Nuclear Structure and Chromatin Organization

Lecture 22

gene structure -

control regions, exons, introns

genes encode

single proteins or different isoforms

gene families arise from 

gene duplication during unequal meiosis recombination

many genes encode functional RNAs that are not translations into proteins but 

preform significant functions, such as rRNA, tRNA, and snRNA

single human cell DNA measures about

2 m in total length, and is contained within nuclei with diameters of less than 10 um - a compaction ratio of greater than 10^5 to 1

each chromosome consists of a single DNA molecules, organized into

increasing levels of condensation from nucleosomes to higher order chromatin folding by histone and nonhistone proteins 

any given portion of highly compacted DNA can be assessed for 

transcription, replication, and repair of damage without the long DNA molecules becoming tangled or broken 

higher eukaryotic DNA consists of 

unique and repeated sequences 

only about 1.5% of human DNA encodes 

proteins and functional RNAs

• the remainder includes regulatory sequences that control gene expression and introns 

about 45% of human DNA is derives from

mobile DNA elements, genetic symbionts that have contributed to the evolution of contempory genomes 

Gene:

the entire nucleic acid sequence that is necessary for the synthesis of a functional gene product (polypeptide or RNA) - protien coding, enhancer, and promotor regions

protein-coding genes with repeats of similar exons separated by introns:

• encode proteins that have repeated domains 

• evolved by tandem duplication of the repeated exon DNA, probably by unequal crossing over during meiosis 

protein-coding genes may be 

solitary or being to a gene family 

gene families encode:

• different proteins that have specific, but similar physiological functions

• heavily used gene products that must be transcribed at high rates 

solitary genes:

25-50% of the protein-coding genes in multicellular organisms are represented only once in the haploid genome 

thousands of genes are transcribed into nonprotein-coding RNAs for

various known and unknown reasons

tandemly repeated rRNA genes have evolved to meet

great cellular demand for their transcripts to makes ribosomes

embryonic cells that divide every 24 hours must maximally transcribe at least

100 copies of the large and small rRNA subunit genes to make the 5-10 million ribosomes necessary for the daughter cells 

simple transcription unit (~10% of human transcripts)

a monocistronic region extending from the 5’ cap site to the 3’ site poly(A) site with introns removed that encodes one protein

simple transcription unit - mutations in the transcription-control region may reduce or prevent 

transcription, thus reducing or eliminating synthesis of the encoded protein 

simple transcription unit - a mutation within an exon may result in 

an abnormal protien with diminished activity 

simple transcription unit - a mutation within an intron that introduces a new splice results in 

an abnormally spliced mRNA encoding a mutated protein 

complex transcription unit - primary transcripts can be processed in

alternative ways 

complex transcription unit - a primary transcript containing alternative splice sites can eb processed into

mRNAs with the same 5’ and 3’ exons but different internal exons to encode protein isoforms 

complex transcription unit - a primary transcript with two poly(A) site can be processed

into mRNAs with alternative 3’ exons 

complex transcription unit - alternative cell type-specific promotors (f or g)yield

mRNA1 in one cell type in which promotor f is activated with a first exon different from the first exon in mRNAs produces in a different cell type in which g is activated 

eukaryotic transcription units - a mutation in control regions or regions within exons shared by the alternative mRNAs affects the

protiens encoded by both alternatively processes mRNAs

eukaryotic transcription units - a mutation in different control regions or in different exons that are unique to one of the alternatively processed mRNAs affects

only the protein translated from that mRNA

mobile DNA elements include

transposons and retrotransposons

mobile DNA elements promote

the generation of gene families by gene duplication

• also promote the formation of complex regulatory regions 

mobile DNA elements - exon shuffling creates 

new versions of genes 

exon duplication by

unequal crossing over during meiosis

exon duplication - parental chromosomes: each parental chromosome contains

one ancestral gene containing three exons and two introns with Li long interspersed element homologous noncoding sequences 5’ and 3’ of the gene as well as in the intron between exons 2 and 3

exon duplication - homologous recombination: between L1 elements displaced relative to each other generate two nonidentical recombinant chromosomes:

• one recombinant chromosome in which the gene now has hour exons (two copies of exon 3)

• one recombinant chromosome in which the gene is missing exon 3

gene duplication by similar unequal crossing over during meiosis:

• each parental chromosome contains one ancestral B-globin gene

• unequal recombination between L1 elements and subsequent independent mutations yield duplicated genes on one chromosome that might encode slightly different proteins 

redundancy - 

duplicate genes retain its function and increase basal transcript levels 

neofunctionalization -

duplicated genes tend to accumulate mutations faster and these mutations may result in new and different functions 

subfunctionalization -

mutation in both copies of the gene lead to functionality of the original gene become distributed among the two copies

gene loss of pseudogene -

the extra copy of the gene may be lost over time do to not being needed, or it may become a psuedogene (the copy of the gene is retained but mutations lead to nonfunctionality)

comparison of related protein sequences from different species yields

clues to evolutionary relationships

deduced mechanism for evolution of tubular genes in existing species:

duplication event occurred before speciation, because the a-tubulin sequences from different species are more alike than are the a-tubulin and b-tubulin sequences within a species 

branch points (nodes, small numbers) represent 

common ancestral genes at the time that two sequences diverged 

homologous genes -

all tubules evolved from common ancestor

orthologous genes -

same function but differ as a result of speciation

paralogous genes -

differ as a result of gene duplication

computer algorithm analysis of DNA and protein sequences can predict

genes, protein functions, and protein family evolutionary relationships

biological complexity is not directly related to 

the number of protein-coding genes  

chromatin contains

nucleosomes of DNA wrapped around histone octamers

histone tail modifications regulate

chromatin structure, X-chromosome inactivation, and gene transcription

chromosome are localized in

non-overlapping “territories” in the interphase nucleus

nucleosome -

DNA wrapped around the histone octamer

10 nm nucleosome filament -

“beads on a string”: nucleosomes linked together by DNA strand

chromosomes and chromatin -

• 30 nm fibers 

• supercoiled DNA loops - anchored loops 

• condensed mitotic chromosome 

chromosomes and chromatin

packaging the genome 

chromsomes consist os

chromatin fibers, composed of DNA and associated proteins

each chromosome contains a

single, continuous DNA

nucleosome

the lowest level of chromosome organization 

the protein component of chromosomes include 

histones, a group of highly conserved proteins 

histones have a high content of 

basic amino acids (positive charge)

each nucleosome is joined by a short stretch of

linker DNA: length varies up to about 80bp

nucleosome consists of eight histone subunit proteins:

the histone octamer

• and 147 bp of DNA that makes 1.7 turns around this core histone assembly 

histone octamer:

two molecules each of histone H2A, H2B, H3 and H4 adopts a disc shape around which the 147bp coil always in a left-handed turn

all 4 histone proteins are small with a

large number of positively charged lysine residues that promote tight association with the negatively charged DNA sugar-phosphate backbone

each histone has a

long, unstructured N-ternimal amino acid tails that extend out from the nucleosome

• play an important roe in regulating higher order of packing

nucleosome core particle function and structure/sequence - 

so deeply conserved that there are only 2 amino acid differences in the H4 protein between humans and pea plants

higher order packing - a fifth histone protein (H1) binds

linker DNA and the DNA wrapped around the octamer

• it pulls nucleosomes together into the regular repeated array that established the 30nm chromatin fiber

higher levels of chromatin structure -

• a 30-nm filament is another level of chromatin packaging, maintained by histone H1

• organized into larger supercoiled loops 

higher levels of chromatin structure (other information) -

• a nucleus 10 mm in diameter can pack 200,000 times this length of DNA within its boundaries 

• packing ratio of the DNA in nucleosomes is approximately 7:1

• assembly of the 30-nm fiber increases the DNA-packing ration to 40:1

• mitotic chromosomes represent the ultimate in chromatin compactness with a ration of 10,000:1

histone modification is one mechanism to

alter the character of nucleosomes

DNA and histones are held together by

noncovalent bonds

ionic bonds between

negatively charged phosphates of the DNA backbone and positively charged residues of the histones 

histones, regulatory protiens, and enzymes dynamically mediate

DNA transcription, compaction, replication, recombination, and repair

changes in nucleosome structure allow

access to DNA

chromatin-remodeling complexes:

hydrolyze ATP and use this energy to slide DNA associated with octamers in order to regulate compaction

chromatin-remodeling complexes - the results make chromatin 

more or less compact - by promoting the expulsion of inclusion of octamers or the exchange of many histone proteins variants 

modification of histone by

acetylation, methylation, phosphorylation, and ubiquitination control of chromatin condensation and function 

histone in a single nucleosome usually contain

several, but not all, tail modifications simultaneously 

histone code:

specific post-translational modification combination in different chromatin regions specifically influence chromatin function by creating or removing chromatin-associated protein binding sites 

N-terminal tails of all 4 primary nucleosome histones are subject to

covalent modifications that loosen or tighten compaction

specific residues in tails of each histone can be targets by a variety of enzymes that promote:

• acetylation

• phosphorlyation

• methylation

• ubiquitination

reversible histone modifications -

• some residues can be modified in more than one way 

• also serve as a docking site to recruit additional chromatin modifiers 

acetylation of lysines neutralize their positive charge, weakening

histone/DNA associations, thereby making DNA more accessible

methylation will prevent

acetylation, resulting in more compact DNA not as accessible for transcription 

depending on the residue, the specific histone proteins and the specific covalent modification these changes may either 

compact or loosen chromatin, thereby promoting gene silencing or expression, respectively 

specific enzymes (methytransferases, acetylates, deacetylases) are tightly controlled in order to 

regionally control chromatin configuration leading to the activation/inactivation of only specific genes 

state of condensation/compaction varies from cell to cell as well as temporally in response to 

diverse cues (environmental stress, nutrient availability, signals from other cells, as well as the differentiated state of the cell) 

most highly condensed interphase chromatin is called

heterochromatin - essentially inactive and without transcription

state of condensation/compaction - concentrated around the

center (centromere) and termini (telomeres) of chromosomes

• with variable regions interspersed along the length 

euchromatin -

variable state of decondensed chromatin, some more relaxed than other, transcriptionally active regions of chromosome

heterochromatin -

methylation of lysine 9 in histone H3 (H3K9me) is principle factor establishing heterochromatin 

H3K9me promotes 

heterochromatin spreading by recruiting specific methyltransferases that modify adjacent nucleosomes 

• will continue to spread until it encounters a barrier DNA sequences 

HP1 (heterochromatin protein 1) contributes to heterochromatin condensation:

• binds to histone H3 N-terminal tails trimethylated at lysine 9 

• associated with other histone-bound HP1 molecules 

HP1 chromoshadow domain binds a histone methyltransferase (H3K9 HMT) that 

methylates lysine 9 of a histone H3 in a adjacent nucleosome, which creates a binding site for another HP1 on the neighboring nucleosome 

• the spreading process continues until it encounters a “boundary element” where several nonhistone proteins are bound to the DNA 

when DNA in heterchromatin is replicated, histone octamers di- or trimethylated at H3 lysine 9 are distributed to both 

daughter chromosomes along with an equal number of newly assembled histone octamers 

the H3K9 HMT associated with the H3K9 di- and trimethylated nucleosomes methylates lysine 9 of the newly assembled nucleosomes, regenerating 

the heterochromatin in both daughter chromosomes 

epigenetic regulation depends on 

factors other than DNA sequence 

epigenetic modifications can be transmitted from

parents to progeny cells and regulate gene expression without altering nucleotide sequence

• X-chromosome is an example 

an epigenetic state can usually be

reversed; X-chromosomes, for example are reactivated prior to formation of gametes 

differences in disease susceptibility and longevity between genetically identical twine may be due, in part, to 

epigenetic difference that appear between the twins as they age