Chromosomes. Ms. Gunjan M. Chaudhari

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1 Chromosomes Ms. Gunjan M. Chaudhari

2 Chromsomes Chromosome structure Chromatin structure Chromosome variations The new cytogenetics

3 Prokaryotic chromosomes Circular double helix Complexed with protein in a structure termed the nucleoid Attached to plasma membrane

4 Eukaryotic Chromosomes Located in the nucleus Each chromosome consists of a single molecule of DNA and its associated proteins The DNA and protein complex found in eukaryotic chromosomes is called chromatin 1/3 DNA and 2/3 protein Complex interactions between proteins and nucleic acids in the chromosomes regulate gene and chromosomal function

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6 Some evidence that chromosomes contain a single DNA molecule Pulsed-field gel electrophoresis - separation of chromosomes Analysis of the complete nucleotide sequence of many genomes now In situ hybridization (below)

7 Karyotype The representation of entire metaphase chromosomes in a cell, arranged in order of size and other characteristics

8 Ideogram Diagramatic representation of a karyotype Individual chromsomes are recognized by -arm lengths p, short q, long -centromere position metacentric sub-metacentric acrocentric telocentric -staining (banding) patterns From Miller & Therman (2001) Human Chromosomes, Springer

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10 Chromsome banding Q (quinicrine) & G (Giemsa) banding preferentially stain AT rich regions R (reverse banding) preferentially stains GC-rich regions C-banding (denaturation & staining) preferentially stains constitutive heterochromatin, found in the centromere regions and distal Yq

11 C-banded karyotype of XY cell From Miller & Therman (2001) Human Chromosomes, Springer

12 Constitutive heterochromatin Tandem, highly repeated short sequences of DNA Non-coding and non-expressing Buoyant density discrete from the bulk of the genome (satellite DNA ) C-banding Late replicating Maintains a highly compacted organization Never transcribed

13 Facultative heterochromatin All types of sequences C-banding negative Late replicating Condensed conformation Not transcribed Includes genes silenced in specific cell types and/or at specific times in development e.g. inactivated X chromosomes

14 Euchromatin Actively expressed sequences More open conformation

15 Fluorescence in situ hybridization (FISH) a The basic elements of fluorescence in situ hybridization are a DNA probe and a target sequence. b Before hybridization, the DNA probe is labelled by various means such as NICK TRANSLATION, RANDOM-PRIMED LABELLING and PCR. Two labelling strategies are commonly used indirect labelling (left panel) and direct labelling (right panel). For indirect labelling, probes are labelled with modified nucleotides that contain a HAPTEN, whereas direct labelling uses the incorporation of nucleotides that have been directly modified to contain a fluorophore. c The labelled probe and the target DNA are denatured to yield ssdna. d They are then combined, which allows the annealing of complementary DNA sequences. e If the probe has been labelled indirectly, an extra step is required for visualization of the non-fluorescent hapten that uses an enzymatic or immunological detection system. Whereas FISH is faster with directly labelled probes, indirect labelling offers the advantage of signal amplification by using several layers of antibodies, and might therefore produce a signal that is brighter compared with background levels. Finally, the signals are evaluated by fluorescence microscopy (not shown). [From Speicher & Carter (2005) Nature Rev Genet 6:782]

16 Fluorescence in situ hybridization (FISH) [From Speicher & Carter (2005) Nature Rev Genet 6:782] a Painting probes stain entire chromosomes. b Regional painting probes can be generated by chromosome microdissection c Centromeric-repeat probes are available for almost all human chromosomes. d Large-insert clones are available for most genomic regions. Subtelomeric probes, which are often used to screen for cryptic translocations that are not usually visible in conventional chromosome-banding analyses, are shown in this example. e Special probe sets can be designed to facilitate diagnosis of known structural rearrangements. In this example, the probe set includes a breakpoint-spanning probe (red) and two breakpoint-flanking probes (green and blue). f Genomic DNA is used as the probe in comparative genomic hybridization (CGH) to establish copy number. An analysis of chromosome 8 is shown as an example. Simultaneous visualization of both test DNA (green region) and normal reference DNA (red region) fluorochromes shows balanced regions in orange (equal amounts of green and red fluorochromes). g For highresolution analysis, DNA fibres can be used as the target for probe hybridization. The simultaneous hybridization of two different probes is shown, labelled green and red. h Microarrays can be used as targets for hybridization to provide resolutions down to the single-nucleotide level. A BAC array is shown, to which test DNA and reference DNA are hybridized. Individual clones show different colours after hybridization depending on whether the corresponding DNA in the test sample is lost (red on the array), gained (green on the array) or neither (yellow on the array).

17 Fluorescence in situ hybridization (FISH) probes on metaphase chromosomes

18 Chromosome-specific paints for FISH

19 Fluorescence in situ hybridization (FISH) metaphase chromosome painting

20 Chromosome maintenance Origins of replication Telomeres Centromeres

21 Origins of replication Multiple origins -every 100 kb on average in humans Heterochromatin is late replicating Replication times correspond to banding patterns Each band replicated independently From Miller & Therman (2001) Human Chromosomes, Springer

22 Telomeres End structures of linear chromosomes Serve to replicate chromosome ends Serve to stabilize chromosome ends (i.e. prevent nonhomologous end joining, NHEJ) G-rich tandem repeats - TTAGG, insects - TTAGGG, vertebrates - TTTAGGG, plants Length is under genetic and developmental control - e.g. 2-5 kb in Arabidopsis, kb in Tobacco, 15 kb in humans Sequence and proteins conserved across taxa, mammals to plants

23 FISH with a telomere-specific probe

24 Telomeres & telomerase in the replication of linear chromosome ends

25 Telomerase Reverse transcriptase & RNA primer Repeating cycles of parental strand extension - build template for lagging strand replication - build up the number of telomeres Abundant in mammalian embryos, stem cells and cancer cells Absent in mammalian somatic cells - telomeres shorten with each cell division - cells cease division and begin senescence Abundant in rapidly dividing and germ-line cells of plants Absent in vegetative tissues of plants

26 Centromeres Primary constriction Kinetochore - spindle fiber attachment Region of sister chromatid cohesion Constitutive heterochromatin Repeat sequences - CENs - 5 to 170 bp e.g. human alphoid satellite repeat No universal centromere repeat, but the same repeat can be found in more than one centromere of a species or between species Centromere repeats can change rapidly in evolution via mutation, new elements, recruitment of other genomic repeats Specific associated proteins e.g. Centromere-specific histone HE (CenH3)

27 A model of centromere structure

28 Chromatin structure Compacts DNA ~ 10,000 X From Miller & Therman (2001) Human Chromosomes, Springer

29 Chromatin structure 11 nm fiber Nucleosomes -147 bp DNA wound on histone core - Histones H3, H4, H2A, H2B (2 each) Internucleosomal spacer -~ 60 bp linker DNA 30 nm fiber Histone H1 (linker) binds and compacts nucleosomes Exact structure is controversial - Solenoid = single helix coiling of 11 nm fiber - Zig-zag stacking of nucleosomes then coiling = double helix of 11 nm fiber From Woodcock (2006) Curr Opin Struct Biol 16:213

30 Chromatin structure 300 nm fiber Loops of 30 nm fibers Attached to protein scaffold Attachment points correspond to boundary elements, isolating regions of differential gene expression Metaphase chromatin Coiling of the 300 nm fiber

31 Chromatin structure histone modifications Post-translational modifications on histone proteins Establish global chromatin structure -heterochromatin vs euchromatin Regulate DNA-based functions - Transcription - Replication, recombination & repair Complex interactions - Not really a simple histone code - The truth is likely to be that any given modification has the potential to activate or repress under different conditions. [From Kouzarides (2007) Cell 128:693]

32 Chromatin structure histone modifications Post-translational modifications on histone proteins alter chromatin structure and, consequently, chromatin function Table 1. Different Classes of Modifications Identified on Histones Chromatin Modifications Residues Modified Functions Regulated Acetylation K-ac Transcription, Repair, Replication, Condensation Methylation (lysines) K-me1 K-me2 K-me3 Transcription, Repair Methylation (arginines) R-me1 R-me2a R-me2s Transcription Phosphorylation S-ph T-ph Transcription, Repair, Condensation Ubiquitylation K-ub Transcription, Repair Sumoylation K-su Transcription ADP ribosylation E-ar Transcription Deimination R > Cit Transcription Proline Isomerization P-cis > P-trans Transcription Overview of different classes of modification identified on histones. The functions that have been associated with each modification are shown. Each modification is discussed in detail in the text under the heading of the function it regulates. [From Kouzarides (2007) Cell 128:693]

33 Chromatin structure histone modifications Post-translational modifications on histone proteins alter chromatin structure and, consequently, chromatin function Figure 1. Recruitment of Proteins to Histones (A) Domains used for the recognition of methylated lysines, acetylated lysines, or phosphorylated serines. (B) Proteins found that associate preferentially with modified versions of histone H3 and histone H4. [From Kouzarides (2007) Cell 128:693]

34 Chromatin structure histone modifications Post-translational modifications on histone proteins The truth is likely to be that any given modification has the potential to activate or repress under different conditions. [Kouzarides (2007) Cell 128:693] Histone acetylation - generally associated with activation of transcription Histone de-acetylation - generally associated with repression of transcription - Histone de-acetylase targeted to methylated CpG islands

35 Chromatin structure histone modifications Post-translational modifications on histone proteins The truth is likely to be that any given modification has the potential to activate or repress under different conditions. [Kouzarides (2007) Cell 128:693] Lysine methyation associated with activation of transcription: H3K4, H3K36, H3K79 Lysine methyation associated with repression of transcription: H3K9, H3K27, H4K20

36 Chromatin structure functional consequences of histone modifications Figure 3. Functional Consequences of Histone Modifications (A) Geneexpression changes are brought about by the recruitment of the NURF complex, which contains a component BRTF recognizing H3K4me and a componentremodeling chromatin. (B) The Crb2 protein of fission yeast is recruited to DNA-repair foci during a DNA-repair response. Crb2 is partly tethered there by association with methylated H4 and phosphorylated H2A. (C) The HBO1 acetyltransferase is an ING5-associated factor and is therefore tethered to sites of replication via methylated H3K4. HBO1 also binds to the MCM proteins found at replication sites. Evidence exists that HBO1 augments the formation of the preinitiation complex and is required for DNA replication. [From Kouzarides (2007) Cell 128:693]

37 Nuclear architecture Chromosome territories aall the chromosome territories that make up the human genome can be visualized simultaneously in intact interphase nuclei, each in a different colour. a A red, green and blue image of the 24 labelled chromosomes (1 22, X and Y) was produced from deconvoluted mid-plane nuclear sections from a three-dimensional stack by superposition of the 7 colour channels. b As in 24-colour KARYOTYPING, each chromosome can be identified by using a combination labelling scheme in which each chromosome is labelled with a different set of fluorochromes. In this way, each chromosome territory can be automatically classified using appropriate software, which assigns the corresponding chromosome number to a territory. If a stack of these images is collected throughout the nucleus, a simultaneous three-dimensional reconstruction of all chromosome territories is possible. Some of the dark regions represent unstained nucleoli. For further details see Ref. 90. [From Speicher & Carter (2005) Nature Rev Genet 6:782]

38 Nuclear architecture Chromosome territories Nonrandom chromosome positioning Gene rich chromosomes toward center Gene poor chromosomes toward periphery Centromeres are not the determining factor Chromosomes with adjacent positions more likely to interact cytolologically

39 Nuclear architecture consequences of chromosome territories Figure 3. Functional Consequences of Global Chromatin Organization (A and B) Spatial clustering of genes on distinct chromosomes facilitates their expression by (A) association with shared transcription and processing sites or (B) physical interactions with regulatory elements on separate chromosomes. (C) The physical proximity of chromosomes contributes to the probability of chromosomal translocations. [From Misteli (2007) Cell 128:787]

40 Nuclear architecture Nuclear factories Figure 1. Compartmentalization of Nuclear Processes Transcription, replication, and DNA repair are compartmentalized. (A) Transcription sites visualized by incorporation of bromo-utp, (B) replication sites visualized by incorporation of bromodutp, and (C) repair sites visualized by accumulation of repair factor 53BP1 at a double-strand break (DSB) are shown. In all cases, components are dynamically recruited from the nucleoplasm as single subunits or small preassembled subcomplexes. (A) is reprinted with permission from Elbi et al., 2002, (B) is courtesy of Rong Wu and David Gilbert at Florida State University, and (C) is courtesy of Evi Soutoglou from the National Cancer Institute, NIH. [From Misteli (2007) Cell 128:787]

41 Model of functional nuclear architecture Figure 3. Structural features that support the chromosome-territory interchromatin-compartment (CT IC) model are shown. These features are drawn roughly to scale on an optical section taken from the nucleus of a living HeLa cell. Although experimental evidence is available to support these features, the overall model of functional nuclear architecture is speculative (see text). [From Cremer & Cremer (2001) Nature Rev Genet 2:292]

42 Model of functional nuclear architecture Figure 3. Structural features that support the chromosome-territory interchromatin-compartment (CT IC) model are shown. These features are drawn roughly to scale on an optical section taken from the nucleus of a living HeLa cell. Although experimental evidence is available to support these features, the overall model of functional nuclear architecture is speculative (see text). a CTs have complex folded surfaces. Inset: topological model of gene regulation23. A giant chromatin loop with several active genes (red) expands from the CT surface into the IC space. b CTs contain separate arm domains for the short (p) and long chromosome arms (q), and a centromeric domain (asterisks). Inset: topological model of gene regulation78, 79. Top, actively transcribed genes (white) are located on a chromatin loop that is remote from centromeric heterochromatin. Bottom, recruitment of the same genes (black) to the centromeric heterochromatin leads to their silencing. c CTs have variable chromatin density (dark brown, high density; light yellow, low density). Loose chromatin expands into the IC, whereas the most dense chromatin is remote from the IC. d CT showing early-replicating chromatin domains (green) and mid-to-late-replicating chromatin domains (red). Each domain comprises 1 Mb. Gene-poor chromatin (red), is preferentially located at the nuclear periphery and in close contact with the nuclear lamina (yellow), as well as with infoldings of the lamina and around the nucleolus (nu). Gene-rich chromatin (green) is located between the gene-poor compartments. e Higher-order chromatin structures built up from a hierarchy of chromatin fibres88. Inset: this topological view of gene regulation27, 68 indicates that active genes (white dots) are at the surface of convoluted chromatin fibres. Silenced genes (black dots) may be located towards the interior of the chromatin structure. f The CT IC model predicts that the IC (green) contains complexes (orange dots) and larger non-chromatin domains (aggregations of orange dots) for transcription, splicing, DNA replication and repair. g CT with 1-Mb chromatin domains (red) and IC (green) expanding between these domains. Inset: the topological relationships between the IC, and active and inactive genes72. The finest branches of the IC end between 100-kb chromatin domains. Top: active genes (white dots) are located at the surface of these domains, whereas silenced genes (black dots) are located in the interior. Bottom: alternatively, closed 100-kb chromatin domains with silenced genes are transformed into an open configuration before transcriptional activation. [From Cremer & Cremer (2001) Nature Rev Genet 2:292]