Chapter 10: Accessing the Genome

Question 1

it is the ______ that is expressed, not individual _____

Answer 1

• genome
• genes

Question 2

functional compartments within the nucleus are not individually enclosed by ______, and so are not visible when the cell is observed using conventional light or electron microscopy techniques

Answer 2

membranes

Question 3

nuclear matrix

Answer 3

• nuclear substructure
• a complex network of protein and RNA fibrils
• permeates the entire nucleus and includes regions defined as the chromosome scaffold

Question 4

chromosome scaffold

Answer 4

• Most of the chromosomal DNA chains within the interphase nucleus are believed to be held on a scaffold or backbone structure made from various proteins, with loops of between 20 and 200 kb extruding from attachment sites
• The scaffold, as well as permitting further compaction, serves to bring the DNA together in organised regions.
• There are many different protein components of these scaffolds, amongst them DNA topoisomerases.
• changes its structure during cell division, resulting in condensation of the chromosomes into their metaphase forms

Question 5

nucleolus

Answer 5

• the center for synthesis and processing of rRNA molecules
• a small dense spherical structure in the nucleus of a cell during interphase

Question 6

migration of nuclear proteins does not occur as rapidly as expected because movement is hindered by the large amounts of DNA and RNA in the nucleus. Proteins involved in genome expression have the freedom needed to move from one activity site to another, as dictated by the changing requirements of the cell. In particular, the _____ _____ continually detach and reattach to their binding sites on the genome

Answer 6

linker histones

Question 7

each chromosome occupies its own space, or ______. These can be visualized by chromosome painting which reveals territories occupied by individual chromosomes. These territories take up the majority of the space within the nucleus, but are separated from one another by ______ regions, within which the enzymes and other proteins involved in expression of the genome are located

Answer 7

• territory
• nonchromatin

Question 8

individual centromeres remain _____ throughout the cell cycle, though there are occasional bursts of relatively slow movement, but remain fairly ______ during the lifetime of a cell

Answer 8

• stationary
• static

Question 9

translocations

Answer 9

• result in a segment of one chromosome becoming attached to another chromosome
• more frequent between certain pairs than others

Question 10

repeated occurrence of the same translocation suggests that the territories of the interacting pair of chromosomes are frequently ______ to one another in the nucleus. There is also evidence that, at least in some organisms, certain chromosomes preferentially occupy territories close to the ______ of the nucleus. Relatively little genome expression occurs in this region, and it is often here that those chromosomes that contain few active genes are found

Answer 10

• close
• periphery

Question 11

More refined microscopic examination has shown that _____ run through chromosome territories, linking different parts of the ______ regions, and providing a means by which the transcription machinery can penetrate into the internal parts of these territories

Answer 11

• channels
• nonchromatin

Question 12

chromatin

Answer 12

the complex of genomic DNA and chromosomal proteins present in the eukaryotic nucleus

Question 13

Chromatin structure is ______, ranging from the two lowest levels of DNA packaging, the ______and the ______ chromatin fiber, to the _____ chromosomes, which represent the most compact form of chromatin in eukaryotes and occur only during nuclear division

Answer 13

• hierarchic
• nucleosome
• 30 nm
• metaphase

Question 14

When nondividing nuclei are examined by light microscopy all that can be seen is a mixture of light- and dark-staining areas within the nucleus. The dark areas are called ______ and contain DNA that is still in a relatively ______ organization, although less than in the ______ structure

Answer 14

• heterochromatin
• compact
• metaphase

Question 15

Two types of heterochromatin are recognized:

Answer 15

• Constitutive heterochromatin
  
  • permanent feature of all cells
  • contains no genes
  • can always be retained in a compact organization
  • includes centromeric and telomeric DNA as well as certain regions of some other chromosomes, i.e. human Y chromosome
• Facultative heterochromatin
  
  • Facultative heterochromatin
  • contain genes that are inactive in some cells or at some periods of the cell cycle. When these genes are inactive, their DNA regions are compacted into heterochromatin

Question 16

Module 8

euchromatin

Answer 16

• parts of the chromosomal DNA where the active genes are located are less compact and permit entry of the expression proteins
• loops of DNA within the euchromatin regions
• loops are between 40 kb and 100 kb in length and predominantly in the form of the 30 nm chromatin fiber
• loops are attached to the nuclear matrix via AT-rich DNA segments called matrix-associated regions (MARs), or scaffold attachment regions (SARs), which are 100 - 1000 bp

Question 17

Module 8

structural domains

Answer 17

The loops of DNA between the nuclear matrix attachment points

Question 18

Module 8

How is a functional domain delineated

Answer 18

• It is delineated by treating a region of purified chromatin with deoxyribonuclease I (DNase I)
• deoxyribonuclease I (DNase I) is a DNA-binding protein that cannot gain access to the more compacted regions of DNA
• Regions sensitive to DNase I extend to either side of a gene or set of genes that is being expressed, indicating that in this area the chromatin has a more open organization

Question 19

there should be a correspondence between structural and functional domains, and this view is supported by the location of some ______, which mark the limits of a structural domain, at the boundary of a ______ domain. But the correspondence does not seem to be complete because some structural domains contain genes that are not ______ at the same time, and the boundaries of some structural domains lie _____ _____

Answer 19

• MARs
• functional
• expressed
• within genes

Question 20

Module 8

insulators

Answer 20

• sequences that are 1–2 kb in length, which mark boundaries of functional domains
• first discovered in Drosophila
• identified in a range of eukaryotes
• best studied are the pair of sequences called scs and scs’ (specialized chromatin structure)
• located either side of the gene in question

Question 21

Module 8

Insulators display two special properties related to their role as the delimiters of functional domains. The first is their ability to overcome the positional effect that occurs during a gene-cloning experiment with a eukaryotic host. Explain.

Answer 21

• positional effect refers to the variability in gene expression that occurs after a new gene has been inserted into a eukaryotic chromosome
• Results from the random nature of the insertion event
• Gene can be inserted in a region of highly packaged chromatin, where it will be inactive, or into an area of open chromatin where it will be expressed
• ability of scs and scs’ to overcome the positional effect was demonstrated by
  
  • placing them either side of a fruit-fly gene
  • When a gene is flanked by the insulators, it was always highly expressed when inserted back into the Drosophila genome
  • in contrast, when it didn’t flank a gene on either side, the gene experienced variable expression
• The deduction is that insulators can bring about modifications to chromatin packaging and establish a functional domain when inserted into a new site in the genome

Question 22

Module 8

Insulators display two special properties related to their role as the delimiters of functional domains. The second is their ability to maintain the independence of each functional domain, preventing “cross-talk” between adjacent domains. Explain.

Answer 22

• If scs or scs’ is excised from its normal location and reinserted between a gene and the upstream regulatory modules that control expression of that gene, then the gene no longer responds to its regulatory modules
• the gene becomes “insulated” from their effects
• This observation suggests that, in their normal positions, insulators prevent the genes within a domain from being influenced by the regulatory modules present in an adjacent domain

Question 23

Module 8

locus control region, or LCR

Answer 23

• DNA sequence responsible for the formation and maintenance of an open functional domain, at least for some domains
• can overcome the positional effect when linked to a new gene that is inserted into a eukaryotic chromosome
• also stimulates the expression of genes contained within its functional domain
• first discovered during a study of the human b-globin genes
• thought to be involved in expression of many genes that are active in only some tissues or during certain developmental stages
• 12 kb in length, positioned upstream of the genes in the 60 kb b-globin functional domain
• ability of mutations in the LCR to cause disease (thalassemia in human globin) is a clear indication that disruption of the LCR results in a loss of gene expression

Question 24

Module 8

locus control region (LCR) DNase I hypersensitive sites

Answer 24

• contains five separate DNase I hypersensitive sites
• these short regions of DNA are cleaved by DNase I more easily than other parts of the functional domain
• these sites are thought to coincide with positions where nucleosomes have been modified or are absent and which are therefore accessible to binding proteins that attach to the DNA
• the positioning of the nucleosomes influences gene expression. nucleosomes with regular spacing, as displayed by the typical “beads-on-a-string” structure and nucleosome whose positioning has changed have exposed a short stretch of DNA
• the presence or absence of nucleosomes is a cause of gene expression, the gene being switched off if nucleosomes cover the assembly site, or switched on if access to the site is open
• these binding proteins, not the DNA sequence of the LCR, control the chromatin structure within the functional domain

Question 25

# Module 8 locus control region (LCR) DNase I hypersensitive sites in genes in the b-globin functional domain

Answer 25

* DNase I hypersensitive sites occur immediately upstream of each of the genes in the b-globin functional domain * at the positions where the transcription initiation complex is assembled on the DNA * DNase I hypersensitive sites are variant components of a functional domain * the different b-type globin genes are expressed at different stages of the human developmental cycle, ε being active in the early embryo, Gγ and Aγ in the fetus, and δ and β in the adult * Only when the gene is active is its assembly position for the transcription initiation complex marked by a hypersensitive site

Question 26

# Module 8 two ways in which chromatin structure can influence genome expression

Answer 26

* The degree of chromatin packaging displayed by a segment of a chromosome determines whether or not genes within that segment are expressed. * If a gene is accessible, then its transcription is influenced by the precise nature and positioning of the nucleosomes in the region where the transcription initiation complex will be assembled

Question 27

# Module 8 Histone proteins can undergo various types of modification, the best studied of these being histone acetylation which is

Answer 27

* the attachment of acetyl groups to lysine amino acids in the N-terminal regions of each of the core molecules * These N termini form tails that protrude from the nucleosome core octamer * their acetylation reduces the affinity of the histones for DNA and possibly also reduces the interaction between individual * nucleosomes, destabilizing the 30 nm chromatin fiber * induces gene expression

Question 28

# Module 8 The histones in heterochromatin are generally ______ whereas those in functional domains are \_\_\_\_\_\_, a clear indication that this type of modification is linked to DNA packaging.

Answer 28

* unacetylated * acetylated

Question 29

* The view on the left is downward from the top of the barrel-shaped octamer * the view on the right is from the side * The two strands of the DNA double helix wrapped around the octamer are shown in brown and green * The octamer comprises a central tetramer of two histone H3 (blue) and two histone H4 (bright green) subunits plus a pair of H2A (yellow)–H2B (red) dimers, one above and one below the central tetramer * Nterminal tails of the histone proteins protrude from the core octamer

Answer 29

Two views of the nucleosome core octamer * The view on the left is downward from the top of the barrel-shaped octamer * the view on the right is from the side * The two strands of the DNA double helix wrapped around the octamer are shown in brown and green * The octamer comprises a central tetramer of two histone H3 (blue) and two histone H4 (bright green) subunits plus a pair of H2A (yellow)–H2B (red) dimers, one above and one below the central tetramer * Nterminal tails of the histone proteins protrude from the core octamer

Question 30

# Module 8 histone acetyltransferases (HATs)

Answer 30

* enzymes that add acetyl groups to histones * induce gene expression * do not work independently, but instead form multiprotein complexes * these complexes are typical of the large multiprotein structures that catalyze and regulate the various steps in genome expression * here are at least five different families of HAT proteins * the presence of proteins within these that have distinct roles (activation of gene transcription, DNA repair) in the initiation of gene expression, indicates that the individual events that result in gene activation are linked with not just histone acetylation

Question 31

Different complexes appear to acetylate different histones and some can also acetylate other proteins involved in genome expression. The enzymes are emerging as versatile proteins that may have diverse functions in

Answer 31

expression, replication, and maintenance of the genome

Question 32

# Module 8 histone deacetylases (HDACs)

Answer 32

* enzymes that can remove acetyl groups from histone tails, overturning the transcription-activating effects of the HATs * repress gene expression * gene silencing * contained in multiprotein complexes along with others that do not have deacetylase activity but which provide ancillary functions essential to the process.

Question 33

Methylation

Answer 33

* Methylation of lysine and arginine residues in the N-terminal regions of histones H3 and H4 * thought to be irreversible * view has been challenged by the discovery of enzymes that demethylate lysine and arginine residues, but it is still accepted that the effects of methylation are relatively long term * Methylation of lysine-9 forms a binding site for the HP1 protein, which induces chromatin packaging and silences gene expression * this is blocked by Methylation of lysine-4 * Methylation of lysine-4 promotes an open chromatin structure and is associated with active genes * Lysine-4 methylation may work hand-in-hand with histone acetylation to activate regions of chromatin

Question 34

Phosphorylatio

Answer 34

* of serine residues in the N-terminal regions of H2A, H2B, H3, and H4 * phosphorylation of histone H3 and of the linker histone has been associated with formation of metaphase chromosomes

Question 35

Ubiquitination

Answer 35

* of lysine residues at the C termini of H2A and H2B * addition of the small, common (“ubiquitous”) protein called ubiquitin or another one called SUMO * ubiquitination of histone H2B is part of the general role that ubiquitin plays in control of the cell cycle

Question 36

Altogether, _____ sites in the N- and C-terminal regions of the four core histones are known to be subject to covalent modification

Answer 36

29

Question 37

histone code

Answer 37

* pattern of chemical modifications specifies which regions of the genome are expressed at a particular time and dictates other aspects of genome biology * such as the repair of damaged sites and coordination of genome replication with the cell cycle * unproven

Question 38

Studies of human chromosomes 21 and 22 have shown that regions within these chromosomes where lysine-4 of histone H3 is trimethylated and lysine-9 and lysine-14 are acetylated correspond to the ______ start points for active genes, and that dimethylated \_\_\_\_\_\_-\_\_\_ is also sometimes found in these regions

Answer 38

* transcription * lysine-4

Question 39

nucleosome remodeling

Answer 39

* chromatin modification that can influence genome expression * modification or repositioning of nucleosomes within a short region of the genome, so that DNA-binding proteins can gain access to their attachment sites * not essential requirement for transcription of all genes * some gene expression proteins can bind to surfaces of nucleosomes or linker DNA instead * in others it is a prerequisite for gene activation * hsp70 gene of Drosophila melanogaster * activated by GAGA protein in response to a heat shock * creates DNase I hypersensitive region upstream of hsp70, indicating nucleosomes in this area have been moved and a segment of naked DNA exposed * doesn't involve covalent alterations to histone molecules * induced by an energy-dependent process that weakens the contact between the nucleosome and the DNA

Question 40

nucleosome remodeling 3 types

Answer 40

* Remodeling: * involves a change in the structure of the nucleosome, but no change in its position * nature of the structural change is not known * when induced in vitro the outcome is a doubling in size of the nucleosome and an increased DNase sensitivity of the attached DNA. * G Sliding, or cis-displacement: physically moves the nucleosome along the DNA. * G Transfer, or trans-displacement: results in the nucleosome being transferred to a second DNA molecule, or to a nonadjacent part of the same molecule

Question 41

DNA Methylation

Answer 41

* chemical changes to the DNA itself * associated with the semipermanent silencing of regions of the genome, possibly entire chromosomes * often the modified state is inherited by the progeny arising from cell division

Question 42

cytosine bases in chromosomal DNA molecules are sometimes changed to 5-methylcytosine by the addition of methyl groups by enzymes called ____ \_\_\_\_\_\_. Cytosine methylation is relatively rare in lower eukaryotes but in vertebrates up to _____ of the total number of cytosine bases in a genome are methylated, and in plants the figure can be as high as \_\_\_\_\_. The methylation pattern is not random, instead being limited to the cytosine in some copies of the sequences ______ and, in plants, \_\_\_\_\_\_.

Answer 42

* DNA methyltransferases * 10% * 30% * 5'–CG–3' * 5'–CNG–3'

Question 43

DNA methyltransferases, two types of methylation activity

Answer 43

* Both methylate DNA at CpG islands and result in repression of gene activity * maintenance methylation * following genome replication * adding of methyl groups to the newly synthesized strand of DNA at positions opposite methylated sites on the parent strand * ensures that the two daughter DNA molecules retain the methylation pattern of the parent molecule * pattern can be inherited after cell division * Dnmt1 - enzyme responsible * de novo methylation * adds methyl groups at totally new positions and so changes the pattern of methylation in a localized region of the genome * Dnmt3a and Dnmt3b - enzyme responsible

Question 44

Genomic imprinting

Answer 44

* uncommon but important feature of mammals * occurs in some insects and plants * only one of a pair of genes, present on homologous chromosomes in a diploid nucleus, is expressed, the second being silenced by methylation * always the same member of a pair of genes that is imprinted and hence inactive * for some genes this is the version inherited from the mother and others the father * 60+ genes in humans/mice have display imprinting * includes both protein-coding and functional RNA genes * Imprinted genes are distributed around the genome but tend to occur in clusters * controlled by imprint control elements * DNA sequences that are found within a few kilobases of clusters of imprinted genes * mediate the methylation of the imprinted regions

Question 45

X Inactivation

Answer 45

* imprinting that leads to almost total inactivation of one of the X chromosomes * If both of X chromosomes were active then proteins coded by genes on the X chromosome might be synthesized at twice the rate in females compared with males * To avoid this one of the female X chromosomes is silenced and is seen in the nucleus as a condensed structure called the Barr body * some 20% escape the process and remain functional. * Silencing occurs early in embryo controlled by the X inactivation center (Xic) present in each X chromsome * initiates the formation of heterochromatin * until the entire chromosome is affected, with the exception of a few short segments containing those genes that remain active * mechanism by which the X chromosomes in the nucleus are counted and the appropriate number inactivated * does not simply count the X chromosomes; it also counts the autosomes and compares the two numbers. * two genes within the X inactivation center, called Tsix and Xite, control the process