Prophase
is the first step of cell division in mitosis. As it occurs after G2 of
interphase, DNA has been already replicated when prophase begins.Fluorescence microscope image of two mouse cell nuclei in prophase (scale bar is 5 μm).
Prophase (from Ancient Greekπρο- (pro-)'before' and φάσις (phásis)'appearance') is the first stage of cell division in both mitosis and meiosis. Beginning after interphase, DNA has already been replicated when the cell enters prophase. The main occurrences in prophase are the condensation of the chromatin reticulum and the disappearance of the nucleolus.
Various DNA stains are used to treat cells such that condensing chromosomes can be visualized as the move through prophase.
The giemsaG-banding technique is commonly used to identify mammalianchromosomes, but utilizing the technology on plant cells was originally difficult due to the high degree of chromosome compaction in plant cells. G-banding was fully realized for plant chromosomes in 1990. During both meiotic and mitotic prophase, giemsa staining can be applied to cells to elicit G-banding in chromosomes. Silver staining, a more modern technology, in conjunction with giemsa staining can be used to image the synaptonemal complex throughout the various stages of meiotic prophase. To perform G-banding, chromosomes must be fixed, and thus it is not possible to perform on living cells.
DNA that was replicated in interphase is condensed from DNA strands with lengths reaching 0.7 μm down to
0.2-0.3 μm. This process employs the condensin complex. Condensed chromosomes consist of two sister chromatids joined at the centromere.
The nucleoli begin to break down in prophase, resulting in the discontinuation of ribosome production. This indicates a redirection of cellular energy from general cellular metabolism to cellular division. The nuclear envelope stays intact during this process.
Meiotic prophase
Meiosis involves two rounds of chromosome segregation and thus undergoes prophase twice, resulting in prophase I and prophase II. Prophase I is the most complex phase in all of meiosis because homologous chromosomes must pair and exchange genetic information. Prophase II is very similar to mitotic prophase.
Prophase I
Prophase
I is divided into five phases: leptotene, zygotene, pachytene,
diplotene, and diakinesis. In addition to the events that occur in mitotic prophase, several crucial events occur within these phases such as pairing of homologous chromosomes and the reciprocal exchange of genetic material between these homologous chromosomes. Prophase I occurs at different speeds dependent on species and sex. Many species arrest meiosis in diplotene of prophase I until ovulation. In humans, decades can pass as oocytes remain arrested in prophase I only to quickly complete meiosis I prior to ovulation.
In the second phase of prophase I, zygotene (from the Greek for "conjugation"), all maternally and paternally derived chromosomes have found their homologous partner. The homologous pairs then undergo synapsis,a process by which the synaptonemal complex (a proteinaceous structure) aligns corresponding regions of genetic information on maternally and paternally derived non-sister chromatids of homologous chromosome pairs. The paired homologous chromosome bound by the synaptonemal complex are referred to as bivalents or tetrads.Sex (X and Y) chromosomes do not fully synapse because only a small region of the chromosomes are homologous.
The nucleolus moves from a central to a peripheral position in the nucleus.
Pachytene
The third phase of prophase I, pachytene (from the Greek for "thick"), begins at the completion of synapsis.Chromatin has condensed enough that chromosomes can now be resolved in microscopy. Structures called recombination nodules form on the synaptonemal complex of bivalents. These recombination nodules facilitate genetic exchange between the non-sister chromatids of the synaptonemal complex in an event known as crossing-over or genetic recombination. Multiple recombination events can occur on each bivalent. In humans, an average of 2-3 events occur on each chromosome.[
Diplotene
In the fourth phase of prophase I, diplotene (from the Greek for "twofold"), crossing-over is completed. Homologous chromosomes retain a full set of genetic information; however, the homologous chromosomes are now of mixed maternal and paternal descent. Visible junctions called chiasmata hold the homologous chromosomes together at locations where recombination occurred as the synaptonemal complex dissolves. It is at this stage where meiotic arrest occurs in many species.
Diakinesis
In
the fifth and final phase of prophase I, diakinesis (from the Greek for
"double movement"), full chromatin condensation has occurred and all
four sister chromatids can be seen in bivalents with microscopy. The rest of the phase resemble the early stages of mitotic prometaphase, as the meiotic prophase ends with the spindle apparatus beginning to form, and the nuclear membrane beginning to break down.
Prophase II
Prophase II of meiosis is very similar to prophase of mitosis. The most noticeable difference is that prophase II occurs with a haploid number of chromosomes as opposed to the diploid number in mitotic prophase. In both animal and plant cells chromosomes may de-condense during telophase I requiring them to re-condense in prophase II. If chromosomes do not need to re-condense, prophase II often proceeds very quickly as is seen in the model organismArabidopsis.
Prophase I arrest
Female mammals and birds are born possessing all the oocytes needed for future ovulations, and these oocytes are arrested at the prophase I stage of meiosis. In humans, as an example, oocytes are formed between three and four months of gestation within the fetus and are therefore present at birth. During this prophase I arrested stage (dictyate), which may last for decades, four copies of the genome
are present in the oocytes. The adaptive significance of prophase I
arrest is still not fully understood. However, it has been proposed
that the arrest of oocytes at the four genome copy stage may provide the
informational redundancy needed to repair damage in the DNA of the germline. The repair process used appears to be homologous recombinational repair Prophase arrested oocytes have a high capability for efficient repair of DNA damages. DNA repair capability appears to be a key quality control mechanism in the female germ line and a critical determinant of fertility.
Differences in plant and animal cell prophase
Arabidopsis thaliana
cell in preprophase, prophase and prometaphase. Preprophase band is
present along the cell wall from images 1–3, is fading in image 4, and
disappears by image 5.
The most notable difference between prophase in plant cells and animal cells occurs because plant cells lack centrioles. The organization of the spindle apparatus is associated instead with foci at opposite poles of the cell or is mediated by chromosomes. Another notable difference is preprophase, an additional step in plant mitosis that results in formation of the preprophase band, a structure composed of microtubules. In mitotic prophase I of plants, this band disappears.
A multiple sequence alignment of five mammalian histone H1 proteins Sequences are the amino acids
for residues 120-180 of the proteins. Residues that are conserved
across all sequences are highlighted in grey. Below each site (i.e.,
position) of the protein sequence alignment is a key denoting conserved
sites (*), sites with conservative replacements (:), sites with semi-conservative replacements (.), and sites with non-conservative replacements ( ).
The discovery of the role of DNA in heredity, and observations by Frederick Sanger of variation between animal insulins in 1949, prompted early molecular biologists to study taxonomy from a molecular perspective. Studies in the 1960s used DNA hybridization and protein cross-reactivity techniques to measure similarity between known orthologous proteins, such as hemoglobin and cytochrome c. In 1965, Émile Zuckerkandl and Linus Pauling introduced the concept of the molecular clock, proposing that steady rates of amino acid replacement could be used to estimate the time since two organisms diverged. While initial phylogenies closely matched the fossil record, observations that some genes appeared to evolve at different rates led to the development of theories of molecular evolution. Margaret Dayhoff's 1966 comparison of ferredoxin sequences showed that natural selection would act to conserve and optimise protein sequences essential to life.
Over many generations, nucleic acid sequences in the genome of an evolutionary lineage can gradually change over time due to random mutations and deletions. Sequences may also recombine or be deleted due to chromosomal rearrangements.
Conserved sequences are sequences which persist in the genome despite
such forces, and have slower rates of mutation than the background
mutation rate.
Conservation can occur in coding and non-coding
nucleic acid sequences. Highly conserved DNA sequences are thought to
have functional value, although the role for many highly conserved non-coding DNA sequences is poorly understood. The extent to which a sequence is conserved can be affected by varying selection pressures, its robustness to mutation, population size and genetic drift. Many functional sequences are also modular, containing regions which may be subject to independent selection pressures, such as protein domains.
Coding sequence
In coding sequences, the nucleic acid and amino acid sequence may be conserved to different extents, as the degeneracy of the genetic code means that synonymous mutations in a coding sequence do not affect the amino acid sequence of its protein product.
The nucleic acid sequence of a protein coding gene may also be conserved by other selective pressures. The codon usage bias in some organisms may restrict the types of synonymous mutations in a sequence. Nucleic acid sequences that cause secondary structure
in the mRNA of a coding gene may be selected against, as some
structures may negatively affect translation, or conserved where the
mRNA also acts as a functional non-coding RNA.
Non-coding sequences important for gene regulation, such as the binding or recognition sites of ribosomes and transcription factors, may be conserved within a genome. For example, the promoter of a conserved gene or operon may also be conserved. As with proteins, nucleic acids that are important for the structure and function of non-coding RNA
(ncRNA) can also be conserved. However, sequence conservation in ncRNAs
is generally poor compared to protein-coding sequences, and base pairs that contribute to structure or function are often conserved instead.
Conserved sequences may be identified by homology search, using tools such as BLAST, HMMER, OrthologR, and Infernal.
Homology search tools may take an individual nucleic acid or protein
sequence as input, or use statistical models generated from multiple sequence alignments of known related sequences. Statistical models such as profile-HMMs, and RNA covariance models which also incorporate structural information,
can be helpful when searching for more distantly related sequences.
Input sequences are then aligned against a database of sequences from
related individuals or other species. The resulting alignments are then
scored based on the number of matching amino acids or bases, and the
number of gaps or deletions generated by the alignment. Acceptable
conservative substitutions may be identified using substitution matrices
such as PAM and BLOSUM.
Highly scoring alignments are assumed to be from homologous sequences.
The conservation of a sequence may then be inferred by detection of
highly similar homologs over a broad phylogenetic range.
Multiple sequence alignment
A sequence logo for the LexA-binding motif of gram-positive bacteria. As the adenosine at position 5 is highly conserved, it appears larger than other characters.
Multiple sequence alignments can be used to visualise conserved sequences. The CLUSTAL
format includes a plain-text key to annotate conserved columns of the
alignment, denoting conserved sequence (*), conservative mutations (:),
semi-conservative mutations (.), and non-conservative mutations ( )
Sequence logos can also show conserved sequence by representing the
proportions of characters at each point in the alignment by height.
Genome alignment
This image from the ECR browser shows the result of aligning different vertebrate genomes to the human genome at the conserved OTX2 gene. Top: Gene annotations of exons and introns of the OTX2 gene. For each genome, sequence similarity (%) compared to the human genome is plotted. Tracks show the zebrafish, dog, chicken, western clawed frog, opossum, mouse, rhesus macaque and chimpanzee genomes. The peaks show regions of high sequence similarity across all genomes, showing that this sequence is highly conserved.
Whole genome alignments (WGAs) may also be used to identify highly conserved regions across species. Currently the accuracy and scalability
of WGA tools remains limited due to the computational complexity of
dealing with rearrangements, repeat regions and the large size of many
eukaryotic genomes. However, WGAs of 30 or more closely related bacteria (prokaryotes) are now increasingly feasible.
Scoring systems
Other approaches use measurements of conservation based on statistical tests that attempt to identify sequences which mutate differently to an expected background (neutral) mutation rate.
The GERP (Genomic Evolutionary Rate Profiling) framework scores
conservation of genetic sequences across species. This approach
estimates the rate of neutral mutation in a set of species from a
multiple sequence alignment, and then identifies regions of the sequence
that exhibit fewer mutations than expected. These regions are then
assigned scores based on the difference between the observed mutation
rate and expected background mutation rate. A high GERP score then
indicates a highly conserved sequence.
LIST (Local Identity and Shared Taxa) is based on the assumption that
variations observed in species closely related to human are more
significant when assessing conservation compared to those in distantly
related species. Thus, LIST utilizes the local alignment identity around
each position to identify relevant sequences in the multiple sequence
alignment (MSA) and then it estimates conservation based on the taxonomy
distances of these sequences to human. Unlike other tools, LIST ignores
the count/frequency of variations in the MSA.
Aminode
combines multiple alignments with phylogenetic analysis to analyze
changes in homologous proteins and produce a plot that indicates the
local rates of evolutionary changes. This approach identifies the
Evolutionarily Constrained Regions in a protein, which are segments that
are subject to purifying selection and are typically critical for normal protein function.
Other approaches such as PhyloP and PhyloHMM incorporate statistical phylogenetics methods to compare probability distributions
of substitution rates, which allows the detection of both conservation
and accelerated mutation. First, a background probability distribution
is generated of the number of substitutions expected to occur for a
column in a multiple sequence alignment, based on a phylogenetic tree.
The estimated evolutionary relationships between the species of
interest are used to calculate the significance of any substitutions
(i.e. a substitution between two closely related species may be less
likely to occur than distantly related ones, and therefore more
significant). To detect conservation, a probability distribution is
calculated for a subset of the multiple sequence alignment, and compared
to the background distribution using a statistical test such as a likelihood-ratio test or score test. P-values generated from comparing the two distributions are then used to identify conserved regions. PhyloHMM uses hidden Markov models to generate probability distributions. The PhyloP software package compares probability distributions using a likelihood-ratio test or score test, as well as using a GERP-like scoring system.
Extreme conservation
Ultra-conserved elements
Ultra-conserved elements or UCEs are sequences that are highly similar or identical across multiple taxonomic groupings. These were first discovered in vertebrates, and have subsequently been identified within widely-differing taxa.[44] While the origin and function of UCEs are poorly understood, they have been used to investigate deep-time divergences in amniotes, insects, and between animals and plants.
Universally conserved genes
The most highly conserved genes are those that can be found in all organisms. These consist mainly of the ncRNAs and proteins required for transcription and translation, which are assumed to have been conserved from the last universal common ancestor of all life.
Sets of conserved sequences are often used for generating phylogenetic trees, as it can be assumed that organisms with similar sequences are closely related.
The choice of sequences may vary depending on the taxonomic scope of
the study. For example, the most highly conserved genes such as the 16S
RNA and other ribosomal sequences are useful for reconstructing deep
phylogenetic relationships and identifying bacterial phyla in metagenomics studies. Sequences that are conserved within a clade but undergo some mutations, such as housekeeping genes, can be used to study species relationships. The internal transcribed spacer (ITS) region, which is required for spacing conserved rRNA genes but undergoes rapid evolution, is commonly used to classify fungi and strains of rapidly evolving bacteria.
Medical research
As
highly conserved sequences often have important biological functions,
they can be useful a starting point for identifying the cause of genetic diseases. Many congenital metabolic disorders and Lysosomal storage diseases
are the result of changes to individual conserved genes, resulting in
missing or faulty enzymes that are the underlying cause of the symptoms
of the disease. Genetic diseases may be predicted by identifying
sequences that are conserved between humans and lab organisms such as mice or fruit flies, and studying the effects of knock-outs of these genes. Genome-wide association studies
can also be used to identify variation in conserved sequences
associated with disease or health outcomes. More than two dozen novel
potential susceptibility loci have been discovered for Alzehimer's
disease.
Functional annotation
Identifying conserved sequences can be used to discover and predict functional sequences such as genes.
Conserved sequences with a known function, such as protein domains, can
also be used to predict the function of a sequence. Databases of
conserved protein domains such as Pfam and the Conserved Domain Database can be used to annotate functional domains in predicted protein coding genes.
A chromomere, also known as an idiomere, is one of the serially aligned beads or granules of a eukaryoticchromosome, resulting from local coiling of a continuous DNA thread. Chromomeres are regions of chromatin that have been compacted through localized contraction. In areas of chromatin with the absence of transcription, condensing of DNA and protein complexes will result in the formation of chromomeres. It is visible on a chromosome during the prophase of meiosis and mitosis. Giant banded (Polytene)
chromosomes resulting from the replication of the chromosomes and the
synapsis of homologs without cell division is a process called
endomitosis. These chromosomes consist of more than 1000 copies of the
same chromatid that are aligned and produce alternating dark and light
bands when stained. The dark bands are the chromomere.
It is unknown when chromomeres first appear on the chromosome. Chromomeres can be observed best when chromosomes are highly condensed.
The chromomeres are present during leptotene phase of prophase I during
meiosis. During zygotene phase of prophase I, the chromomeres of
homologs align with each other to form homologous rough pairing
(homology searching). These chromomeres helps provide a unique identity
for each homologous pairs. They appear as dense granules during leptotene stage
There are more than 2000 chromomeres on 20 chromosomes of maize.
Polytene chromosome of Drosophila. (b) Displays the chromomeric and interchromomeric bands of the chromosome.
Physical properties
Chromomeres
are organized in a discontinuous linear pattern along the condensed
chromosomes (pachytene chromosomes) during the prophase stage of meiosis. The linear pattern of chromomeres is linked to the arrangement of genes along the chromosome. Chromomeres contain genes and sometimes clusters of genes within their structure. Aggregates of chromomeres are known as chromonemata.
Cohesive proteins SMC3 and hRAD21(plays a role in sister chromatidcohesion)
are found within chromomeres at high concentrations, and maintain the
proper structure of chromomeres. The protein XCAP-D2 is also present at
high concentrations within the chromomere, and acts as a condensin component. High concentrations of tandem repeats of the heterochromatin protein HP1β builds up within the chromomere. In regions where loops attach to chromomeres, there is hyperacetylation of histone 4. The extra acetylation loosens chromatin from a condensed form, making it more accessible to proteins involved in transcription.
Functions
Chromomeres
are known as the structural subunit of a chromosome. The arrangement of
chromomere structure can aid in control of gene expression.
Maps of chromomeres can be made for use in genetic and
evolutionary studies. Chromomeric maps can be used to locate the exact
position of genes on a chromosome. Chromomeric maps can be used to
analyze chromosome aberrations, and find correlations between the
aberrations and their effects on genes near breakpoints.Lampbrush chromosomes and chromomeres
Chromomeres display different properties and behaviours when associated with lampbrush
chromosomes. Although found all across the lampbrush chromosome, they
are not organized in a clear pattern along as they are in normal
pachytene chromosomes of meiosis.
The two sister chromatids of a lampbrush chromosome separate fully,
forming lateral loops that extend from chromomeres, and act as
transcription complexes. The lateral loops are areas where the
chromosomes are transcriptionally active.
The loops define where the chromomere will be positioned. The
chromomere is an organization of regions of non-transcribed chromatin.
These regions of chromatin that have not been transcribed are located at
the ends of the loops that were formed by the sister chromatids of a
lampbrush chromosome.
Each chromomere can have up to several pairs of loops from lampbrush
chromosomes originating from it, as well as micro-loops that cannot be
detected with a light microscope.
Chromomeres of lampbrush chromosomes act as structural units, and
are not considered to be genetic units participating in transcription.
The arrangement of a chromosome into chromomeric units happens almost
simultaneously with the start of transcription.
The loop structures extending from chromomeres are maintained by a high
level of transcriptional activity and structural proteins of the
chromosome. Chromatin within the chromomere are held in position by a
variety of histone modifications and epigenetic markers.
Methyl-CpG-binding protein 2
(MeCP2) is a protein that binds to methylated DNA. MeCP2 has been found
to associate most strongly with transcriptionally inactive chromomere
domains. MeCP2 binding patterns to chromomere domains are proportional
to the density of chromomeric DNA found on a chromosome. The binding of
MeCP2 to chromomeric regions represents regions of chromatin that are
highly dynamic on the lampbrush chromosome.
A nucleosome is the basic structural unit of DNA packaging in eukaryotes. The structure of a nucleosome consists of a segment of DNA wound around eight histone proteins and resembles thread wrapped around a spool. The nucleosome is the fundamental subunit of chromatin.
Each nucleosome is composed of a little less than two turns of DNA
wrapped around a set of eight proteins called histones, which are known
as a histone octamer. Each histone octamer is composed of two copies each of the histone proteins H2A, H2B, H3, and H4.
DNA must be compacted into nucleosomes to fit within the cell nucleus. In addition to nucleosome wrapping, eukaryotic chromatin is further compacted by being folded into a series of more complex structures, eventually forming a chromosome. Each human cell contains about 30 million nucleosomes.
Nucleosomes are thought to carry epigenetically inherited information in the form of covalent modifications of their core histones.
Nucleosome positions in the genome are not random, and it is important
to know where each nucleosome is located because this determines the
accessibility of the DNA to regulatory proteins.
Nucleosomes were first observed as particles in the electron microscope by Don and Ada Olins in 1974, and their existence and structure (as histone octamers surrounded by approximately 200 base pairs of DNA) were proposed by Roger Kornberg. The role of the nucleosome as a regulator of transcription was demonstrated by Lorch et al. in vitro in 1987 and by Han and Grunstein and Clark-Adams et al. in vivo in 1988.
The nucleosome core particle consists of approximately 146 base pairs (bp) of DNA wrapped in 1.67 left-handed superhelical turns around a histone octamer, consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. Core particles are connected by stretches of linker DNA,
which can be up to about 80 bp long. Technically, a nucleosome is
defined as the core particle plus one of these linker regions; however
the word is often synonymous with the core particle. Genome-wide nucleosome positioning maps are now available for many model organisms and human cells.
Linker histones such as H1
and its isoforms are involved in chromatin compaction and sit at the
base of the nucleosome near the DNA entry and exit binding to the linker
region of the DNA. Non-condensed nucleosomes without the linker histone resemble "beads on a string of DNA" under an electron microscope.
In contrast to most eukaryotic cells, mature sperm cells largely use protamines to package their genomic DNA, most likely to achieve an even higher packaging ratio. Histone equivalents and a simplified chromatin structure have also been found in Archaea, suggesting that eukaryotes are not the only organisms that use nucleosomes.
Structure
Structure of the core particle
The crystal structure of the nucleosome core particle consisting of H2A , H2B , H3 and H4 core histones, and DNA. The view is from the top through the superhelical axis.
Overview
Pioneering
structural studies in the 1980s by Aaron Klug's group provided the
first evidence that an octamer of histone proteins wraps DNA around
itself in about 1.7 turns of a left-handed superhelix. In 1997 the first near atomic resolution crystal structure of the nucleosome was solved by the Richmond group at the ETH Zurich, showing the most important details of the particle. The human alpha satellitepalindromic DNA
critical to achieving the 1997 nucleosome crystal structure was
developed by the Bunick group at Oak Ridge National Laboratory in
Tennessee. The structures of over 20 different nucleosome core particles have been solved to date,
including those containing histone variants and histones from different
species. The structure of the nucleosome core particle is remarkably
conserved, and even a change of over 100 residues between frog and yeast
histones results in electron density maps with an overall root mean square deviation of only 1.6Å.
The nucleosome core particle (NCP)
The nucleosome core particle (shown in the figure) consists of about 146 base pair of DNA wrapped in 1.67 left-handed superhelical turns around the histone octamer, consisting of 2 copies each of the core histones H2A, H2B, H3, and H4. Adjacent nucleosomes are joined by a stretch of free DNA termed linker DNA (which varies from 10 - 80 bp in length depending on species and tissue type). The whole structure generates a cylinder of diameter 11 nm and a height of 5.5 nm.
Apoptotic DNA laddering. Digested chromatin is in the first lane; the second contains DNA standard to compare lengths.Scheme of nucleosome organizationThe crystal structure of the nucleosome core particle (PDB: 1EQZ)
Nucleosome core particles are observed when chromatin in interphase
is treated to cause the chromatin to unfold partially. The resulting
image, via an electron microscope, is "beads on a string". The string is
the DNA, while each bead in the nucleosome is a core particle. The
nucleosome core particle is composed of DNA and histone proteins.
Partial DNAse digestion of chromatin
reveals its nucleosome structure. Because DNA portions of nucleosome
core particles are less accessible for DNAse than linking sections, DNA
gets digested into fragments of lengths equal to multiplicity of
distance between nucleosomes (180, 360, 540 base pairs etc.). Hence a
very characteristic pattern similar to a ladder is visible during gel electrophoresis of that DNA. Such digestion can occur also under natural conditions during apoptosis ("cell suicide" or programmed cell death), because autodestruction of DNA typically is its role.
Protein interactions within the nucleosome
The
core histone proteins contains a characteristic structural motif termed
the "histone fold", which consists of three alpha-helices (α1-3)
separated by two loops (L1-2). In solution, the histones form H2A-H2B
heterodimers and H3-H4 heterotetramers. Histones dimerise about their
long α2 helices in an anti-parallel orientation, and, in the case of H3
and H4, two such dimers form a 4-helix bundle stabilised by extensive
H3-H3' interaction. The H2A/H2B dimer binds onto the H3/H4 tetramer due
to interactions between H4 and H2B, which include the formation of a
hydrophobic cluster.
The histone octamer is formed by a central H3/H4 tetramer sandwiched
between two H2A/H2B dimers. Due to the highly basic charge of all four
core histones, the histone octamer is stable only in the presence of DNA
or very high salt concentrations.
Histone - DNA interactions
The nucleosome contains over 120 direct protein-DNA interactions and several hundred water-mediated ones.
Direct protein - DNA interactions are not spread evenly about the
octamer surface but rather located at discrete sites. These are due to
the formation of two types of DNA binding sites within the octamer; the
α1α1 site, which uses the α1 helix from two adjacent histones, and the
L1L2 site formed by the L1 and L2 loops. Salt links and hydrogen bonding
between both side-chain basic and hydroxyl groups and main-chain amides
with the DNA backbone phosphates form the bulk of interactions with the
DNA. This is important, given that the ubiquitous distribution of
nucleosomes along genomes requires it to be a non-sequence-specific
DNA-binding factor. Although nucleosomes tend to prefer some DNA
sequences over others,
they are capable of binding practically to any sequence, which is
thought to be due to the flexibility in the formation of these
water-mediated interactions. In addition, non-polar interactions are
made between protein side-chains and the deoxyribose groups, and an
arginine side-chain intercalates into the DNA minor groove at all 14
sites where it faces the octamer surface.
The distribution and strength of DNA-binding sites about the octamer
surface distorts the DNA within the nucleosome core. The DNA is
non-uniformly bent and also contains twist defects. The twist of free
B-form DNA in solution is 10.5 bp per turn. However, the overall twist
of nucleosomal DNA is only 10.2 bp per turn, varying from a value of 9.4
to 10.9 bp per turn.
Histone tail domains
The
histone tail extensions constitute up to 30% by mass of histones, but
are not visible in the crystal structures of nucleosomes due to their
high intrinsic flexibility, and have been thought to be largely
unstructured.
The N-terminal tails of histones H3 and H2B pass through a channel
formed by the minor grooves of the two DNA strands, protruding from the
DNA every 20 bp. The N-terminal
tail of histone H4, on the other hand, has a region of highly basic
amino acids (16–25), which, in the crystal structure, forms an
interaction with the highly acidic surface region of a H2A-H2B dimer of
another nucleosome, being potentially relevant for the higher-order
structure of nucleosomes. This interaction is thought to occur under
physiological conditions also, and suggests that acetylation of the H4 tail distorts the higher-order structure of chromatin.
Higher order structure
The current chromatin compaction model
The organization of the DNA that is achieved by the nucleosome cannot
fully explain the packaging of DNA observed in the cell nucleus.
Further compaction of chromatin into the cell nucleus is necessary, but it is not yet well understood. The current understanding is that repeating nucleosomes with intervening "linker" DNA form a 10-nm-fiber, described as "beads on a string", and have a packing ratio of about five to ten. A chain of nucleosomes can be arranged in a 30 nm fiber, a compacted structure with a packing ratio of ~50 and whose formation is dependent on the presence of the H1 histone.
A crystal structure of a tetranucleosome has been presented and
used to build up a proposed structure of the 30 nm fiber as a two-start
helix.
There is still a certain amount of contention regarding this model, as it is incompatible with recent electron microscopy data.
Beyond this, the structure of chromatin is poorly understood, but it
is classically suggested that the 30 nm fiber is arranged into loops
along a central protein scaffold to form transcriptionally active euchromatin. Further compaction leads to transcriptionally inactive heterochromatin.
Dynamics
Although
the nucleosome is a very stable protein-DNA complex, it is not static
and has been shown to undergo a number of different structural
re-arrangements including nucleosome sliding and DNA site exposure.
Depending on the context, nucleosomes can inhibit or facilitate
transcription factor binding. Nucleosome positions are controlled by
three major contributions: First, the intrinsic binding affinity of the
histone octamer depends on the DNA sequence. Second, the nucleosome can
be displaced or recruited by the competitive or cooperative binding of other protein factors. Third, the nucleosome may be actively translocated by ATP-dependent remodeling complexes.
Nucleosome sliding
When
incubated thermally, nucleosomes reconstituted onto the 5S DNA
positioning sequence were able to reposition themselves translationally
onto adjacent sequences.
This repositioning does not require disruption of the histone octamer
but is consistent with nucleosomes being able to "slide" along the DNA in cis. CTCF
binding sites act as nucleosome positioning anchors so that, when used
to align various genomic signals, multiple flanking nucleosomes can be
readily identified.
Although nucleosomes are intrinsically mobile, eukaryotes have evolved a
large family of ATP-dependent chromatin remodelling enzymes to alter
chromatin structure, many of which do so via nucleosome sliding.
Nucleosome sliding is one of the possible mechanism for large scale
tissue specific expression of genes. The transcription start site for
genes expressed in a particular tissue, are nucleosome depleted while,
the same set of genes in other tissue where they are not expressed, are
nucleosome bound.
DNA site exposure
Nucleosomal
DNA is in equilibrium between a wrapped and unwrapped state. DNA within
the nucleosome remains fully wrapped for only 250 ms before it is
unwrapped for 10-50 ms and then rapidly rewrapped, as measured using
time-resolved FRET.
This implies that DNA does not need to be actively dissociated from the
nucleosome but that there is a significant fraction of time during
which it is fully accessible. Introducing a DNA-binding sequence within
the nucleosome increases the accessibility of adjacent regions of DNA
when bound.
This propensity for DNA within the nucleosome to "breathe" has
important functional consequences for all DNA-binding proteins that
operate in a chromatin environment. In particular, the dynamic breathing of nucleosomes plays an important role in restricting the advancement of RNA polymerase II during transcription elongation.
Nucleosome free region
Promoters
of active genes have nucleosome free regions (NFR). This allows for
promoter DNA accessibility to various proteins, such as transcription
factors. Nucleosome free region typically spans for 200 nucleotides in S. cerevisiae
Well-positioned nucleosomes form boundaries of NFR. These nucleosomes
are called +1-nucleosome and −1-nucleosome and are located at canonical
distances downstream and upstream, respectively, from transcription
start site. +1-nucleosome and several downstream nucleosomes also tend to incorporate H2A.Z histone variant.
Modulating nucleosome structure
Eukaryotic
genomes are ubiquitously associated into chromatin; however, cells must
spatially and temporally regulate specific loci independently of bulk
chromatin. In order to achieve the high level of control required to
co-ordinate nuclear processes such as DNA replication, repair, and
transcription, cells have developed a variety of means to locally and
specifically modulate chromatin structure and function. This can involve
covalent modification of histones, the incorporation of histone
variants, and non-covalent remodelling by ATP-dependent remodeling
enzymes.
Histone post-translational modifications
Histone tails and their function in chromatin formation
Since they were discovered in the mid-1960s, histone modifications have been predicted to affect transcription.
The fact that most of the early post-translational modifications found
were concentrated within the tail extensions that protrude from the
nucleosome core lead to two main theories regarding the mechanism of
histone modification. The first of the theories suggested that they may
affect electrostatic interactions between the histone tails and DNA to
"loosen" chromatin structure. Later it was proposed that combinations of
these modifications may create binding epitopes with which to recruit
other proteins.
Recently, given that more modifications have been found in the
structured regions of histones, it has been put forward that these
modifications may affect histone-DNA and histone-histone
interactions within the nucleosome core. Modifications (such as
acetylation or phosphorylation) that lower the charge of the globular
histone core are predicted to "loosen" core-DNA association; the
strength of the effect depends on location of the modification within
the core.
Some modifications have been shown to be correlated with gene silencing;
others seem to be correlated with gene activation. Common modifications
include acetylation, methylation, or ubiquitination of lysine; methylation of arginine; and phosphorylation of serine. The information stored in this way is considered epigenetic,
since it is not encoded in the DNA but is still inherited to daughter
cells. The maintenance of a repressed or activated status of a gene is
often necessary for cellular differentiation.
Histone variants
Although
histones are remarkably conserved throughout evolution, several variant
forms have been identified. This diversification of histone function is
restricted to H2A and H3, with H2B and H4 being mostly invariant. H2A
can be replaced by H2AZ (which leads to reduced nucleosome stability) or H2AX (which is associated with DNA repair and T cell differentiation), whereas the inactive X chromosomes
in mammals are enriched in macroH2A. H3 can be replaced by H3.3 (which
correlates with activate genes and regulatory elements) and in centromeres H3 is replaced by CENPA.
ATP-dependent nucleosome remodeling
A number of distinct reactions are associated with the term ATP-dependent chromatin remodeling. Remodeling enzymes have been shown to slide nucleosomes along DNA, disrupt histone-DNA contacts to the extent of destabilizing the H2A/H2B dimer and to generate negative superhelical torsion in DNA and chromatin. Recently, the Swr1 remodeling enzyme has been shown to introduce the variant histone H2A.Z into nucleosomes.
At present, it is not clear if all of these represent distinct
reactions or merely alternative outcomes of a common mechanism. What is
shared between all, and indeed the hallmark of ATP-dependent chromatin
remodeling, is that they all result in altered DNA accessibility.
Studies looking at gene activation in vivo and, more astonishingly, remodeling in vitro
have revealed that chromatin remodeling events and transcription-factor
binding are cyclical and periodic in nature. While the consequences of
this for the reaction mechanism of chromatin remodeling are not known,
the dynamic nature of the system may allow it to respond faster to
external stimuli. A recent study indicates that nucleosome positions
change significantly during mouse embryonic stem cell development, and
these changes are related to binding of developmental transcription
factors.
Dynamic nucleosome remodelling across the Yeast genome
Studies in 2007 have catalogued nucleosome positions in yeast and shown that nucleosomes are depleted in promoter regions and origins of replication.
About 80% of the yeast genome appears to be covered by nucleosomes and the pattern of nucleosome positioning clearly relates to DNA regions that regulate transcription, regions that are transcribed and regions that initiate DNA replication. Most recently, a new study examined dynamic changes
in nucleosome repositioning during a global transcriptional
reprogramming event to elucidate the effects on nucleosome displacement
during genome-wide transcriptional changes in yeast (Saccharomyces cerevisiae). The results suggested that nucleosomes that were localized to promoter regions are displaced in response to stress (like heat shock).
In addition, the removal of nucleosomes usually corresponded to
transcriptional activation and the replacement of nucleosomes usually
corresponded to transcriptional repression, presumably because transcription factor
binding sites became more or less accessible, respectively. In general,
only one or two nucleosomes were repositioned at the promoter to effect
these transcriptional changes. However, even in chromosomal regions
that were not associated with transcriptional changes, nucleosome
repositioning was observed, suggesting that the covering and uncovering
of transcriptional DNA does not necessarily produce a transcriptional
event. After transcription, the rDNA region has to protected from any
damage, it suggested HMGB proteins play a major role in protecting the
nucleosome free region.
DNA Twist Defects
DNA
twist defects are when the addition of one or a few base pairs from one
DNA segment are transferred to the next segment resulting in a change
of the DNA twist. This will not only change the twist of the DNA but it
will also change the length.
This twist defect eventually moves around the nucleosome through the
transferring of the base pair, this means DNA twists can cause
nucleosome sliding.
Nucleosome crystal structures have shown that superhelix location 2 and
5 on the nucleosome are commonly found to be where DNA twist defects
occur as these are common remodeler binding sites.
There are a variety of chromatin remodelers but all share the existence
of an ATPase motor which facilitates chromatin sliding on DNA through
the binding and hydrolysis of ATP.
ATPase has an open and closed state. When the ATPase motor is changing
from open and closed states, the DNA duplex changes geometry and
exhibits base pair tilting.
The initiation of the twist defects via the ATPase motor causes tension
to accumulate around the remodeler site. The tension is released when
the sliding of DNA has been completed throughout the nucleosome via the
spread of two twist defects (one on each strand) in opposite directions.
Nucleosome assembly in vitro
Diagram of nucleosome assembly
Nucleosomes can be assembled in vitro by either using purified native or recombinant histones.One standard technique of loading the DNA around the histones involves the use of salt dialysis.
A reaction consisting of the histone octamers and a naked DNA template
can be incubated together at a salt concentration of 2 M. By steadily
decreasing the salt concentration, the DNA will equilibrate to a
position where it is wrapped around the histone octamers, forming
nucleosomes. In appropriate conditions, this reconstitution process
allows for the nucleosome positioning affinity of a given sequence to be
mapped experimentally.
Disulfide crosslinked nucleosome core particles
A recent advance in the production of nucleosome core particles with enhanced stability involves site-specific disulfide crosslinks. Two different crosslinks can be introduced into the nucleosome core particle. A first one crosslinks the two copies of H2A via an introduced cysteine (N38C) resulting in histone octamer
which is stable against H2A/H2B dimer loss during nucleosome
reconstitution. A second crosslink can be introduced between the H3
N-terminal histone tail and the nucleosome DNA ends via an incorporated
convertible nucleotide.
The DNA-histone octamer crosslink stabilizes the nucleosome core
particle against DNA dissociation at very low particle concentrations
and at elevated salt concentrations.
Nucleosome assembly in vivo
Steps in nucleosome assembly
Nucleosomes are the basic packing unit of genomic DNA built from
histone proteins around which DNA is coiled. They serve as a scaffold
for formation of higher order chromatin structure as well as for a layer
of regulatory control of gene expression. Nucleosomes are quickly
assembled onto newly synthesized DNA behind the replication fork.
H3 and H4
Histones H3 and H4 from disassembled old nucleosomes are kept in the vicinity and randomly distributed on the newly synthesized DNA. They are assembled by the chromatin assembly factor 1 (CAF-1) complex, which consists of three subunits (p150, p60, and p48).
Newly synthesized H3 and H4 are assembled by the replication coupling
assembly factor (RCAF). RCAF contains the subunit Asf1, which binds to
newly synthesized H3 and H4 proteins.
The old H3 and H4 proteins retain their chemical modifications which
contributes to the passing down of the epigenetic signature. The newly
synthesized H3 and H4 proteins are gradually acetylated at different
lysine residues as part of the chromatin maturation process.
It is also thought that the old H3 and H4 proteins in the new
nucleosomes recruit histone modifying enzymes that mark the new
histones, contributing to epigenetic memory.
H2A and H2B
In contrast to old H3 and H4, the old H2A and H2B
histone proteins are released and degraded; therefore, newly assembled
H2A and H2B proteins are incorporated into new nucleosomes.
H2A and H2B are assembled into dimers which are then loaded onto
nucleosomes by the nucleosome assembly protein-1 (NAP-1) which also
assists with nucleosome sliding.
The nucleosomes are also spaced by ATP-dependent nucleosome-remodeling
complexes containing enzymes such as Isw1 Ino80, and Chd1, and
subsequently assembled into higher order structure.
Histone H1 is one of the five main histoneprotein families which are components of chromatin in eukaryotic cells. Though highly conserved, it is nevertheless the most variable histone in sequence across species.
Structure
A diagram showing where H1 can be found in the nucleosome
Metazoan H1 proteins feature a central globular "winged helix" domain and long C- and short N-terminal
tails. H1 is involved with the packing of the "beads on a string"
sub-structures into a high order structure, whose details have not yet
been solved. H1 found in protists and bacteria, otherwise known as nucleoproteins HC1 and HC2 (PfamPF07432, PF07382), lack the central domain and the N-terminal tail.
H1 is less conserved than core histones. The globular domain is the most conserved part of H1.
Function
Unlike the other histones, H1 does not make up the nucleosome
"bead". Instead, it sits on top of the structure, keeping in place the
DNA that has wrapped around the nucleosome. H1 is present in half the
amount of the other four histones, which contribute two molecules to
each nucleosome bead. In addition to binding to the nucleosome, the H1
protein binds to the "linker DNA" (approximately 20-80 nucleotides in
length) region between nucleosomes, helping stabilize the zig-zagged
30 nm chromatin fiber. Much has been learned about histone H1 from studies on purified chromatin
fibers. Ionic extraction of linker histones from native or
reconstituted chromatin promotes its unfolding under hypotonic
conditions from fibers of 30 nm width to beads-on-a-string nucleosome
arrays.
It is uncertain whether H1 promotes a solenoid-like
chromatin fiber, in which exposed linker DNA is shortened, or whether
it merely promotes a change in the angle of adjacent nucleosomes,
without affecting linker length However, linker histones have been demonstrated to drive the compaction of chromatin fibres that had been reconstituted in vitro using synthetic DNA arrays of the strong '601' nucleosome positioning element.
Nuclease digestion and DNA footprinting experiments suggest that the
globular domain of histone H1 localizes near the nucleosome dyad, where
it protects approximately 15-30 base pairs of additional DNA. In addition, experiments on reconstituted chromatin reveal a characteristic stem motif at the dyad in the presence of H1.
Despite gaps in our understanding, a general model has emerged wherein
H1's globular domain closes the nucleosome by crosslinking incoming and
outgoing DNA, while the tail binds to linker DNA and neutralizes its
negative charge.
Many experiments addressing H1 function have been performed on
purified, processed chromatin under low-salt conditions, but H1's role
in vivo is less certain. Cellular studies have shown that overexpression
of H1 can cause aberrant nuclear morphology and chromatin structure,
and that H1 can serve as both a positive and negative regulator of
transcription, depending on the gene. In Xenopus
egg extracts, linker histone depletion causes ~2-fold lengthwise
extension of mitotic chromosomes, while overexpression causes
chromosomes to hypercompact into an inseparable mass.
Complete knockout of H1 in vivo has not been achieved in multicellular
organisms due to the existence of multiple isoforms that may be present
in several gene clusters, but various linker histone isoforms have been
depleted to varying degrees in Tetrahymena,
C. elegans, Arabidopsis, fruit fly, and mouse, resulting in various
organism-specific defects in nuclear morphology, chromatin structure,
DNA methylation, and/or specific gene expression.
Dynamics
While
most histone H1 in the nucleus is bound to chromatin, H1 molecules
shuttle between chromatin regions at a fairly high rate.
It is difficult to understand how such a dynamic protein could be
a structural component of chromatin, but it has been suggested that the
steady-state equilibrium within the nucleus still strongly favors
association between H1 and chromatin, meaning that despite its dynamics,
the vast majority of H1 at any given timepoint is chromatin bound.
H1 compacts and stabilizes DNA under force and during chromatin
assembly, which suggests that dynamic binding of H1 may provide
protection for DNA in situations where nucleosomes need to be removed.
Cytoplasmic factors appear to be necessary for the dynamic
exchange of histone H1 on chromatin, but these have yet to be
specifically identified.
H1 dynamics may be mediated to some degree by O-glycosylation and
phosphorylation. O-glycosylation of H1 may promote chromatin
condensation and compaction. Phosphorylation during interphase has been
shown to decrease H1 affinity for chromatin and may promote chromatin
decondensation and active transcription. However, during mitosis
phosphorylation has been shown to increase the affinity of H1 for
chromosomes and therefore promote mitotic chromosome condensation.
The H1 family in animals includes multiple H1 isoforms that can be
expressed in different or overlapping tissues and developmental stages
within a single organism. The reason for these multiple isoforms remains
unclear, but both their evolutionary conservation from sea urchin to
humans as well as significant differences in their amino acid sequences
suggest that they are not functionally equivalent. One isoform is histone H5, which is only found in avian erythrocytes, which are unlike mammalian erythrocytes in that they have nuclei. Another isoform is the oocyte/zygotic
H1M isoform (also known as B4 or H1foo), found in sea urchins, frogs,
mice, and humans, which is replaced in the embryo by somatic isoforms
H1A-E, and H10 which resembles H5. Despite having more negative charges than somatic isoforms, H1M binds with higher affinity to mitotic chromosomes in Xenopus egg extracts.
Post-translational modifications
Like
other histones, the histone H1 family is extensively
post-translationally modified (PTMs). This includes serine and threonine
phosphorylation, lysine acetylation, lysine methylation and
ubiquitination. These PTMs serve a variety of functions but are less well studied than the PTMs of other histones.
