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13.2: Genes and Chromatin in Eukaryotes - Biology


Chromosomes and chromatin are a uniquely eukaryotic association of DNA with more or less protein. Bacterial DNA (and prokaryotic DNA generally) is relatively ‘naked’ – not visibly associated with protein.

The electron micrograph of an interphase cell (below) reveals that the chromatin can itself exist in various states of condensation.

Chromatin is maximally condensed during mitosis, forming chromosomes. During interphase, chromatin exists in more or less condensed forms, called Heterochromatin and euchromatin respectively. Transition between these chromatin forms involve changes in the amounts and types of proteins bound to the chromatin, and can that can occur during gene regulation, i.e., when genes are turned on or off. Active genes tend to be in the more dispersed euchromatin so that enzymes of replication and transcription have easier access to the DNA. Genes that are inactive in transcription are heterochromatic, obscured by additional chromatin proteins present in heterochromatin. We’ll be looking at some experiments that demonstrate this in a later chapter.

We can define three levels of chromatin organization in general terms:

1. DNA wrapped around histone proteins form nucleosomes in a "beads on a string" structure.

2. Multiple nucleosomes coil (condense), forming 30 nm fiber (solenoid) structures.

3. Higher-order packing of the 30 nm fiber leads to formation of metaphase chromosomes seen in mitosis & meiosis.

The levels of chromatin structure were determined in part by selective isolation and extraction of interphase cell chromatin, followed by selective chemical extraction of chromatin components. The steps are:

· Nuclei are first isolated from the cells.

· The nuclear envelope gently ruptured so as not to physically disrupt chromatin structure.

· the chromatin can be gently extracted by one of several different chemical treatments (high salt, low salt, acid...).

The levels of chromatin structure are illustrated below.

Salt extraction dissociates most of the proteins from the chromatin. When a low salt extract is centrifuged and the pellet resuspended, the remaining chromatin looks like beads on a string. DNA-wrapped nucleosomes are the beads, which are in turn linked by uniform lengths of metaphorical DNA “string’ ( # 1 in the illustration above). A high salt chromatin extract appears as a coil of nucleosomes, or 30 nm solenoid fiber (# 2 above). Other extraction protocols revealed other aspects of chromatin structure shown in #s 3 and 4 above. Chromosomes seen in metaphase of mitosis are the ‘highest order’, most condensed form of chromatin.

The 10 nm filament of nucleosome ‘beads-on-a-string’ remaining after a low salt extraction can be seen in an electron microscope as shown below.

When these nucleosome necklaces were digested with the enzyme deoxyribonuclease (DNAse), the DNA between the ‘beads’ was degraded, leaving behind shortened 10nm filaments after a short digest period, or just single beads the beads after a longer digestion (below).

Roger Kornberg (son of Nobel Laureate Arthur Kornberg who discovered the first DNA polymerase enzyme of replication) participated in the discovery and characterization of nucleosomes while he was still a post-doc! Electrophoresis of DNA extracted from these digests revealed nucleosomes separated by a “linker” DNA stretch of about 80 base pairs. DNA extracted from the nucleosomes was about 147 base pairs long. This is the DNA that had been wrapped around the proteins of the nucleosome.

172 Nucleosomes-DNA & Protein

After separating all of the proteins from nucleosomal DNA, five proteins were identified (illustrated below).

Histones are basic proteins containing many lysine and arginine amino acids. Their positively charged side chains enable these amino acids bind the acidic, negatively charged phosphodiester backbone of double helical DNA. The DNA wraps around an octamer of histones (2 each of 4 of the histone proteins) to form the nucleosome. About a gram of histones is associated with each gram of DNA. After a high salt chromatin extraction, the structure visible in the electron microscope is the 30nm solenoid, the coil of nucleosomes modeled in the figure below.

As shown above, simply increasing the salt concentration of an already extracted nucleosome preparation will cause the ‘necklace’ to fold into the 30nm solenoid structure.

173 Chromatin Structure: Dissecting Chromatin

As you might guess, an acidic extraction of chromatin should selectively remove the basic histone proteins, leaving behind an association of DNA with non-histone proteins. This proved to be the case. An electron micrograph of the chromatin remnant after an acid extraction of metaphase chromosomes is shown on the next page.

DNA freed of the regularly spaced histone-based nucleosomes, loops out, away from the long axis of the chromatin. Dark material along this axis is a protein scaffolding that makes up what’s left after histone extraction. Much of this protein is topoisomerase, an enzyme that prevents DNA from breaking apart under the strain of replication.

174 Histones and Non-Histone Proteins


Gene Regulation in Eukaryotes

Let us make an in-depth study of the gene regulation in eukaryotes. After reading this article you will learn about: 1. Chromatin Modification 2. Control of Transcription by Hormones 3. Regulation of Processing of mRNA 4. Control of Life Span of mRNA 5. Gene Amplification 6. Post Translation Regulation and 7. Post Transcription Gene Silencing.

Introduction to Gene Regulation:

The expression of genes can be regulated in eukaryotes by all the principles as those of prokaryotes. But there are many additional mechanisms of control of gene expression in eukaryotes as genome is much bigger. The genes are present in the nucleus where mRNA is synthesized. The mRNA is then exported to cytoplasm where translation takes place.

In eukaryotes, the organization is multicellular and specialized into tissues and organs. The cells are differentiated and cells of a tissue generally produce a specific protein involving a particular set of genes. All other genes become permanently shut off and are never transcribed.

Structural features of eukaryotes that influence the gene expression are the presence of nucleosomes in chromatin, heterochromatin and the presence of the split genes in chromosomes.

As compared to prokaryotic genes, the eukaryotic genes have many more regulatory binding sites and they are controlled by many more regulatory proteins. Regulatory sequences can be present thousands of nucleotides away from the promoter, may lie upstream and downstream. These regulatory sequences act from a distance. The intervening DNA loops out, so that the regulatory sequence and promoter come to lie near each other.

Most of the regulation of gene control occurs at the initiation of transcription level. Initiation of translation also influences gene regulation immensely.

Chromatin Modification:

The genome of eukaryotes is wrapped in histone proteins to form nucleosomes. This condition leads to partial concealment of genes and reduces the expression of genes.

The packing of DNA with histone octomers is not permanent. Any portion of DNA can be released from the octomer whenever DNA binding proteins have to act on it. These DNA binding proteins or enzymes recognize their binding sites on DNA only when it is released from histone octomer or when present on linker DNA. The DNA is unwrapped from nucleosomes.

This unwrapping of DNA from nucleosomes is performed by nucleosome modifier enzymes or nucleosome remodelling complex. They act in various ways. They may remodel the structure of octomer or slide the octomer along DNA, thus uncover the DNA binding sites for the action of regulatory proteins. Thus the genes are activated.

Some of these nucleosome modifiers add acetyl groups (acetylation) to the tails of histones, thus loosen the DNA wrapping and in the process exposes the DNA binding sites. All these lead to the expression of genes. Similarly, deacetylation by deacetylases causes inactivation of DNA.

Nucleosomes are entirely absent in the regions that are active in transcription like rRNA genes.

Dense form of chromatin is called heterochromatin in eukaryotes. It leads to gene inhibition or gene silencing. Heterochromatin is densely packaged part of chromatin which does not allow gene expression. Densely packaged chromatin cannot be easily transcribed. Some enzymes make the chromatin more dense. Telomeres and contromeres are in the form of heterochromatin.

In higher animals about 50% of the genome is in the form of heterochromatin. Enzymes are capable of changing the density of chromatin by chemically modifying the tails of histones. This affects transcription.

In this way, both activation and repression of transcription is performed by modification of chromatin into heterochromatin and euchromatin.

Methylation of certain sequences of DNA prevents the transcription of genes in mammals. It has been observed that genes, which are heavily methylated are not transcribed, therefore not expressed. DNA methylase enzymes cause methylation of certain DNA sequences thereby silencing of genes.

Control of Transcription by Hormones:

Various intercellular and intracellular signals regulate the gene expression.

Hormones exercise considerable control over transcription. Hormones are extracellular substances synthesized by endocrine glands. They are carried to the distant target cells. Various hormones like insulin, estrogen, progesterone, testosterone etc. often act by “switching on” transcription of DNA.

The hormone on entering a target cell forms a complex with the receptor present in the cytoplasm. This hormone-receptor complex enters the nucleus and binds to a particular chromosome by means of specific proteins. This initiates the transcription. Hormone-receptor complex can enhance or suppress the expression of genes.

It has been observed in chickens that when hormone estrogen is injected, the oviduct responds by synthesizing mRNA, which is responsible for synthesis of albumen. The hormone directly binds to DNA and acts as an inducer.

Regulation of Processing of mRNA:

Genes of eukaryotes have non-coding regions (introns) in between coding regions (exons). Such genes are called split genes. The entire gene is transcribed to produce mRNA which is called precursor mRNA or primary transcript (pre-mRNA). Before translation takes place, the introns are spliced out by excision and discarded. This is known as processing of mRNA and the processed mRNA is called mature mRNA. This takes part in protein synthesis. Mature mRNA is considerably smaller than precursor mRNA.

Higher eukaryotes have various mechanisms by which pre-mRNA is processed in alternate or differential ways to produce different mRNAs which encode different proteins. Multiple proteins are produced from one gene by alternate mRNA processing. Many cells take advantage of different splicing pathways to alter the expression of genes and synthesize different polypeptides. Alternate mRNA splicing increases the number of proteins expressed by a single eukaryotic gene.

Alternate processing of pre-mRNA is accomplished by exon skipping, by retaining certain introns etc.

These alternate processing pathways are highly regulated.

In drosophilla mRNA is processed in four different ways, therefore produces four different kinds of muscle protein myosin. Different kind of myosin is produced in larva, pupa and late embryonic stages.

Control of Life Span of mRNA:

In prokaryotes the life span of an mRNA molecule is very brief, lasting only for a minute or less. The mRNA immediately degenerates after the protein synthesis.

But as the mRNA in eukaryotes is transported to cytoplasm through the nucleopores, this mRNA is repeatedly translated. This repeated translation of mRNA is achieved by increasing the life span of mRNA. In a highly differentiated cell, single mRNA molecule having long life span is able to produce large amount of single protein. Life span of a eukaryotic mRNA varies from a few hours to several days.

Chicken oviduct cells have a single copy of ovalbumen gene but produce large amount of albumen.

Silk gland of silkworm produces a very long thread made of protein fibroin, which forms cocoon. Silk gland is a single polyploid cell. It produces large number of mRNA molecules, which have long life span of several days.

Gene Amplification:

A mechanism exists in various organisms whereby the number of genes is increased many fold without mitosis division. This is called gene amplification.

During amplification DNA repeatedly undergoes replication without mitotic separation into daughter DNA molecules or chromatids. This enables the cell to produce large amount of protein in a short time.

Post Translation Regulation:

In prokaryotes, a single polycistronic mRNA molecule codes for many different proteins. But in eukaryotes having mono-cistronic mRNA, synthesis of different proteins is achieved in a different way. A single mRNA yields a large polypeptide called polyprotein. This polyprotein is then cleaved in alternate ways to produce different proteins. Each protein is regarded as the product of a single gene. In this system, there are many cleaving sites on the polyprotien.

Post Transcription Gene Silencing:

Many small RNAs exist in eukaryotes that play their role in silencing of genes. These small RNAs act on mRNA resulting in disruption of translation. These small RNAs are micro RNAs (miRNAs), small interfering RNAs (siRNAs) and many others.


Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana

Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Figures

Identification of co-expressed physical clusters…

Identification of co-expressed physical clusters in stamen development. ( A ) Schematic view…

Chromosomal position and gene composition…

Chromosomal position and gene composition of the clusters. ( A ) The position…

Expression levels in floral buds…

Expression levels in floral buds of different developmental stages. ( A ) Relative…

Chromosomal de-condensation estimated by DNA…

Chromosomal de-condensation estimated by DNA FISH. Biotin- (orange) or DIG- (green) labelled probes…


Histone Acetylation and Nucleosome Assembly

When examined by electron microscopy at low ionic strength, nucleosomal chromatin resembles a string of beads with diameters of about 10 nm and linker DNA extended between adjacent nucleosomes (Fig. 13-1). Each nucleosome in chromosomes is typically associated with about 200 base pairs of DNA. With subtraction of 166 base pairs for two turns around the histone octamer, this leaves 34 base pairs of linker DNA between adjacent nucleosomes. Linker DNA can vary widely in length in different tissues and cell types.

A fifth histone, H1 or linker histone, is thought to bind to linker DNA at the side of each nucleosome core where the DNA molecule enters and exits the structure (Fig. 13-5). H1 histones have a “winged helix” central domain flanked by unstructured basic domains at both the N- and C-termini (Fig. 13-3). Mammals have at least eight variant forms (called subtypes) of H1 histones (H1a–e, H1 0 , H1t, and H1oo). The amino acid sequences of these variants differ by 40% or more. Of these, H1 0 is found in cells entering the nondividing Go state (see Chapter 41), while H1t and H1oo are found exclusively in developing sperm and oocytes, respectively.

(A, PDB file: 1KX5. B, PDB file: 1HST.)


Stem Cell Proliferation and Differentiation

David C. Klein , Sarah J. Hainer , in Current Topics in Developmental Biology , 2020

13.2 Bivalent promoters mark lowly expressed but poised genes in ES cells

Overall, ES cell chromatin is enriched for active histone modifications such as H3K4me3 and H3K9ac (reviewed in Meshorer & Misteli, 2006 ). Pluripotent chromatin is, however, distinguishable by a subset of gene promoters (between 3000 and 4000) ( Li, Lian, Dai, Xiang, & Dai, 2013 ) marked with both active (H3K4me3) and repressive (H3K27me3) histone modifications, placed, respectively, by complexes in the Trithorax and Polycomb (PRC2) groups ( Fig. 7 ). The first evidence of bivalent chromatin marks in murine ES cells was identified using ChIP and tiling oligonucleotide arrays where a large region of H3K27me3 was accompanied by smaller patches of H3K4me3 ( Azuara et al., 2006 Bernstein et al., 2006 ). These bivalent promoters mark lowly expressed genes in undifferentiated cells, and are therefore poised for activation in response to developmental signaling ( Azuara et al., 2006 Bernstein et al., 2006 Voigt, Tee, & Reinberg, 2013 ). Transcription of these genes is repressed, but not blocked: bivalent genes remain open to transcription factors and are therefore poised for upregulation upon differentiation as required by the organism ( Voigt et al., 2013 ). During differentiation, one mark is typically lost from the promoter while the other becomes enriched depending on whether the gene is expressed or silenced. In ES cells, bivalent chromatin is resolved by SWI/SNF-mediated eviction of PcG proteins ( Stanton et al., 2017 ) and by ASF1A-driven clearing of H3K27me3 ( Gao, Gan, Lou, & Zhang, 2018 ). Eviction of the repressive H3K27me3 mark and the PcG proteins responsible for its placement allows derepression of bivalent lineage specification genes ( Stanton et al., 2017 ). Consistent with their poised state, bivalent genes tend to display low levels of DNA methylation, another repressive epigenetic mark, with the CpG islands of germline and Polycomb genes becoming increasingly methylated as ES cells differentiate and commit to specific lineages ( Mohn et al., 2008 Vastenhouw & Schier, 2012 ). Bivalent genes are largely lineage specification genes (such as members of the SOX, FOX, PAX, IRX, and POU/OCT families Bernstein et al., 2006 ) and remain accessible but not expressed in ES cells. Bivalent chromatin marks are largely specific to stem cells (including ES cells, iPSCs, and hematopoietic stem cells Cui et al., 2009 Harikumar & Meshorer, 2015 ). Bivalent genes have, however, been identified in non-stem cells, including differentiated T cells and some cancer cell types ( Bapat et al., 2010 Barski et al., 2007 Lin et al., 2015 McGarvey et al., 2008 Rodriguez et al., 2008 Roh, Cuddapah, Cui, & Zhao, 2006 ). While bivalent promoters were initially thought to be restricted to developmentally-regulated genes to enable fast transition between active and repressed expression states, it is clear that bivalency is more complex, as bivalent promoters have been identified in different gene families in numerous different cell types. Furthermore, regulation of the bivalent state is carried out by a wide variety of different proteins and regulators that extend well beyond the Trithorax and Polycomb group members that place the epigenetic marks of bivalency. Some important regulators include nucleosome remodeling complexes such as Tip60-p400, esBAF, CHD7, and NuRD ( Alajem et al., 2015 Fazzio et al., 2008b Ho, Jothi, et al., 2009 Lei et al., 2015 Reynolds, Salmon-Divon, et al., 2012 Schnetz et al., 2010 ). Tip60-p400 significantly co-localizes with H3K4me3, particularly near the TSS, and the complex mostly acts to repress gene expression in ES cells ( Fazzio et al., 2008b ). Knockdown of Tip60-p400 results in deregulation of 4% of genes, most of which are upregulated interestingly, many of those found to be upregulated were classical bivalent early-differentiation genes, which are normally silenced in ES cells ( Fazzio et al., 2008b ). The esBAF subunit BAF60A was mapped by ChIP-seq to reveal a distribution similar to that of H3K27me3 around TSSs, with significant enrichment at promoters of bivalent genes after BAF60A depletion, these marks were found to be significantly redistributed genome-wide ( Alajem et al., 2015 ). While esBAF tends to function as an activator of gene expression and PRC2 as a repressor, the two function synergistically to properly regulate expression of differentiation-associated genes ( Ho et al., 2011 ). CHD7 associates with a PcG cluster, containing SUZ12, RING1B, and EZH2, suggesting a connection between CHD7 and H3K27me3 ( Schnetz et al., 2010 ). Finally, the NuRD ATPase CHD4 removes acetyl groups from H3K27 in ES cells, thereby allowing the subsequent recruitment of PcG proteins and H3K27me3 ( Reynolds, Salmon-Divon, et al., 2012 ). CHD4 also interacts with the H3K4 demethylase LSD1, which occupies a majority of active genes and approximately two thirds of bivalent genes ( Whyte et al., 2012 ). Nucleosome remodeling factors play critical roles in cell fate decisions and maintenance of stem cell characteristics while their relationships with bivalency have been discussed, the next section will substantially expand upon these roles as well as other functions of nucleosome remodeling factors in ES cells.


Euchromatin and Heterochromatin

Chromatin within a cell may be compacted to varying degrees depending on a cell's stage in the cell cycle.

In the nucleus, chromatin exists as euchromatin or heterochromatin. During interphase of the cycle, the cell is not dividing but undergoing a period of growth.

Most of the chromatin is in a less compact form known as euchromatin. More of the DNA is exposed in euchromatin allowing replication and DNA transcription to take place.

During transcription, the DNA double helix unwinds and opens to allow the genes coding for proteins to be copied. DNA replication and transcription are needed for the cell to synthesize DNA, proteins, and organelles in preparation for cell division (mitosis or meiosis).

A small percentage of chromatin exists as heterochromatin during interphase. This chromatin is tightly packed, not allowing gene transcription. Heterochromatin stains more darkly with dyes than does euchromatin.


14.5 DNA Replication in Eukaryotes

By the end of this section, you will be able to do the following:

  • Discuss the similarities and differences between DNA replication in eukaryotes and prokaryotes
  • State the role of telomerase in DNA replication

Eukaryotic genomes are much more complex and larger in size than prokaryotic genomes. Eukaryotes also have a number of different linear chromosomes. The human genome has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are replicated during the S phase of the cell cycle. There are multiple origins of replication on each eukaryotic chromosome humans can have up to 100,000 origins of replication across the genome. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic replication. In yeast, which is a eukaryote, special sequences known as autonomously replicating sequences (ARS) are found on the chromosomes. These are equivalent to the origin of replication in E. coli.

The number of DNA polymerases in eukaryotes is much more than in prokaryotes: 14 are known, of which five are known to have major roles during replication and have been well studied. They are known as pol α, pol β, pol γ, pol δ, and pol ε.

The essential steps of replication are the same as in prokaryotes. Before replication can start, the DNA has to be made available as a template. Eukaryotic DNA is bound to basic proteins known as histones to form structures called nucleosomes. Histones must be removed and then replaced during the replication process, which helps to account for the lower replication rate in eukaryotes. The chromatin (the complex between DNA and proteins) may undergo some chemical modifications, so that the DNA may be able to slide off the proteins or be accessible to the enzymes of the DNA replication machinery. At the origin of replication, a pre-replication complex is made with other initiator proteins. Helicase and other proteins are then recruited to start the replication process (Table 14.2).

PropertyProkaryotesEukaryotes
Origin of replicationSingleMultiple
Rate of replication1000 nucleotides/s50 to 100 nucleotides/s
DNA polymerase types514
TelomeraseNot presentPresent
RNA primer removalDNA pol IRNase H
Strand elongationDNA pol IIIPol α, pol δ, pol ε
Sliding clampSliding clampPCNA

A helicase using the energy from ATP hydrolysis opens up the DNA helix. Replication forks are formed at each replication origin as the DNA unwinds. The opening of the double helix causes over-winding, or supercoiling, in the DNA ahead of the replication fork. These are resolved with the action of topoisomerases. Primers are formed by the enzyme primase, and using the primer, DNA pol can start synthesis. Three major DNA polymerases are then involved: α, δ and ε. DNA pol α adds a short (20 to 30 nucleotides) DNA fragment to the RNA primer on both strands, and then hands off to a second polymerase. While the leading strand is continuously synthesized by the enzyme pol ε, the lagging strand is synthesized by pol δ. A sliding clamp protein known as PCNA (proliferating cell nuclear antigen) holds the DNA pol in place so that it does not slide off the DNA. As pol δ runs into the primer RNA on the lagging strand, it displaces it from the DNA template. The displaced primer RNA is then removed by RNase H (AKA flap endonuclease) and replaced with DNA nucleotides. The Okazaki fragments in the lagging strand are joined after the replacement of the RNA primers with DNA. The gaps that remain are sealed by DNA ligase, which forms the phosphodiester bond.

Telomere replication

Unlike prokaryotic chromosomes, eukaryotic chromosomes are linear. As you’ve learned, the enzyme DNA pol can add nucleotides only in the 5' to 3' direction. In the leading strand, synthesis continues until the end of the chromosome is reached. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no way to replace the primer on the 5’ end of the lagging strand. The DNA at the ends of the chromosome thus remains unpaired, and over time these ends, called telomeres, may get progressively shorter as cells continue to divide.

Telomeres comprise repetitive sequences that code for no particular gene. In humans, a six-base-pair sequence, TTAGGG, is repeated 100 to 1000 times in the telomere regions. In a way, these telomeres protect the genes from getting deleted as cells continue to divide. The telomeres are added to the ends of chromosomes by a separate enzyme, telomerase (Figure 14.16), whose discovery helped in the understanding of how these repetitive chromosome ends are maintained. The telomerase enzyme contains a catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and DNA nucleotides complementary to the RNA template are added on the 3' end of the DNA strand. Once the 3' end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. Thus, the ends of the chromosomes are replicated.

Telomerase is typically active in germ cells and adult stem cells. It is not active in adult somatic cells. For their discovery of telomerase and its action, Elizabeth Blackburn, Carol W. Greider, and Jack W. Szostak (Figure 14.16) received the Nobel Prize for Medicine and Physiology in 2009.

Telomerase and Aging

Cells that undergo cell division continue to have their telomeres shortened because most somatic cells do not make telomerase. This essentially means that telomere shortening is associated with aging. With the advent of modern medicine, preventative health care, and healthier lifestyles, the human life span has increased, and there is an increasing demand for people to look younger and have a better quality of life as they grow older.

In 2010, scientists found that telomerase can reverse some age-related conditions in mice. This may have potential in regenerative medicine. 2 Telomerase-deficient mice were used in these studies these mice have tissue atrophy, stem cell depletion, organ system failure, and impaired tissue injury responses. Telomerase reactivation in these mice caused extension of telomeres, reduced DNA damage, reversed neurodegeneration, and improved the function of the testes, spleen, and intestines. Thus, telomere reactivation may have potential for treating age-related diseases in humans.

Cancer is characterized by uncontrolled cell division of abnormal cells. The cells accumulate mutations, proliferate uncontrollably, and can migrate to different parts of the body through a process called metastasis. Scientists have observed that cancerous cells have considerably shortened telomeres and that telomerase is active in these cells. Interestingly, only after the telomeres were shortened in the cancer cells did the telomerase become active. If the action of telomerase in these cells can be inhibited by drugs during cancer therapy, then the cancerous cells could potentially be stopped from further division.


The Forms of DNA

Figure 4.13.2 Forms of DNA.

Except when a eukaryotic cell divides, its nuclear DNA exists as a grainy material called chromatin . Only once a cell is about to divide and its DNA has replicated does DNA condense and coil into the familiar X-shaped form of a chromosome , like the one shown below.

Figure 4.13.3 Diagram of a chromosome showing that in a chromosome with the typical “X” shape, it is comprised of two identical pieces of DNA, each called a chromatid.

Most cells in the human body have two pairs of 23 different chromosomes, for a total of 46 chromosomes. Cells that have two pairs of chromosomes are called diploid. Because DNA has already replicated when it coils into a chromosome, each chromosome actually consists of two identical structures called sister chromatids . Sister chromatids are joined together at a region called a centromere .


Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana

Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation.

© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Figures

Identification of co-expressed physical clusters…

Identification of co-expressed physical clusters in stamen development. ( A ) Schematic view…

Chromosomal position and gene composition…

Chromosomal position and gene composition of the clusters. ( A ) The position…

Expression levels in floral buds…

Expression levels in floral buds of different developmental stages. ( A ) Relative…

Chromosomal de-condensation estimated by DNA…

Chromosomal de-condensation estimated by DNA FISH. Biotin- (orange) or DIG- (green) labelled probes…


Contents

Some coactivators indirectly regulate gene expression by binding to an activator and inducing a conformational change that then allows the activator to bind to the DNA enhancer or promoter sequence. [2] [7] [8] Once the activator-coactivator complex binds to the enhancer, RNA polymerase II and other general transcription machinery are recruited to the DNA and transcription begins. [9]

Histone acetyltransferase Edit

Nuclear DNA is normally wrapped tightly around histones, making it hard or impossible for the transcription machinery to access the DNA. This association is due primarily to the electrostatic attraction between the DNA and histones as the DNA phosphate backbone is negatively charged and histones are rich in lysine residues, which are positively charged. [10] The tight DNA-histone association prevents the transcription of DNA into RNA.

Many coactivators have histone acetyltransferase (HAT) activity meaning that they can acetylate specific lysine residues on the N-terminal tails of histones. [4] [7] [11] In this method, an activator binds to an enhancer site and recruits a HAT complex that then acetylates nucleosomal promoter-bound histones by neutralizing the positively charged lysine residues. [7] [11] This charge neutralization causes the histones to have a weaker bond to the negatively charged DNA, which relaxes the chromatin structure, allowing other transcription factors or transcription machinery to bind to the promoter (transcription initiation). [4] [11] Acetylation by HAT complexes may also help keep chromatin open throughout the process of elongation, increasing the speed of transcription. [4]

Acetylation of the N-terminal histone tail is one of the most common protein modifications found in eukaryotes, with about 85% of all human proteins being acetylated. [12] Acetylation is crucial for synthesis, stability, function, regulation and localization of proteins and RNA transcripts. [11] [12]

HATs function similarly to N-terminal acetyltransferases (NATs) but their acetylation is reversible unlike in NATs. [13] HAT mediated histone acetylation is reversed using histone deactetylase (HDAC), which catalyzes the hydrolysis of lysine residues, removing the acetyl group from the histones. [4] [7] [11] This causes the chromatin to close back up from their relaxed state, making it difficult for the transcription machinery to bind to the promoter, thus repressing gene expression. [4] [7]

Examples of coactivators that display HAT activity include CARM1, CBP and EP300. [14] [15]

Corepression Edit

Many coactivators also function as corepressors under certain circumstances. [5] [9] Cofactors such as TAF1 and BTAF1 can initiate transcription in the presence of an activator (act as a coactivator) and repress basal transcription in the absence of an activator (act as a corepressor). [9]

Biological significance Edit

Transcriptional regulation is one of the most common ways for an organism to alter gene expression. [16] The use of activation and coactivation allows for greater control over when, where and how much of a protein is produced. [1] [7] [16] This enables each cell to be able to quickly respond to environmental or physiological changes and helps to mitigate any damage that may occur if it were otherwise unregulated. [1] [7]

Associated disorders Edit

Mutations to coactivator genes leading to loss or gain of protein function have been linked to diseases and disorders such as birth defects, cancer (especially hormone dependent cancers), neurodevelopmental disorders and intellectual disability (ID), among many others. [17] [5] Dysregulation leading to the over- or under-expression of coactivators can detrimentally interact with many drugs (especially anti-hormone drugs) and has been implicated in cancer, fertility issues and neurodevelopmental and neuropsychiatric disorders. [5] For a specific example, dysregulation of CREB-binding protein (CBP)—which acts as a coactivator for numerous transcription factors within the central nervous system (CNS), reproductive system, thymus and kidneys—has been linked to Huntington's Disease, leukaemia, Rubinstein-Taybi syndrome, neurodevelopmental disorders and deficits of the immune system, hematopoiesis and skeletal muscle function. [14] [18]

As drug targets Edit

Coactivators are promising targets for drug therapies in the treatment of cancer, metabolic disorder, cardiovascular disease and type 2 diabetes, along with many other disorders. [5] [19] For example, the steroid receptor coactivator (SCR) NCOA3 is often overexpressed in breast cancer, so the development of an inhibitor molecule that targets this coactivator and decreases its expression could be used as a potential treatment for breast cancer. [15] [20]

Because transcription factors control many different biological processes, they are ideal targets for drug therapy. [14] [21] The coactivators that regulate them can be easily replaced with a synthetic ligand that allows for control over an increase or decrease in gene expression. [14]

Further technological advances will provide new insights into the function and regulation of coactivators at a whole-organism level and elucidate their role in human disease, which will hopefully provide better targets for future drug therapies. [14] [15]

To date there are more than 300 known coregulators. [15] Some examples of these coactivators include: [22]


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