CHROMATIN
The p packaging g g of Eukaryotic y DNA into chromosomes
Human Chromosome number 2n = 46 2C DNA content 7.0 pg 1C DNA content 3 x 109 bp Smallest chromosome 4.6 4 6 x 107 bp 1.4 1 4 cm in length
The packaging of Eukaryotic DNA into chromosomes
Base pairs
Compact Size
DNA
Length
Compaction
0.33 x 1.1 nm
1 bp b
0.33 nm DNA
1x
Nucleosome
Disk 1 3/4 turn of DNA (146 bp)+ linker DNA
6- 11 nm
200 bp
66 nm DNA
6-11 x
Chromatin fiber
Approx. 6 nucleosomes per ‘turn’ of 11 nm
30 nm diameter
1200 bp
400 nm DNA
35x
DNA domain
Anchored DNA loop p
1 replicon?
60 nm x 0.5 m
60 kbp
20m DNA
35 x
Mitotic chromosome
2 chromatids, 1m thick
2 DNA molecules
10 m long
2 x 130 Mbp
2 x 43 mm DNA
10,000x
Nucleus (human)
2 x 23 =46 chromosomes
92 DNA molecules
10 m ball b ll
12 000 Mb 12,000 Mbp
4 m DNA
400 000 x 400,000
Compaction of DNA by histones Compaction by chromosome scaffold/nuclear matrix
Importance p of DNA foldingg
DNA folding is essential for orderly packing of long DNA molecule l l into i t the th nucleus l
Importance p of DNA foldingg
DNA folding is essential for orderly packing of long DNA molecule l l into i t the th nucleus l The manner in which the region of the genome is folded in a particular cell can determine the activity of the genes in that region
Classes of pprotein associated with DNA
Histones Non-histone chromosomal pprotein ((nuclear matrix or scaffold))
Chromatin – DNA that is associated with histones DNA histone complex forms an 11 nm fiber or 11 nm beads on fiber The packing ratio of 11 nm fiber is 10:1
Characteristics of histones Nucleosomal histones are relatively small proteins comprised of ~100 – 135 a.a. H1 (non-nucleosomal (non nucleosomal histone) is comprised of 215 amino acids. Histones contain a very high proportion of positively charged amino acids (lysine and argenine). Positive charge helps in binding with DNA. Histones possibly remain bound with DNA at all times.
Composition of histones Protein
Class
Number of a.a. % of basic a.a.
H1
Very lysine rich
213
30
H2A
Lysine, arginine rich
129
23
H2B
Moderately lysine rich
125
24
H3
Arginine rich
135
24
H4
Arginine, glycine rich
102
27
Nucleosomal histones
Responsible for folding DNA into nucleosomes H2A, H2A H2B H2B, H3 H3, and d H4 are nucleosomal l l hi histones t Highly g y conserved amongg species p The H4 a.a. sequences of pea and bovine differ by only two sequences
Nucleosome The fundamental DNA packing unit Gives chromatin its beads on a string appearance The length of DNA associated with nucleosome was determined by digestion of DNA between the nucleosome beads with micrococcal nuclease Double stranded DNA fragments 146 bp remain that were protected from digestion by association with histones
Nucleosome 11 nm disc shaped particles (“beads”) Each particle contains eight histone molecules (octomeric histone core) Th The octomeric t i core consists i t off two t copies i eachh off H2A, H2A H2B, H2B H3 and H4 The octomer forms a protein core around which the 146 bp double stranded DNA fragment is wound
H4 H3 H2A H2B
white green light blue d k blue dark bl
111 nm
Each core histone dimer has 6 DNA binding surfaces that organize 3 DNA turns
The histone octomer organizes g 145 bpp of DNA in 1 ¾ helical turn of DNA
48 nm of DNA packaged in a disc of 6 x 11 nm
6 nm
Nucleosome H1 is not included in the nucleosome core nor is it involved in the coiling of the DNA helix about the histone octomer H1 is bound to the DNA where the double helix enters and leaves the nucleosome core Approximately pp y 166 base ppairs are bound to the nucleosome: A. 146 (±1) base pairs are tightly bound to the core particle and B. the remaining 20 base pairs are associated with the H1 histone The DNA between two nucleosomes is called the linker segment This linker segment gives unfolded chromatin a beads segment. beadson-a-string appearance E Eachh nucleosome l bead b d is i separated t d from f the th nextt by b a region i off linker DNA ~60 bp long (range 0 to 100 bp)
~60 bp associated with the linker DNA
146±1 bp b associated i t d with the histone core
20 bp associated with the H1 histone
30 nm Chromatin fiber Packing ratio of the 11 nm fiber into the 30 nm fiber is ~5:1 The extended beads on string form is rarely adopted in nature since the chromatin must be kept in a highly compacted state for packaging purposes H1 appears to be responsible for packing nucleosomes into the 30 nm fiber A A.
H1 consists of a globular central region linked to extended amino-terminal amino terminal and carboxyl-terminal arms
B.
the globular portion binds to a unique site on each nucleosome, and the arms probably extend to cover the linker DNA near the point where it joins the nucleosome bead as well as to contact the histone cores of adjacent nucleosomes
C C.
H1 pulls the nucleosomes together into a regular repeating array
D.
H1 tends to bind DNA in clusters of eight or more H1 molecules (cooperative binding)
The packaging of Eukaryotic DNA into chromosomes
Base pairs
Compact Size
DNA
Length
Compaction
0.33 x 1.1 nm
1 bp b
0.33 nm DNA
1x
Nucleosome
Disk 1 3/4 turn of DNA (146 bp)+ linker DNA
6 x 11 nm
200 bp
66 nm DNA
6-11 x
Chromatin fiber
Approx. 6 nucleosomes per ‘turn’ of 11 nm
30 nm diameter
1200 bp
400 nm DNA
35x
DNA domain
Anchored DNA loop p
1 replicon?
60 nm x 0.5 m
60 kbp
20m DNA
35 x
Mitotic chromosome
2 chromatids, 1m thick
2 DNA molecules
10 m long
2 x 130 Mbp
2 x 43 mm DNA
10,000x
Nucleus (human)
2 x 23 =46 chromosomes
92 DNA molecules
10 m ball b ll
12 000 Mb 12,000 Mbp
4 m DNA
400 000 x 400,000
Compaction of DNA by histones Compaction by chromosome scaffold/nuclear matrix
Specificity of histones Purified histones will, in vitro, form nucleosomes with any DNA molecule Subtle alterations of the nucleosomal organization of chromatin i selected in l d regions i off the h genome may modulate d l DNA functions f i Modification of histones may occur by: A. B. C. D. E. F. G.
Histone acetylase and deacetylase Histone phosphoryaltion Histone methylation ADP-ribosylation of histones Ubiquitin binding to H2A Synthesis of histone variants Heterogeneity in H1
Interphase chromosomes Euchromatin – Extended chromatin, light staining Heterochromatin – More condensed and more darkly staining Studies that allow visualization of individual genes, selected chromosome domains,, and entire single g chromosomes in interphase nuclei determined that: 1.
Even genetically active regions, chromatin can be highly folded and confined t discrete to di t spatially ti ll limited li it d nuclear l domains d i
2.
Whole individual chromosomes are organized as finite morphological entities in interphase
3.
Some chromosomal domains are non-randomly arranged in a cell in a typespecific manner
Interphase chromosomes The loop domain was considered to encompass small genetic units of ~30 30 kb (basic unit of chromatin folding of 30 nm wide solenoid fiber) g levels of foldingg correspond p to: Higher 1.Larger transcriptional and replication units that define band-like chromosome domains (0.03 to > 3.0 Mb) 2.Constitutive i i heterochromatic h h i coiled il d domains d i off ~9 Mbb
The highest and most complex level of genome organization manifests itself as the massive regions of dense heterochromatin and more extended euchromatin that morphologically characterize interphase nuclei
Interphase chromosomes Active genetic regions are recognized by nuclease accessibility in open chromatin configurations 1.The chromatin structure may determine accessibility for interaction with diffusible cytoplasmic factors 2.Highly condensed metaphase chromosomes are nearly completely heterochromatic and exhibit negligible gene transcription
Characteristics of heterochromatin Remains condensed during interphase Inactive in DNA transcription Replicated very late in the S-phase S phase of each cell cycle
Classes of heterochromatin Constitutive heterochromatin remains condensed in all cells of the organism g 1.
Relatively resistant to decondensation in interphase
2.
Contains relatively simple, serially repeated DNA sequences (i.e. satellite DNA)
3.
Found adjacent to centeromeres in most eukaryotes, and some conserved telomeric repeats
Facultative heterochromatin is condensed only in some cells, but not in others 1.
Does not contain large amounts of highly repeated DNA sequences
2.
Does not stain differentially in mitotic DNA
Interphase chromosomes Classes of noncoding DNA are non-randomly organized in chromosomes of mammalian species. species The domains they define have similar characteristics with respect to: 1.Transcription p capacity p y 2.Temporal order of replication 3.Higher order of folding
M Metaphase t h chromosomes h provide id a reference f point i t for f cytogeneticists Chromosomal domains identified by their molecular properties are related to landmarks of differentially stained cytogenetic bands
Metaphase chromosomes Contain far fewer nonhistone proteins than chromosomes of interphase nuclei The metaphase scaffold (nuclear scaffold=nuclear matrix=proteinaceous structures that remain after membrane and histone extraction) consists of mainly a 170,000 MW protein identified as topoisomerase II 1.
Topo.II occurs with frequency of 3 copies per 70 kb loop of DNA
2 2.
Probably P b bl hhas a role l in i stabilizing bili i the h bases b off the h DNA loops l in i metaphase h chromosomes
3.
Required for chromosome condensation (evidence from yeast mutants)
Matrix Attachment Regions (MARs)
Dispersed DNA sequences in the genome that interact specifically with the nuclear scaffold
Binding activity along with their dispersal throughout genomes is thought to organize chromatin into loop domains
MARs are hypothesized yp to pplay y an important p role in DNA replication p and have been demonstrated to influence gene expression Their ability to boost the expression of transgenes makes them a valuable genetic ge et c engineering e g ee g tool too Flanking MARs may insulate transgenes from the influences of surrounding chromatin
Matrix Attachment Regions g ((MARs))
Extraction using Lithium diiodosalicylate (LIS) is used to identify Scaffold Attachment Regions (SARs)
Salt extraction is used to identify MARs
Matrix is used to define the nuclear structure during interphase
Scaffold is sometimes limited to metaphase chromosomes
MAR is sometimes limited to specific A-T rich attachment sites
Matrix Attachment Regions ( MARs)
Characteristics of MARs Ability to bind nuclear matrices I. Matrices of one species p are able to bind MARs of another species. p They are evolutionarily highly conserved II. Homology between MARs is usually not great enough for crosshybridization MARs are usually found in non-transcribed regions Most MAR sequences are highly A-T rich (>70%), but A-T richness alone l is i nott sufficient ffi i t for f matrix t i binding bi di MARs are typically 300-1000 bp in length MARs may be related to gene expression
M i Attachment Matrix A h R Regions i ( MARs) MAR ) Regions R i off MARs MAR may appear tto be b enriched i h d for f transcribe t ib sequences Transcriptionally active domains are typically unpackaged from a 30 nm to a 10 nm fiber, leaving DNA accessibly to in vitro degradation by DNAse I Association of MARs with topoisomerase II may allow supercoiling within chromatin loops that may be critical in preparing chromatin for transcription Hi Highh mobility bili group (HMG) proteins i found f d iin actively i l expressedd chromatin h i and H1 histones found in inactive 30 nm fibers HMG-I/Y is found in histone H1 depleted (expressed) chromatin HMG and H1 both have affinity for MARs but are not typically components of the nuclear matrix MARs may be b the h gateway for f expression-dependent i d d proteins i
Glossary of terms Chromatin: DNA that is associated with histones generation. Nucleosome: The fundamental DNA packing unit Constitutive heterochromatin: remains condensed in all cells of the organism Facultative heterochromatin: is condensed only in some cells, but not in others Nuclear scaffold: The metaphase scaffold; nuclear matrix; proteinaceous structures that remain after membrane and histone extraction Matrix Attachment Regions (MARs): dispersed DNA sequences in the genome that interact specifically with the nuclear scaffold
Glossary of terms Chromatin: DNA that is associated with histones generation. Nucleosome: The fundamental DNA packing unit
