Zhou Chromosome And Chromatin 1 New

Chromosome Chromatin Telomeres 周金秋 [email protected] Istitute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Fall, 2005

I. Chromosome and Chromatin II. Centromere III. Telomeres

I. Chromosome and Chromatin II. Centromere III. Telomeres

Genome size in different organisms

Genomes and gene number

6000

19,000

13,500

32

12

8

20,000 25

30,000 36

40

25,000 30,000 46

10

Compacting DNA into chromosomes is essential 3 x 109 bp 3.4 Å ~1.0 meters / haploid genome ~2.0 meters / per cell ~10 micrometers - nucleus diameter ~ 1 micrometer – condensed chromosomes >10,000 fold

History of chromatin and chromosomes 1673 1665

Van Leeuwenhoek (Dutch) Hooke (England)

Microorganisms Cell

Early 1800s

Brown (Scottland)

Nucleus (little nut)

1866

Mendel (Austria)

Mendel’s Law

1869

Miescher (Swiss)

Nuclein (C, H, O, N, P)

1857

Perkin (England)

aniline purple (mauveine dye)

1879

Flemming (Germany)

Chromatin, chromosome, Mitosis (thread 1882)

1887

Van Beneden (Belgium)

Chromosome No

1900

De Vries (Neitherland) Correns (Germany) Tschermak (Austria)

Re-discovery of Mendel law

1902-1903

Sutton (USA) Boveri (Germany)

Chromosomes are paired and may be the carriers of heredity. Mendel's "factors" (genes) are located on chromosomes

1920s-1930s

Morgan (USA)

Morgan Law

1938

Mullar (USA)

Mutation of chromosome

1928

Griffith (Britain)

Transforming principle

1943

Avery (Canada)

Genetic material - DNA

A-T rich G band

Centromere

Large rDNA

Chromosome karyotype (human)

Chromosome spread and FISH

Visualize chromosomes (FISH)

Fluorescent antibody-tagged DNA probes hybridize to their complementary sequences in the chromosomes.

Human cells contain 23 pairs of Chromosomes. For each pair of chromosomes, one is maternal and one is paternal – homologous chromosomes Sex chromosomes are nonhomologous chromosomes, X from mom, Y from dad.

Visualize gene(s) (FISH)

Chromosome spread (FISH)

Visualize gene(s) (FISH)

Visualize gene(s) (FISH)

Chromosome Translocation (FISH)

Function of chromosome and chromatin • Storage of genetic information • Precise segregation of replicated DNA into two daughter cells • Platform for transcription, replication, recombination and repair

Problem(s) How to retrieve genetic information from DNA packaged in chromosomes?

I. Chromosome and Chromatin II. Centromere III. Telomeres

Chromatin in different cell-stage

Interphase chromatin

A mitotic chromosome

Euchromatin:

(1) delicate (2) active (3) at the nucleus interior

,

Heterochromatin: (1) darkly staining (2) tightly packaged, (3) genetically inactive. (4) at the nucleus periphery

Constitutive heterochromatin: fixed and irreversible Centromere, Telomeres (Movie 1)

Facultative heterochromatin: able to return to the euchromatin inactive X chromosome

Chromatin composition: DNA Histone proteins

Stable association

Nonhistone: HMG proteins residual proteins phosphoproteins RNA species lipid species

Nucleosome Nucleosome:

a nucleosome core particle + linker DNA (180-200 bp) + a linker histone

Nucleosome core particle: histone octamer (2x H2A, H2B, H3, H4) + 146 bp DNA

Nucleosome: unit of chromatin

A) 30 nm fibers B) beads on a string-nucleosome From interphase nucleus

Nucleosome: unit of chromatin

Histone depleted metaphase chromosomes

Nucleosomes can be isolated by digesting with nucleases that cut between the nucleosomes in a region called the linker

Nucleosome-octamer 2 each H2A, H2B, H3, H4

Histones - highly basic proteins Protein

Molecular weight 21

Major Amino acid ++ Lys

13.8

Lys

H2b

13.8

Lys

H3

15.4

Arg/Lys

H4

11.4

Arg/Lys

H1 H2a

The position of the core histone in the nucleosomes Nucleosome core particle

Crystal structure of the mono-nucleosome

From Luger et al Nature 389: 251 - 260 (1997)

142 hydrogen bonds between DNA and nucleosome, mostly between phosphodiester bonds and amino acid backbone of histones

Is DNA in the nucleosome different from DNA in solution? 1. DNA (146 bp) is wrapped in 1.75 left-handed superhelical turns 2. One side of DNA is in contact with histone octamer 3. DNA helical turns in a nucleosome have an average number of base pairs per helical turn of 10.2 vs 10.5 of DNA in solution

Histone tail interactions with DNA

From Luger et al Nature 389: 251 - 260 (1997)

Histone fold3 alpha helices and 2 folds

N terminal tails are subject to covalent modification-important for transcription

Histone self-assembly

Modification of histone tail

Silent chromatin

Gene silencing and silent chromatin Gene silencing: gene silencing acts in a regional rather than promoter- or sequence-specific manner to generate large domains or DNA that are usually inaccessible to DNA binding proteins: RNA polymerase, cellular recombination machinary, exogenous enzymes (dam methyltransferase and restriction endonuclease).

Silent chromatin domain is persistent through mitotic and meiotic cell divisions such that a particular chromatin structure (DNA and its associated proteins) is replicated during the process of chromosome duplication. This mode of inheritance, commonly referred to as epigenetic inheritance, is believed to underlie cellular memory mechanisms that maintain cell identity and stable patterns of gene expression in eukaryotes.

Silent chromatin and heterochromatin Silent chromatin shares the central properties of general inaccessibility and epigenetic in heritance with heterochromatin. Therefore, although silent chromatin domains, unlike heterochromatin, are not always cytologically distinguished, they are often referred to as heterochromatic. Gene silencing and heterochromatin are often associated with repetitive DNA sequences and may be involved in stabilizing such sequences.

Biochemical nature of silent chromatin/heterochromatin 1.

Histone H3 methylated at lysine 9 (H3-mLys9)

2.

Hypoacetylation of lysine residues

3.

cytosine methylation, the most common form of DNA modification in eukaryotes.

Richards and Elgin (2002) Cell 108:489

Telomere heterochromatin/Position effect

Telomere looping

Rap1p

Nucleosome

Sir3p

Sir4p

Strahl-Bolsinger et al Genes & Development 1997

Telomere silencing of ADE2 Telomere position effect ADE2 “OFF” = RED ADE2

ADE2 “ON” = WHITE

Wildtype, ADE2 gene near telomere is silenced

Lack of a telomere binding protein reduces/disrupts telomere silencing Ivessa et at, (2002) G&D16:1383

Assembly of silent chromatin in budding yeast (example of biochemical study) nucleosomes

protective cap

telosome

report gene

Rap 1

Sir2/3/4 complex

Ku

Cdc13

Chromatin immunoprecipitation (ChIP) DNA-binding proteins are crosslinked to DNA with formaldehyde in vivo.

Isolate the chromatin. Shear DNA along with bound proteins into small fragments.

Bind antibodies specific to the DNA-binding protein to isolate the complex by precipitation. Reverse the cross-linking to release the DNA and digest the proteins.

Use PCR to amplify specific DNA sequences to see if they were precipitated with the antibody.

Sir2/3/4 interacts with telomeric DNA Sir2/Sir3 and DNA interaction requires Sir4

Luo et al (2002) Genes & Development 16:1528

Sir2/Sir3/Sir4 binding to telomeric DNA decreases at telomere distal regions

Luo et al (2002) Genes & Development 16:1528

Sir2 and Sir3 binding at the telomeric end require the enzymatic activity of Sir2

Model for assembly of silent chromatin in budding yeast

Moazed (2001) Mol Cell 8:489

Model for assembly of silent chromatin domain in fission yeast

Moazed (2001) Mol Cell 8:489

The Swi6/HP1 silencing complex is conserved in the fission yeast, S. pombe, and metazoans

Moazed (2001) Mol Cell 8:489

Silencing components in different systems

Richards and Elgin (2002) Cell 108:489

Euchromatin and heterochromatin

Richards and Elgin (2002) Cell 108:489

Chromosome Packing

How linear DNA molecule is packaged into a compact chromosome?

Interphase chromatin

A mitotic chromosome

?

Interphase

M phase

“Beads on a string” to 30 nm Chromatin Fiber?

Histone H1 - linker of nucleosomes

Zigzag model 10 nm nucleosome ------ 30 nm fiber

Solenoid Model six to eight nucleosomes per turn

Linker histones in higher order chromatin compaction

Further Compaction? Chromatin in the interphase nucleus is believed to organized into discrete domains defined by sites of attachment to the nuclear matrix.

Scaffold attachment regions (SARs) • Regions of the chromosomes with sequences specific for topoisomerase, HMG protein, and histone H1 binding • Found only in untranscribed regions of the eukaryotic chromosomes • Spaced along the chromosomes, with the intervening regions containing one or more genes? • Highly AT rich (65%) and may be several hundred bp long

How linear DNA molecule is packaged into a compact chromosome?

???

I. Chromosome and Chromatin II. Centromere III. Telomeres

Centromere

Centromere

Figure 23-38, p. 1094, Molecular Cell Biology, 3rd ed., Lodish, et al.

Centromere is a region of a eukaryotic chromosome where the kinetochore is assembled. It is the site where spindle fibers of the mitotic spindle attach to the chromosome during mitosis. It is the site at which a chromatid and its identical sister attach together during the process of cell division. It is a chromosomal locus that ensures delivery of one copy of each chromosome to each daughter at cell division. In most eukaryotes, the centromere has no defined DNA sequence. It typically consists of large arrays of repetitive DNA where the sequence within individual repeat elements is similar but not identical.

Kinetochore, the protein complex assembled at each centromere, serves as the attachment site for spindle microtubules and the site at which motors generate forces to power chromosome movement.

Functions of centromere • Required for chromosome stability • Sister chromatid pairing • Mitotic and meiotic spindle attachment • Chromosome movement • Cell cycle checkpoint control

The structure of centromere • • • • •

1 centromere / chromosome Structural complex Kinetochore - spindle fiber attachment DNA at yeast centromeres is relatively simple Human centromere is a family of highly repeated, tandemly arrayed ‘satellite’ DNA which measure 300-5,000 kb in length Repeat sequences • Specific associated proteins

Centromere DNA

Fukagawa.(2004) Chromosome Res. 12: 557–567

Centromere DNA

Bjerling and Ekwall. (2002) Braz J Med Biol Res. 35: 499-507

Centromere proteins

Bjerling and Ekwall. (2002) Braz J Med Biol Res. 35: 499-507

Budding yeast kinetochore proteins and their homologues

Essential genes in red nonessential genes in black metazoan homologue in black (right column) S. pombe homologue in blue.

Cheeseman et al. (2002) J Cell Biol. 157: 199-203

S. pombe Centromere

- Swi6 and Cnp1 localization

SpCENP-A

,Kniola et al, (2001) MBC 12:2767–2775

S. pombe Centromere

- Ndc80 and Cnp1 localization

Kniola et al, (2001) MBC 12:2767–2775

Clr4p and Rik1p are required for centromere localization of Swi6p

Ekwall et al, (1996) J. Cell. Sci. 109,2637–2648.

Centromere dysfunction in clr4 and rik1 mutants

WT

clr4

rik1 Ekwall et al, (1996) J. Cell. Sci. 109,2637–2648.

Centromere-associated proteins in yeast

Mellone and Allshire. (2003) Curr Opin Genet Dev. 13:191–198

Centromere DNA of higher eukaryotes • The universal presence of a great abundance of tandemly repeated DNA • The size of centromere DNA varies from several hundreds of kilobases to tens of megabases on each chromosome • Lack of sequence conservation

CENP-A proteins form a higher order structure in Drosophila and humans Drosophila CID (green, CENP A homologue) ROD (red, outer kinetochore protein, CENP E homologue)

Human CENP A (green, histone H3-like protein) CENP E (red, outer kinetochore protein)

Human CENP A (green, histone H3-like protein) CENP C (red, inner kinetochore protein)

Blower et al, (2002) Developmental Cell 2:319–330

CID, CENP-A H2A, H2B, H3 in Extended Chromatin Fibers H2AB (green) CID (red)

H2AB (green, continuous) CID (red, discontinuous) CID (red) Histone H3 (green) CID (red) Histone PH3 (green) Human CENP-A (green) H3 (red), interspersed Blower et al, (2002) Developmental Cell 2:319–330

Models for 3D organization of centromeric chromatin in Drosophila and humans

Solenoid model

Looping model

Blower et al, (2002) Developmental Cell 2:319–330

Organization of a human or mouse centromere

Choo. (2000) Trends Cell Biol. 10: 182-188

Centromeric organization of fission yeast and human.

Yanagida (2005) Phil. Trans. R. Soc. B 360, 609–621

I. Chromosome and Chromatin II. Centromere III. Telomeres

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