein complex was found to bind to the LCR, causing the chromatin domain to unravel and making the DNA more accessible to transcription factors, thus enhancing gene expression.

DNA sequencing studies have demonstrated a common feature in several genes whose altered expression leads to severe human genetic disease. For example, the gene that causes myotonic dystrophy has a large number of repeating nucleotide triplets in the DNA region immediately adjacent to the protein-encoding segment. Physical studies have shown that this results in the formation of unusually stable nucleosomes, since these repeated sequences create the strongest naturally occurring sites for association with the core histones. It has been suggested that these highly stable nucleosomes are unusually resistant to the unwinding and of the DNA that must occur in order for gene expression to begin. RNA polymerase is the enzyme that makes an RNA transcript of the gene, and its movement through the protein-coding portion of the gene is inhibited if the DNA is unable to dissociate from the nucleosomes. Thus, although the necessary protein product would be normal and functional if it could be made, it is a problem with chromatin unpackaging that leads to reduced gene expression that ultimately leads to clinical symptoms of the disease. Both mild and severe forms of myotonic dystrophy are known, and an increase in the clinical severity correlates exactly with an increased number of nucleotide triplet repeats in the gene. Similar triplet repeats have been found in the genes responsible for Kennedy disease, Huntington’s disease (Huntington’s chorea), spinocerebellar ataxia type I, fragile X syndrome, and dentatorubral-pallidoluysian atrophy.

Fascinating and unexpected research results have suggested that a central event in the determination of gender in mammals depends on local folding of DNA within the chromosome. Molecular biologists Peter Goodfellow and Robin Lovell-Badge successfully cloned a human gene from the Y chromosome that determines maleness. This SRY gene (named from the sex-determining region of the Y chromosome) encodes a protein that selectively recognizes a specific DNA sequence and helps assemble a chromatin complex that activates other male-specific genes. More specifically, binding of the SRY protein causes the DNA to bend at a specific angle and causes conformation that facilitates the assembly of a protein complex to initiate the cascade of gene activation leading to male development. If the bend is too tight or too wide, gene expression will not occur, and the embryo will develop as a female.

The unifying lesson to be learned from these examples of DNA packaging and disease is that DNA sequencing studies and the construction of human genetic maps will not by themselves provide all the answers to questions concerning human variation and genetic disease. An understanding of human genetics at the molecular level depends not only on the primary DNA sequence but also on the three-dimensional organization of that DNA within the chromosome. Compelling genetic and biochemical evidence has left no doubt that the packaging process is an essential component of regulated gene expression.

Key terms

• chromatinthe material that makes up chromosomes; a complex of fibers composed of DNA, histone proteins, and nonhistone proteins
• histone proteinssmall, basic proteins that are complexed with DNA in chromosomes and that are essential for chromosomal structure and chromatin packaging
• nonhistone proteinsa heterogeneous group of acidic or neutral proteins found in chromatin that may be involved with chromosome structure, chromatin packaging, or the control of gene expression
• nucleosomethe basic structural unit of chromosomes, consisting of 146 base pairs of DNA wrapped around a core of eight histone proteins

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