203342 1 chromosomes schmeier 2015

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DNA, CHROMATIN & CHROMOSOMES Sebastian Schmeier

Learning objectives

DNA, building blocks and DNA structure (recap) Composition of chromatin Histones and DNA packaging Nucleosome structure Nucleosome sliding

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Basic molecular genetic processes 3

Molecular Cell Biology, Lodish, 7th edition

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DNA

Figure 3.7. The structure of DNA 2000 by Geoffrey M. Cooper The Cell: A molecular approach.

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Genetic information

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Genetic information

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http://openclipart.org/detail/95191/genetic-code-by-j_alves

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DNA-Helices 7

B-DNA is the predominant configuration found in cells

Whats wrong with these pictures? 8

The actual picture 9


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Levels of chromatin packaging 10

Why is eukaryotic DNA packaged? 11

Why is eukaryotic DNA packaged? The length of DNA in the nucleus is far greater than the

size of the compartment in which it is contained. To fit into this compartment the DNA has to be condensed

in some manner.

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Why is eukaryotic DNA packaged? Each of us has enough DNA to reach from here to the sun

and back, more than 300 times. Each cell contains ~2.5 meter of DNA Each human body contains around estimated 100 trillion cells The distance to the sun is ~150 million km

shortest human chromosome has 48 million bp of DNA about ~10 times the genome size of E. coli (4.6 million bp)

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Levels of chromatin packaging

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Nucleosome ~146-148 bp of DNA ~1.65 turns in left handed supercoil (12 helical turns) 10.2 base pair periodicity (10.5 in

solution). Wrapping distorts the DNA Slightly (two 80 bp superhelical loops)

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Molecular Biology of the Cell, Bruce Alberts, 5th Editition

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Histones 16


Structure of the histone fold H2A and H2B form a dimer

Histones 17

Histone octamer assembly 18


2x

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Is nucleosome positioning random? 19


Where are the n-termini of the core histones?

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Question 1: What is the function of histone N-termini ? Question 2: Are all N-termini functionally equivalent ?

Luger, Mader, Richmond, Sargent & Richmond Nature 389, 251-260 (1997)

The chromatosome 21

Figure 4.9. Structure of a chromatosome (A) The nucleosome core particle consists of 146 base pairs of DNA wrapped 1.65 turns around a histone octamer consisting of two molecules each of H2A, H2B, H3, and H4. A chromatosome contains two full turns of DNA (166 base pairs) locked in place by one molecule of H1. (B) Model of the nucleosome core particle. The DNA backbones are shown in brown and turquoise. The histones are shown in blue (H3), green (H4), yellow (H2A), and red (H2B). (B, from K. Luger et al., 1997. Nature 389: 251.)

Core histone octamer + 1 Linker Histone + 2 full turns of DNA (168 bp)

The Cell: A molecular approach. Geoffrey M. Cooper

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Nucleosome and DNA structure is not fixed but dynamic

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Dynamic nucleosomes 23


DNA in an isolated nucleosome is surprisingly dynamic

rapidly uncoiling and then rewrapping around its nucleosome core

Unwrapping occurs at a rate of 4 times per second

Most of the DNA is accessible in this manner

Dynamic nucleosomes (2) 24


A large Varity of chromatin remodeling complexes exist in eukaryotic cells

Remodeling complex catalysis nucleosome sliding through repeated cycles of ATP hydrolysis

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How does the packaging of DNA influences genes transcription?

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How does the packaging of DNA influences genes transcription?

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Lenhard B et al., Metazoan promoters: emerging characteristics and insights into transcriptional regulation, Nature Reviews Genetics 2012

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HIGH level of histone H1 No gene transcription

Reduced level of histone H1 Gene transcription possible

Chromatin fiber

+ charged N termini (bind DNA on neigboring nucleosomes)

highly acetylated core histones

(especially H3 and H4)

30 nm chromatin fiber

11 nm (beads)


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10nm chromatin fiber 30

Individual nucleosomes (beads on a string) connected by a linker DNA

No H1 histones DNA accessible for transcription of genes

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Zig-zag ribbon stacked on top of each other like coins that forms a helix

H1 histone to stabilize the structure DNA not accessible for gene

transcription


Zig-zag ribbon stacked on top of each other like coins that forms a helix

H1 histone to stabilize the structure DNA not accessible for gene

transcription



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Protein scaffold 34

Figure 9-34. Electron micrograph of a histone-depleted metaphase chromosome prepared from HeLa cells by treatment with a mild detergent. A nonhistone protein scaffolding (dark structure) is visible from which long loops of DNA extend. [From J. R. Paulson and U. K. Laemmli, 1977, Cell 12:817. Copyright 1977 M.I.T.]

50,000Xs 150,000Xs

Chromatin loops In situ hybridization of

interphase cells was carried out with several different probes specific for sequences separated by known distances in linear, cloned DNA.

Measurements of the distances between different hybridized probes, distinguished by color, showed that some sequences (e.g., A and B), separated from one another by millions of base pairs, are located near each other.

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Chromatin loops

Protein scaffold: Non-histone proteins that organize long chromatin loops

scaffold-associated regions (SARs) or matrix-attachment regions (MARs)

S/MARs have been mapped by digesting histone-depleted chromosomes with restriction enzymes and then recovering the fragments that remain associated with the digested histone-depleted preparation

The distances between probes are consistent with chromatin loops ranging in size from 1 million to 4 million base pairs in mammalian interphase cells.

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Chromatin loops

S/MARs are found between transcription units Genes are located primarily within the chromatin loops Some S/MARs may function as insulators, that is, DNA

sequences of tens to hundreds of base pairs that separate transcription units (gene sets) from each other.

Proteins regulating transcription of one gene cannot influence the transcription of a neighboring gene(s) that is separated from it by an insulator.

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Is the Scaffold/matrix attachment region dynamic?

Heng et al. Single and multiple copies of the S/MAR-containing constructs

were introduced into various host genomes of transgenic mice and transfected cell lines.

By utilizing FISH to visualize directly the localization of S/MARs on the nuclear matrix or chromatin loop

These in vivo integration events provided a system to study the association and integration patterns of each introduced S/MAR

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Heng et al. Chromatin loops are selectively anchored using scaffold/matrix-attachment regions. Journal of Cell Science. 117, 999-1008, 2004

Fluorescence in situ hybridization Technique developed by biomedical researchers in the early 1980s Used to detect and localize the presence or absence of specific DNA

sequences on chromosomes. FISH uses fluorescent probes that bind to only those parts of the

chromosome with which they show a high degree of sequence complementarity

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Fluorescence microscopy can be used to find out where the fluorescent probe is bound to the chromosomes

FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification

https://en.wikipedia.org/wiki/File:FISH_%28technique%29.gif

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1005Chromatin loops selectively anchored by S/MAR

typically encountered in the genome. Thus, it could be arguedthat the potential binding sites are saturated when the S/MARsare arranged in tandem. These alternatives were resolved bydetermining whether the endogenous anchors were selectivelyutilized and whether loop regions move along the nuclearmatrix.

We examined a large contiguous 300 kb genomic regionfrom human chromosome 8. It was reasonable to expect thatseveral endogenous S/MARs would be contained within theregion because, on average, one S/MAR is present at every60-90 kb of genomic DNA (Boulikas, 1995). If the loops arefixed, then loops from different nuclei should display thesame configuration when the same sequence on each loop waspainted. By contrast, if the loop is a dynamic structure, withthe ability to use alternative attachment sites, then one shouldobserve different color configurations for each nucleus. Twoadjacent BACs from human chromosome 8 that spanned thisregion were randomly selected and used as probes. Two-colorFISH analysis was used to visualize the configurations of twoprobes on the chromatin loops. Two probes were biotin anddigoxigenin labeled respectively and detected by green(FITC) and red (rhodamin) color. If the loop was fixed on thenuclear matrix, then the same color configuration should bepreserved. If the color configuration changes, then this isindicative and consistent with dynamic loop movement. Asshown in Fig. 6, the color configuration of these two BACprobes varied among nuclei. The majority of signals (53%)are displayed as V-shaped configuration FISH signals (Fig.6A), where both green and red signals form the two portionsof the loop. Two other configurations can also be detected(Fig. 6B,C). In one case, the green signal is locatedproximally whereas the red signal is distal. In the other case,the red signal is located proximally and the green signal isdistal. These cases respectively represent 20 and 27% of thetotal of analyzed FISH images. This suggests that theendogenous chromatin loops of this genomic locus are notstationary with respect to the nuclear matrix. By contrast, ithas been shown that some sequences (e.g. centromeric andtelomeric sequences) and sperm MAR-containing sequencesare tightly anchored on their nuclear matrix (Schmid et al.,2001; Ratsch et al., 2002; Sumer et al., 2003). In addition, intransgenic animal models, most (i.e. over 97%) of theinsertions are detected on the nuclear matrix. This isconsistent with a fixed-anchor characteristic. In accord withthe above, it is reasonable to postulate that some S/MAR

anchors are fixed, representing the structural anchors,whereas others are transmutable.

DiscussionS/MARs are necessary but not sufficient to anchor aloopThe transgenic and transfection studies reported above areconsistent with the view that S/MARs are necessary for loopformation for both meiotic and mitotic chromatin domains, butare selectively used. The correlation of the abnormal size ofchromatin loops in the absence of S/MARs has been previouslydocumented in meiotic chromosomes (Heng et al., 1994). Onecould argue that the observation that not every inserted S/MARcopy serves as an anchor simply reflects the out-titration ofthe 100,000 endogenous binding sites. However, this is veryunlikely considering that single-copy integrants that wereexamined by FISH revealed that 10% of the inserted S/MARswere not associated with the nuclear matrix but were associatedwith the loop portion. It is clear that a cell has to be tolerantto at least a twofold increase in the number of attachmentsduring the natural course of the cell cycle.

We have previously shown that loop size is constrainedaccording to the chromosomal region (Heng et al., 1996).When the same DNA fragments were integrated into differentregions of the chromosome, the loop size was significantlydifferent. Smaller loops were observed towards the telomere,but were observed as larger loops when integrated away fromthe telomeric position. This indicates that not every anchor wasused in the formation of the larger loops. These preliminaryobservations further showed that, for a given genomicfragment, meiotic loops are longer than interphase mitoticloops, yet loops from metaphase chromosomes were smallerthan interphase loops (H.H.Q.H. et al., unpublished). It isapparent that these changes in size require a change of anchors.This loop movement indicates that a switching or changingoccurs among the S/MARs, with some S/MARs being selectedfor attachment to the nuclear matrix whereas others are not. Itdoes not simply reflect the limitation in the number of availableS/MAR-binding sites and must be related to the structural andfunctional requirements of the cell. It is thus reasonable toconclude that S/MARs are necessary to form chromatin loopsusing S/MARs as anchors but are not sufficient for loopformation; that is, their formation is contingent upon bothcellular requirements and the presence of the S/MARs in a

Fig. 6. Two-color FISH showing the loopconfiguration changes of a 300 kb genomicregion. (A) The V-shaped configuration ofboth red and green color probes wereanchored on the nuclear matrix. (B,C) Thelinear-shaped configuration with only oneprobe (red or green) anchored on thenuclear matrix whereas the adjacent probewas not anchored on the matrix. Theexplanation for the configuration changes isthat the anchor was not fixed.


FISH (Fluorescence in situ hybridization) revealed that 10% of the inserted S/MARs were not associated with the nuclear matrix but were associated with the loop portion.

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1005Chromatin loops selectively anchored by S/MAR

typically encountered in the genome. Thus, it could be arguedthat the potential binding sites are saturated when the S/MARsare arranged in tandem. These alternatives were resolved bydetermining whether the endogenous anchors were selectivelyutilized and whether loop regions move along the nuclearmatrix.

We examined a large contiguous 300 kb genomic regionfrom human chromosome 8. It was reasonable to expect thatseveral endogenous S/MARs would be contained within theregion because, on average, one S/MAR is present at every60-90 kb of genomic DNA (Boulikas, 1995). If the loops arefixed, then loops from different nuclei should display thesame configuration when the same sequence on each loop waspainted. By contrast, if the loop is a dynamic structure, withthe ability to use alternative attachment sites, then one shouldobserve different color configurations for each nucleus. Twoadjacent BACs from human chromosome 8 that spanned thisregion were randomly selected and used as probes. Two-colorFISH analysis was used to visualize the configurations of twoprobes on the chromatin loops. Two probes were biotin anddigoxigenin labeled respectively and detected by green(FITC) and red (rhodamin) color. If the loop was fixed on thenuclear matrix, then the same color configuration should bepreserved. If the color configuration changes, then this isindicative and consistent with dynamic loop movement. Asshown in Fig. 6, the color configuration of these two BACprobes varied among nuclei. The majority of signals (53%)are displayed as V-shaped configuration FISH signals (Fig.6A), where both green and red signals form the two portionsof the loop. Two other configurations can also be detected(Fig. 6B,C). In one case, the green signal is locatedproximally whereas the red signal is distal. In the other case,the red signal is located proximally and the green signal isdistal. These cases respectively represent 20 and 27% of thetotal of analyzed FISH images. This suggests that theendogenous chromatin loops of this genomic locus are notstationary with respect to the nuclear matrix. By contrast, ithas been shown that some sequences (e.g. centromeric andtelomeric sequences) and sperm MAR-containing sequencesare tightly anchored on their nuclear matrix (Schmid et al.,2001; Ratsch et al., 2002; Sumer et al., 2003). In addition, intransgenic animal models, most (i.e. over 97%) of theinsertions are detected on the nuclear matrix. This isconsistent with a fixed-anchor characteristic. In accord withthe above, it is reasonable to postulate that some S/MAR

anchors are fixed, representing the structural anchors,whereas others are transmutable.

DiscussionS/MARs are necessary but not sufficient to anchor aloopThe transgenic and transfection studies reported above areconsistent with the view that S/MARs are necessary for loopformation for both meiotic and mitotic chromatin domains, butare selectively used. The correlation of the abnormal size ofchromatin loops in the absence of S/MARs has been previouslydocumented in meiotic chromosomes (Heng et al., 1994). Onecould argue that the observation that not every inserted S/MARcopy serves as an anchor simply reflects the out-titration ofthe 100,000 endogenous binding sites. However, this is veryunlikely considering that single-copy integrants that wereexamined by FISH revealed that 10% of the inserted S/MARswere not associated with the nuclear matrix but were associatedwith the loop portion. It is clear that a cell has to be tolerantto at least a twofold increase in the number of attachmentsduring the natural course of the cell cycle.

We have previously shown that loop size is constrainedaccording to the chromosomal region (Heng et al., 1996).When the same DNA fragments were integrated into differentregions of the chromosome, the loop size was significantlydifferent. Smaller loops were observed towards the telomere,but were observed as larger loops when integrated away fromthe telomeric position. This indicates that not every anchor wasused in the formation of the larger loops. These preliminaryobservations further showed that, for a given genomicfragment, meiotic loops are longer than interphase mitoticloops, yet loops from metaphase chromosomes were smallerthan interphase loops (H.H.Q.H. et al., unpublished). It isapparent that these changes in size require a change of anchors.This loop movement indicates that a switching or changingoccurs among the S/MARs, with some S/MARs being selectedfor attachment to the nuclear matrix whereas others are not. Itdoes not simply reflect the limitation in the number of availableS/MAR-binding sites and must be related to the structural andfunctional requirements of the cell. It is thus reasonable toconclude that S/MARs are necessary to form chromatin loopsusing S/MARs as anchors but are not sufficient for loopformation; that is, their formation is contingent upon bothcellular requirements and the presence of the S/MARs in a

Fig. 6. Two-color FISH showing the loopconfiguration changes of a 300 kb genomicregion. (A) The V-shaped configuration ofboth red and green color probes wereanchored on the nuclear matrix. (B,C) Thelinear-shaped configuration with only oneprobe (red or green) anchored on thenuclear matrix whereas the adjacent probewas not anchored on the matrix. Theexplanation for the configuration changes isthat the anchor was not fixed.


S/MARs are necessary for loop formation for both meiotic and mitotic chromatin domains, but are selectively used

Loop movement indicates that a switching or changing occurs among the S/MARs, with some S/MARs being selected for attachment to the nuclear matrix whereas others are not.

S/MAR anchors are necessary but not sufficient for chromatin loops to form

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Current model

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https://en.wikipedia.org/wiki/File:SMARs_facultative.png

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Summary The nucleosome is an octamer containing Histones H2a,

H2b, H3 and H4 as an octamer (2 of each) 145-147 bp of DNA wraps around the octamer ~1.7 times Between each nucleosome is a length of DNA the

linker. Histone H1 associates with the linker and lies within the

chromatin fiber. The level of chromatin condensation is regulated Actively transcribed genes are characterised by a loose

chromatin structure and H1 depletion the DNA is available for transcription.

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Summary cont. The chromatin in transcriptionally inactive regions of DNA

within cells is thought to exist in a condensed, 30-nm fiber form and higher-order structures built from it

The chromatin in transcriptionally active regions of DNA within cells is thought to exist in an open, extended form.

Chromatin in its looped form is anchored to a protein scaffold through S/MARs

S/MARs are found between transcription units and function as gene insulators

S/MARs are now thought to be used selectively with the help of transcription factors

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References Molecular Cell Biology, Lodish, 7th Edition

Page: 117 118: Structure of Nucleic Acids Page: 256 258, 265 266: Structural Organization of Eukaryotic

Chromosomes Page 266 268: Morphology and Functional Elements of

Eukaryotic Chromosomes

Molecular Biology of the Cell, Alberts, 5th Edition Page: 215 216

http://www.dnaftb.org/dnaftb/29/concept/index.html Go through chapter 29 DNA is packaged in a chromosome.

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HIGHER-ORDER CHROMATIN STRUCTURES

Learning objectives

Chromosome structure Centromeres Kinetochores Telomeres and telomerases The formation and existence of different forms of chromatin

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Why do you think is the packaging of the DNA highest in the metaphase of Mitosis?

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https://en.wikipedia.org/wiki/Mitosis

Time-lapse video of mitosis in a Drosophila melanogaster embryo.

Why do you think is the packaging of the DNA highest in the metaphase of Mitosis? When eukaryotic cells divide, genomic DNA must be

equally partitioned into both daughter cells. To not confuse the chromosome division they get packed

as small as possible.

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Chromosome overview

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F I G U R E 6 - 3 9 : Ty p i c a l m e t a p h a s e chromosome. As seen in this scanning electron micrograph, the chromosome has replicated and comprises two chromatids, each containing one of two ident ical DNA molecules. The centromere, where chromatids are attached at a constriction, is required for their separation late in mitosis. Special telomere sequences at the ends function in preventing chromosome shortening. [Andrew Syred/Photo Researchers, Inc.]


https://www.ncbi.nlm.nih.gov/About/primer/genetics_cell.html

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Chromosomes

Any eukaryotic chromosome must contain three functional elements in order to replicate and segregate correctly: replication origins at which DNA polymerases and

other proteins initiate synthesis of DNA the centromere, the constricted region required for

proper segregation of daughter chromosomes the two ends, or telomeres.

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Chromosomes (2) The yeast transformation studies demonstrated the

functions of these three chromosomal elements and established their importance for chromosome function.

Replication of DNA begins from sites that are scattered throughout eukaryotic chromosomes.

The yeast genome contains many ~100-bp sequences, called autonomously replicating sequences (ARSs), that act as replication origins.

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Chromosomes (3) 54

Insertion of an ARS (replication origin) into a circular plasmid allows the plasmid to replicate

only about 5-20 percent of progeny cells contain the plasmid because mitotic segregation of the plasmids is faulty. Molecular Cell Biology, Lodish, 7th edition, Page 271

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Chromosomes (4) 55

plasmids that also carry a CEN sequence, derived from the centromeres of yeast chromosomes, segregate almost equally to both mother and daughter cells during mitosis

Molecular Cell Biology, Lodish, 7th edition, Page 271

Chromosomes (5) 56

If circular plasmids containing an ARS and CEN sequence are cut once with a restriction enzyme, the resulting linear plasmids do not produce LEU+ colonies unless they contain special telomeric (TEL) sequences ligated to their ends


The centromere 57

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The centromere (2) 58

Figure 4.16. The centromere of a metaphase chromosome The centromere is the region at which the two sister chromatids remain attached at metaphase. Specific proteins bind to centromeric DNA, forming the kinetochore, which is the site of spindle fiber attachment. 2000 by Geoffrey M. Cooper

links sister chromatids Physical role is to act as the site of

assembly of the kinetochore During mitosis, spindle fibers

attach to the centromere via the kinetochore.

The kinetochore Centromere based highly

complex multiprotein-structure responsible for chromosome segregation

Kinetochore microtubules directly connect to the chromosomes, at the kinetochores during cell division to pull sister chromatids apart

Signals to the cell cycle machinery when all chromosomes have adopted correct attachments to the spindle

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FIGURE 6-45 Kinetochore-microtubule interaction in S. cerevisiae: Diagram of the centromere-associated CBF3 complex and its associated Ndc80 complexes that associate with a ring of Dam1 proteins at the end of a spindle microtubule. The Ndc80 complexes initially make lateral interactions with the side of a spindle microtubule (top) and then associate with the Daml ring, making an end-on attachment (bottom) to the microtubule.


The kinetochore (2) bla

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Kinetochores and diseases Deregulation of kinetochore-microtubule attachment

dynamics in the cell cycle have been linked to diseases where chromosome mis-segregation plays a central role. e.g. Mosaic Variegated Aneuploidy (MVA) cells with too few or

too many chromosomes Potentially, in MVA, sister chromatids lose cohesion prematurely,

well before sister kinetochores are able to bi-orient and establish sufficiently stable attachments with microtubules.

MVA can be caused by mutations in Bub1B, that encodes BubR1 BubR1 phosphorylates important spindle assembly checkpoint

(SAC) proteins and is thought be necessary to maintain SAC signalling

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Curr Opin Cell Biol. 2012 February; 24(1): 6470.

Kinetochores and cancer The effect of individual gene mutations on kinetochore-

microtubule (kMT) attachment dynamics is largely an unexplored field

Many proteins were shown to be overexpressed in cancer with the putative consequence of increasing kMT stability.

It is unclear however if these genes are initial triggers of chromosome mis-segregation and chromosome instability

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Curr Opin Cell Biol. 2012 February; 24(1): 6470.

The telomeres 63

Protecting the terminus: t-loops and telomere end-binding proteins C.Wei and C. M. Price* CMLS, Cell. Mol. Life Sci. 60 (2003) 22832294

Chromosomes of eukaryotes are linear Each end is sealed by a specialized region, the telomere Two features characterize the telomere:

Telomerase activity to compensate for replication-related loss of nucleotides at the chromosome ends

telomeric DNA loop formation to stabilize the chromosome ends

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The telomeres (2) 812 bases at the end of the lagging

strand template cannot be synthesized by DNA polymerase because the primer it requires cannot be attached beyond the end of the template strand

812 nucleotides will be lost at the chromosome ends

Some organisms compensate for this loss by adding telomeric repeats to the ends of the chromosome during the replication cycle

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Organism Telomeric repeat sequence

Yeasts

Saccharomyces cerevisiae

G1 3T

Protozoans

Tetrahymena GGGGTT

Dictyostelium G1 8A Plant

Arabidopsis AGGGTTT

Mammal

Human AGGGTT 2000 by Geoffrey M. Cooper

The telomeres (3) Synthesis of the lagging

strand requires RNA primers for replication

The most 5 side of the strand gets lost because the primer position does not get synthesised

Loss of DNA at the 5 end of each strand with each round of replication

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.

(;} FOCUS ANIMATION: Telomere Replication

FIGURE 6-46 Standard DNA replication leads to loss of DNA at the 5' end of each strand of a linear DNA molecule. Replication of the right end of a linear DNA is shown; the same process occurs at the left end (shown by inverting the figure). As the replication fork approaches the end of the parental DNA molecule, the leading strand can be synthesized all the way to the end of the parental template strand without the loss of deoxyribonucleotides. However, since synthesis of thP lagging strand requires RNA primers, the right end of the lagging daughter DNA strand would remain as ribonucleotides which are removed and therefore cannot serve as the template for a replicative DNA polymerase. Alternative mechanisms must be utilized to prevent successive shortening of the lagging strand with each round of replication. [Adapted from the Nobel Assembly at the Karolinska lnst1tute.]

of proteins homologous to those that interact with S. cerevi-siae centromeres bind to these complex S. pombe centromeres and in turn bind the much longer S. pombe chromosomes to several microtubules of the mitotic spindle apparatus. In plants and animals, cemromeres are megabases in length and are composed of multiple repeats of simple-sequence DNA. In humans, centromeres contain 2- to 4-megabase arrays of a 171-bp simple-sequence DNA called alphoid DNA that is bound by nuclcosomes containing the CENP-A histone H3 variant, as well as other repeated simple-sequence DNA.

In higher eukaryotes, a complex protein structure called the kinetochore assembles at ccntromeres and associates with mul-tiple mitotic spindle fibers during mitosis (see Figure 18-39). Homologs of many of the proteins found in the yeasts occur in humans and other higher eukary-otes. For those yeast proteins where clear homologs arc not evident in higher cells based on amino acid sequence compari-sons (such as the Dam l complex), alternative complexes with similar properties have been proposed to function at kineto-chores that are bound to multiple spindle microrubules. The function of the centromere and kinetochore proteins that bind to it during the segregation of sister chromatids in mitosis and meiosis is described in Chapters 18 and 19.

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes Sequencing of telomcres from multiple organisms, including humans, has shown that most arc repetitive oligomers with a high G content in the strand with its 3' end at the end of the chromosome. The telomere repeat sequence in humans and other vertebrates is TTAGGG. These simple sequences are repeated at the very termini of chromosomes for a total of a few hundred base pairs in yeasts and protozoans, and a few thousand base pairs in vertebrates. The 3' end of the G-rich strand extends 12-16 nucleotidcs beyond the 5' end of the complementary C-rich strand. This region is bound by specific proteins that protect the ends of linear chromosomes from attack by exonucleases.

lagging strand DNA synthesis <

# .... / '------------- 3' RNA primer Parent strands

-'< .. Leading strand DNA synthesis

;:==:::-:::::;:;;;::::::-::::::;;;:::==:-:: 3' ...... tJ ...... _ ...... 5' Polymerase Primer /

....,.3' 5'

! 3' / -- \.:. ;; ...

ligation Gap fill-in / 3' I

! I 5'

Primer removal

===========;s:--:- 3' 5' Shortened end / I ==========::;t:-=3' I 5' I Gap not

filled

The need for a specialized region at the ends of eukaryotic chromosomes is apparent when we consider that all known DNA polymerases elongate DNA chains at the 3' end, and all require an RNA or DNA primer. As the replication fork ap-proaches the end of a linear chromosome, synthesis of the leading strand continues to the end of the DNA template strand, completing one daughter DNA double helix. How-ever, because the lagging-strand template is copied in a dis-continuous fashion, it cannot be replicated in its entirety (Figure 6-46). When the final RNA primer is removed, there is no upstream strand onto which DNA polymerase can build to fill the resulting gap. Without some special mechanism, the daughter DNA strand resulting from lagging-strand synthesis would be shortened at each cell division.

The problem of telomere shortening is solved by an en-zyme that adds telomeric (TEL) repeat sequences to the ends of each chromosome. The enzyme is a protein-RNA complex called telomere terminal transferase, or telomerase. Because the sequence of the telomcrase-associared RNA, as we will see, serves as the template for addition of deoxyribonucleo-tides to the ends of telomeres, the source of the enzyme and not the source of the telomeric DNA primer determines the sequence added. This was proven by transforming Tetrahy-mena with a mutated form of the gene encoding the tclomerase-

RNA. The resulting telomerase added a DNA sequence complementary to the mutated RNA sequence to the

6.7 Morphology and Functional Elements of Eukaryotic Chromosomes 273


(;} FOCUS ANIMATION: Telomere Replication

FIGURE 6-47 Mechanism of action of telomerase. The single-stranded 3' terminus of a telomere is extended by telomerase, counteracting the inability of the DNA replication mechanism to synthesize the extreme terminus of linear DNA. Telomerase elongates this single-stranded end by a reiterative reverse-transcription mecha-nism. The action of the telomerase from the protozoan Tetrahymena, which adds a T2G4 repeat unit, is depicted; other telomerases add slightly different sequences. The telomerase contains an RNA template (red) that base-pairs to the 3' end of the lagging-strand template. The telomerase catalytic site then adds deoxyribonucleotides TIG (blue) using the RNA molecule as a template (step Dl. The strands of the resulting DNA-RNA duplex are then thought to slip (translocate) relative to each other so that the TIG sequence at the 3' end of the replicating DNA base-pairs to the complementary RNA sequence in the telomerase RNA (step fl ). The 3' end of the replicating DNA is again extended by telomerase (step J)). Telomerases can add multiple repeats by repetition of steps fl and D . DNA polymerase a-primase can prime synthesis of new Okazaki fragments on this extended template strand. The net result prevents shortening of the lagging strand at each cycle of DNA replication [From c.w. Greider and E. H. Blackburn, 1989, Nature 337:331.]

ends of telomeric primers. Thus telomerase is a specialized form of a reverse transcriptase that carries its own internal RNA tem-plate to direct DNA synthesis. These experiments also earned the Nobel Prize in Physiology and Medicine for the discovery and characterization of the mechanism of telomerase.

Figure 6-4 7 depicts how telomerase, by reverse transcrip-tion of its associated RNA, elongates the 3' end of the single-stranded DNA at the end of the G-rich strand mentioned above. Cells frsm1 knockout mice that cannot produce the telomerase-associated RNA exhibit no telomerase acti\'ity, and their telomeres shorten successively with each cell gen-eration. Such mice can breed and reproduce normall y for three generations before the long telomere repeats become substantially eroded. Then, the absence of telomere DNA results in adverse effects, includ ing fusion of chromosome termini and chromosomal loss. By the fourth generation, the reproductive potential of these knockout mice declines, and they cannot produce offspring after the sixth generation.

The human genes expressing the telomerase protein and the telomerase-associated RNA are active in germ

cells and stem cells, but are turned off in most cells of adult tissues that replicate only a limited number of times, or will never replicate again (such cells are called postmitotic). How-ever, these genes are activated in most human cancer cells, where telomerase is required for th(' mu ltiple cell divisions necessary to form a tumor. This phenomenon has stimulated a search for inhibitors of human te lomerase as potential therapeutic agents for treating cancer.

While telomcrase prevents telomere shorten ing in most cukar}otcs, some organisms use alternative strategies. Drosophila

274 CHAPTER 6 Genes, Genomics, and Chromosomes

)

EI0"9'';"" 1 D )

T""''o"Uo" 1 fJ )

Elo"g'Uo" 1 II )

species maintain telomere lengths by the regulated insertion of non-L TR retrotransposons into telomeres. This is one of the few instances in which a mobi le element has a specific function in its host organism.

KEY CONCEPTS of Section 6.7 Morphology and Functional Elements of Eukaryotic Chromosomes During metaphase, eukaryotic chromosomes become suf-ficiently condensed that they can be visualized individually in the light microscope.

T he chromosomal karyotype is characteristic of each spe-cies. Closely related species can have dramatically different karyotypes, indicating that simila r genetic information can be organized on chromosomes in d ifferent ways. Banding analysis and chromosome painting are used to identify the different human metaphase chromosomes and to detect t ranslocations and deletions (see Figure 6-41 ). Analysis of chromosomal rearrangements and regions of conserved synteny between related species allows scientists

Mechanism of action of telomerase (enzyme)

Telomerase from the protozoan Tetrahymena, which adds a T2G4

Telomerase contains RNA template (red) that base-pairs to the 3' end of the lagging-strand template. The telomerase adds nucleotides TTG (blue) using the RNA molecule as a template

The strands of the resulting DNA-RNA duplex are then thought to translocate relative to each other so that the TTG sequence at the 3' end of the replicating DNA base-pairs to the complementary RNA sequence in the telomerase RNA

The 3' end of the replicating DNA is again extended by telomerase

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Telomeres and cancer The human genes expressing the telomerase protein and

the telomerase-associated RNA are active in germ cells and stem cells

Telomerases are turned off in most cells of adult tissues that replicate only a limited number of times, or will never replicate again

These genes (telomerases) are activated in most human cancer cells where telomerase is required for the multiple cell divisions necessary to form a tumor

This phenomenon has stimulated a search for inhibitors of human telomerase as potential therapeutic agents for treating cancer Cutting-edge research

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Summary Three chromosomal functional elements are needed in

order to replicate and segregate correctly: replication origins, centromeres, telomeres

Spindle fibers attach at the kinetochore at the centromere during cell division to pull sister chromatids apart

Telomerases compensate for the loss of DNA by adding telomeric repeats to the ends of the chromosomes during the replication cycle

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CHROMATIN REMODELING

Learning objectives Different forms of histone modifications Understand how enzymes regulate gene expression by modulating and responding to the epigenetic code

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HIGH level of histone H1 No gene transcription

Reduced level of histone H1 Gene transcription possible

Chromatin fiber

+ charged N termini (bind DNA on neigboring nucleosomes)

highly acetylated core histones

(especially H3 and H4)

30 nm chromatin fiber

11 nm (beads)

Heterochromatin versus euchromatin 74

FIGURE 6-33: Heterochromatin versus euchromatin. (a) In this electron micrograph of a bone marrow stem cell, the dark-staining areas in the nucleus (N) outside the nucleolus (n) are heterochromatin. The light-staining, whitish areas are euchromatin. Part (a) P. C. Cross and K. L. Mercer, 1993, Cell and Tissue Ul t rastructure, W. H. Freeman and Company, p. 165


Heterochromatin versus euchromatin (2)

Heterochromatin does not fully de-condense following mitosis and remains in a compacted state during interphase

Heterochromatin includes centromeres and telomeres of chromosomes, as well as transcriptionally inactive genes.

Euchromatin contains areas which are in a less compacted state during interphase and stain lightly with DNA dyes.

Most transcribed regions of DNA are found in euchromatin.

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Heterochromatin versus euchromatin (3)

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DNase digests DNA The Southern blot shows the

transcriptionally active DNA from 14-day globin-synthesizing cells was sensitive to DNase digestion

The inactive DNA from MSB cells was resistant to digestion.

Globin gene is shielded from DNase digestion

Heterochromatin is less susceptible to digestion then euchromatin

What controls the condensation of the chromatin?

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Histone octamer assembly 78


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Where are the n-termini of the core histones?

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Question 1: What is the function of histone N-termini ? Question 2: Are all N-termini functionally equivalent ?

Luger, Mader, Richmond, Sargent & Richmond Nature 389, 251-260 (1997)


Histone modifications

The histone proteins of the nucleosome core contain a flexible N-terminus of 19-39 residues extending from the globular structure of the nucleosome

Modifications of histone tails control chromatin condensation and function

Histone tails are subject to multiple post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination

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Histone modifications (2) 81


Summary of post-translational modif icat ions observed in human histones. Histone-tail sequences are shown in the one-letter amino acid code. The main portion of each histone is depicted as an oval. These modifications do not all occur simultaneously on a single histone molecule. Rather, specific combinations of a few t h e s e m o d i f i c a t i o n s a r e observed on any one histone

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Histone modifications (3)

Histone core

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Histone modifications (4) One particular histone never has all of these modifications simultaneously

BUT the histones in a single nucleosome usually contain several of these modifications simultaneously

Histone modifications control the condensation, or compaction, of chromatin and its ability to be transcribed, replicated, and repaired

All modifications are reversible Each side-chain modification is created by a specific enzyme, each removal by a different enzyme

83

Histone modifications (5) Histone-tail lysines (K) undergo reversible acetylation

and deacetylation Creation: e.g. Histone acetyl transferases (HATs) Removal: e.g. Histone deacetylase complexes (HDACs)

Histone-tail lysines (K) undergo reversible methylation and demethylation

Creation: e.g. Histone methyl transferases Removal: e.g. Histone demethylase

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Human histone acetyltransferases

domains, including a bromodomain and three cysteinehistidine rich domains (TAZ, PHD and ZZ) that are be-lieved to mediate proteinprotein interaction.

The MYST family of HAT proteins are grouped to-gether on the basis of their close sequence similarities,including a particular highly conserved 370 residueMYST domain, which uses an acetyl-cysteine intermedi-ate in the acetylation reaction, so the catalytic mecha-nism involved is different from that shared by theother families of HATs (reviewed in [9]). The membersof the MYST family are involved in a wide range of reg-ulatory functions including transcriptional activation,transcriptional silencing, dosage compensation and cellcycle progression (Table 1). Besides the MYST domain,many members contain a cysteine-rich, zinc-bindingdomain within the HAT regions and N-terminalchromodomains.

As with bromodomains, chromodomains have beenfound in many other chromatin regulators, includingremodelling factors and histone methyltransferases. Re-cently, it has been shown that the chromodomain ofthe heterochromatin protein 1 (HP1) and the yeastCHD1 protein (Chromo-ATPase/Helicase-DNA bind-ing domain 1) can respectively recognise methylatedK9 and K4 residues within the histone H3 tail [1012].Hence, it is not unreasonable to speculate that some

chromodomain containing HATs might be recruited tochromatin by histone methylation.

Since the addition of an acetyl group to a lysine res-idue creates a new surface for protein association, andmany transcription factors and chromatin regulatorsbind directly or indirectly acetylated histones, the main-tenance of a specific histone acetylation pattern is crucialto cell proliferation. Consequently, it is not surprisingthat mutations or chromosomal translocations involvingHAT genes result in development of malignancies (Ta-ble 4).

Several human histone acetyltransferases have beenfound to be involved in translocations where the resul-tant protein displays a !gain-of-function" by deregulat-ing HAT activity on histones or targeting lysineacetylation to new substrates. The p300 and CREBbinding protein (CBP) genes are located on chromo-somes 16p13 and 22q13, respectively, and are foundrearranged in chromosomal translocations associatedwith leukaemia or treatment-related myelodysplasticsyndrome. CBP fusion partners are the histone acetyl-transferases Monocytic Leukaemia Zinc finger protein(MOZ) and MOZ related factor (MORF) [13]; andmixed lineage leukaemia (MLL), which encodes a K4H3 methyltransferase (reviewed in [14]). The MLL gene,located at 11q23, is fused to the p300 and CBP genes

Table 1Human histone acetyl transferases (HATs)

Family Substrate Complex Role

GNATPCAF H3/H4, TAT, E1A, p53, PCAF, AR PCAF T. CoactivatorGCN5L H3/H4, TAFs STAGA, TFTC T. CoactivatorELP3 H3/H4 Elongator T. ElongationP300/CBPP300 H2A/H2B/H3/H4, p53, EIA, TAT, AR T. CoactivatorCBP H2A/H2B/H3/H4, TFs, EIA T. CoactivatorMYSTTip60 H3/H4/H2A, AR TIP60 T. ActivationMOF (MYST1) H3/H4/H2A MAF2 T. ActivationMOZ (MYST3) H3/H4 T. ActivationMORF (MYST4) H3/H4 T. ActivationHBO1 (MYST2) H3/H4 T. Corepressor; DNA replication

Transcription factorsATF2 H4/H2B T. ActivatorTAF1 (TAFII250) H3/H4 TFIIB T. FactorTFIIIC90 (GTF3C4) H3 T. Initiation

Nuclear hormone-relatedSRC-l (NCOAl) H3/H4 NCOA T. CoactivatorACTR H3/H4 PCAF/P300 T. CoactivatorOthersCIITA (HMC2TA) 114 T. CoactivatorCDYL H4 Protamine ! histoneHAT1 H4/H2A Histone deposition

GNAT: GCN5-related acetyltransferase; PCAF: EP300/CREBP-associated factor; TAT: tyrosine aminotransferase; AR: androgen receptor; TAFs:TATA box-associated factors; ACTR: activin receptor; CBP: CREB-binding protein; p300: e1a-binding protein p300; GCN5L: general control ofamino-acid synthesis 5-like 2; GTF3C4: general transcription factor 3c, polypeptide 4; HBO1: histone acetyltransferase binding to ORC; MYST:MOZ, YBF2/SAS3, SAS2, TIP60 protein family; MOZ: monocytic leukemia zinc finger protein; MORF: MOZ-related factor; NCOA1/2: nuclearreceptor coactivator 1 and 2; SRC, steroid receptor coactivators; HATl: histone aceayl transferase 1.

H. Santos-Rosa, C. Caldas / European Journal of Cancer 41 (2005) 23812402 2385

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Chromatin modifier enzymes, the histone code and cancer. Santos-Rosa et al. 2005:41(16) European Journal of Cancer

Histone modifications (6) Acetylation of lysines on the N-terminal tails tends to loosen

chromatin structure, removing its positive charge thereby reducing the affinity of the tails for adjacent nucleosomes E.g. When H4 lysine 16 is acetylated, the chromatin tends to form the

less condensed "beads-on-a-string conformation conducive for transcription and replication

Methylation of lysine amino groups prevents acetylation thus maintaining their positive charge

Phosphorylation of serine and threonine side chains can introduce two negative charges

However, most important is the ability of histone modifications to attract specific proteins to a specific stretch of chromatin

86

Histone modification cross-talk 87


bla

modifications in the establishment and preservation ofcorrect gene expression patterns and how deregulationand mis-targeting of these histone modifications con-tributes to the development of malignancies.

2. Histone post-translational modifications and thehistone code

A variety of post-translational modifications occuron the amino terminal tail, as well as on residues locatedat exposed sites within the globular domain of thehistones. These post-translational modifications includephosphorylation, acetylation, ubiquitination, methyla-tion and SUMOylation (Fig. 1). Such modificationson histones can create or stabilise binding sites for reg-ulatory proteins, like transcription factors, proteins in-volved in chromatin condensation or DNA repair.Histone modifications may also have the oppositeeffect, disrupting or occluding chromatin-binding sites.Accordingly, there are modifications that co-exist andwork sequentially in a cooperative manner but areincompatible with others in the same nucleosome. Thatis the case for methylation of Lysine 4 H3 (K4 H3), acet-ylation of Lysine 14 H3 (K14 H3) and phosphorylationof Serine 10 H3 (S10 H3), all involved in transcription

activation and incompatible with the generally inhibi-tory H3 Lysine 9 methylation (Fig. 2, H3).

Furthermore, the role of a particular modification intranscriptional signalling may also be influenced by thedegree and stability of the modification. Lysine residuesmay be modified with one, two, or three methyl groups,and the status of histone methylation determines iftranscription of certain genes is activated or repressed[4, 5; reviewed in 6].

Distinct histone modifications, on one or more tails,act sequentially or in combination to form a histonecode that is read by proteins containing specific inter-acting domains: bromodomain and chromodomain.These proteins are the effectors that initiate downstreambiological responses such as chromosome condensation,DNA repair or transcription activation/repression(reviewed in [7]). Examples of recruitment of chromo-and bromo-domain containing proteins, leading to dif-ferent transcriptional read outs are shown in Fig. 3.Thus, although the basic composition of the nucleosomemay be the same over long stretches of chromatin, thespecific palette of modifications on nucleosomes createslocal structural and functional diversity delimiting chro-matin subdomains.

The molecular basis for how the epigenetic informa-tion carried in histone tail modifications is memorised

A2HK

11 9

S GRG K GGQ K VRGVPFQLGARSSRTKAKARA

AU

P A

3HA-N RTK RATQ KS GGT K PA RK LQ ATK AA KR S VGGTAPA K 4 2 9 01 41 71 81 23 62 72 82 63

PM PMM MM M MA

A A A

K21 0

B2HAPEP K AS KPAP KGSK TVAK K KQA K KRKKGD5 21 41 51 02 42

A A A A A

U-N

P

M M

K97

|

4HSGRGK GG K GLG K AGG K RHR K TIGQINDRLV1 3 5 8 21 61 02

P MM A A A A

Histone modifications cross-talk

Fig. 2. Interplay between different post-translational modifications. Compatible modifications (those which facilitate other modifications to occurand/or can co-exist) are represented by green arrows. Incompatible modifications (those which negatively affect other modification and/or can notco-exist) are shown in red.

H. Santos-Rosa, C. Caldas / European Journal of Cancer 41 (2005) 23812402 2383

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The histone code

Specific combinations of the histone modifications in different chromatin regions have been suggested to constitute a histone code

The number of possible distinct markings on individual nucleosomes is enormous

This code is thought to influence chromatin function by creating or removing binding sites of chromatin-associated proteins

88

The histone code (2) 89


(A)A space-filling model of an ING PHD domain bound to a histone tail (green with the trimethyl group highlighted in yellow)

(B)A ribbon model showing how the N-terminal six amino acids in the H3 tail are recognize

Protein module recognises methylated lysines



Several protein modules act together in form of a code-reader complex

The particular histone modification combination together with the code-reader complex attracts additional protein complexes that execute a specific biological function

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Example of some specific meanings of the histone code

(A) The modifications on the histone H3 N-terminal tail are shown

(B) The H3 tail can be marked by different combinations of modifications

Each conveys a specific meaning to the stretch of chromatin where this combination occurs.

Only a few of the meanings are known including the four examples shown

Model for the formation of heterochromatin

92

Higher eukaryotes express a number of proteins containing a so called chromodomain that binds to histone tails when they are methylated at specific lysines, e.g. heterochromatin protein (HP1)

HP1 binds to histone H3 N-terminal tails tri-methylated at lysine 9

HP1 has a second domain (chromoshadow) which binds other chromoshadow domains ! results in a condensed chromatin structure


Model for the formation of heterochromatin (2)

93


Heterochromatin condensation can spread along a chromosome because HP1 binds a histone methyltransferase (HMT) that methylates lysine 9 of histone H3.

This creates a binding site for HP1 on the neighboring nucleosome. The spreading process continues until a "boundary element" is

encountered. Boundary elements so far characterized are generally regions in

chromatin where several non-histone proteins bind to DNA, possibly blocking histone methylation on the other side of the boundary

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The image cannot be displayed. Your computer may not have enough memory to open the image, or the image may have been corrupted. Restart your computer, and then open the file again. If the red x still appears, you may have to delete the image and then insert it again.

Establishment of transcriptionally active chromatin (1) Establishment of transcriptionally active chromatin by lysine 4 H3 methylation 1. Set1p methylates lysine 4 H3. 2. Methylated lysine 4 recruits the

chromatin remodelling factor Chd1p in physical association with histone acetyltransferases.

3. Acetylation of lysine residues prevents repressive modifications to occur and recruits transcription activators.

94


Establishment of transcriptionally active chromatin (2) Establishment of transcriptionally active chromatin by lysine acetylation: 1. GCN5 acetylates several residues

within histones H3 and H4 2. Acetylated lysines recruit the

chromatin remodelling complex SWI/SNF.

3. SWI/SNF, via its ATPase activity, displaces and twists nucleosomes exposing DNA areas for interaction with the transcription machinery.

95


is unknown. Interestingly, biochemical data have sug-gested histones H3 and H4 are deposited into nascentnucleosomes as heterodimers [8]. This opens the possi-bility that the existing epigenetically coded H3/H4 di-mers are divided on the two daughter strands, therebyforming the basis for an epigenetic memory imprint.

3. Histone acetylation and cancer

Acetylation of the e-amino group of lysine residuesoccurs on the four histones (Fig. 1). Broadly, acetylationof histones is linked to transcriptional activation. There-fore it is not surprising that many of the enzymesresponsible for acetylation of histones at different resi-dues where first known as transcriptional co-activatorsand later as enzymes. Most histone acetyltransferasestake part in huge multiprotein complexes involved in lo-cus targeting, thus providing chromosomal domainspecificity in addition to the substrate specificity dis-played by each individual acetyltransferase.

Based on sequence similarity histone acetyl transfer-ases (HATs) can be organised into families, which seemto display different mechanisms of histone substratebinding and catalysis (Table 1).

The Gcn5/PCAF family of HAT proteins (GNATs)function as co-activators for a subset of transcriptionalactivators. They contain a HAT domain of around160 residues and directly C-terminal to the HAT domaina conserved bromodomain, which has been shown torecognise and bind acetyl-lysine residues. The wide dis-tribution of the bromodomain among enzymes thatacetylate, methylate or remodel chromatin highlightthe importance of lysine acetylation in self-maintenanceof a transcriptional active state and recruitment of othersources of chromatin modifying enzymes (reviewed in[7]).

The p300/CBP family is another major group of nu-clear HATs that has been extensively characterised (Ta-ble 1). The members of this family are more globalregulators of transcription; contain a considerably largerHAT domain of about 500 residues, and other protein

Fig. 3. Recruitment of bromo- and chromo-domain containing proteins by histone modifications. (a) Establishment of silent chromatin(heterochromatin) by lysine 9 H3 methylation: (1) SuV39H1 methylates lysine 9 H3. (2) Methylated Lysine 9 recruits the heterochromatin proteinHP1 in physical association with SuV39H1. (3) Methylation of adjacent nucleosomes by SuV39H1 causes the spreading of the heterochromatin. (b)Establishment of transcriptionally active chromatin by lysine 4 H3 methylation: (1) Set1p methylates lysine 4 H3. (2) Methylated lysine 4 recruits thechromatin remodelling factor Chd1p in physical association with histone acetyltransferases. (3) Acetylation of lysine residues prevents repressivemodifications to occur and recruits transcription activators. (c) Establishment of transcriptionally active chromatin by lysine acetylation: (1) GCN5acetylates several residues within histones H3 and H4. (2) Acetylated lysines recruit the chromatin remodelling complex SWI/SNF. (3) SWI/SNF, viaits ATPase activity, displaces and twists nucleosomes exposing DNA areas for interaction with the transcription machinery.

2384 H. Santos-Rosa, C. Caldas / European Journal of Cancer 41 (2005) 23812402

Summary Heterochromatin is compacted inactive genes

Euchromatin is loose actively transcribed genes

Histone tails are subject to multiple post-translational modifications e.g. acetylation, methylation, phosphorylation, and ubiquitination

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Summary (2) Histone modifications control the condensation, or compaction of chromatin and its ability to be transcribed, replicated, and repaired.

Heterochromatin is marked with an epigenetic code, it does not depend on the sequence of bases in DNA that maintains the repression of associated genes in replicated daughter cells

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