Unit 10: Chromatin and Gene Regulation Flashcards
Nucleosome
the basic structural subunit of chromatin, containing about ~200 bp of DNA and an octamer of positively charged histone proteins
Histone tails
flexible amino- or carboxy- terminal regions of the core histones that extend beyond the surface of the nucleosome
-histone tails are sites of extensive post-translational modification
Fill in the blanks: The eukaryotic DNA is organized into ________, where the genetic material is organized in a hierarchical manner.
The eukaryotic DNA is organized into CHROMATIN, where the genetic material is organized in a hierarchical manner.
The basic structure unit of chromatin is called a ?
nucleosome
Fill in the blanks: Histones form the core of the _________, with the DNA wrapped around it lying on the surface.
Histones form the core of the NUCLEOSOME with the DNA wrapped around it lying on the surface.
10nm fiber
a linear array of nucleosomes generated by unfolding from the natural condition of chromatin
What are linker histones?
a family of histones (such as histone H1) that are not components of the nucleosome core but stabilize histone tails
-linker histones bind nucleosomes and/or linker DNA and promote 30nm fiber formation.
What is the organizational level of a nucleosome in order
1) The nucleosome
2) 10nm fiber coil to for a fiber w/30 nanometer diameter, which is a coil of chromosomes
3) this results further 40 fold compaction of the DNA - the structure of this fiber requires the histone tails and is stabilized by linker histones
4) the 30nm fiber is further compacted into interphase chromatin or miotic chromosomes
30nm fiber
a coil of nucleosomes
-it is the basic level of organization of nucleosomes in chromatin
what is a nonhistone?
are any structural proteins found in a chromosomes other than the histones.
Their functions include:
- gene expression
- gene control
- higher order structure
TRUE OR FALSE: DNA has twice as much protein as chromatin
FALSE:
the chromatin contains almost twice as much protein as DN
What is the role micrococcal nuclease (MNase)?
MNase clips/cleaves the region between nucleosomes called linker DNA.
What happens when linker DNA is cleaved?
-it releases individual nucleosomes
What do we see when chromatin is treated w/MNase and the produces are run on a gel?
-we see integral multiples of a unit length of about 200 base pairs. So fragment sizes would be 200 bp or multiples of 200.
Fill in the blank: More than ____% of DNA is recovered in nucleosomes or multimers following cleavage by MNase.
95%
what is the typical size of the DNA in a nucleosome?
about 200 basepairs but can vary between 154 and 260 basepairs. The average value may be different in different tissues in an organism or even between different parts of the genome w/in a single cell
Fill in the blanK: nucleosomal DNA is divided into two regions: the _____ DNA and the ________ DNA, depending on its susceptibility to MNase
nucleosomal DNA is divided into the CORE DNA and the LINKER DNA, depending on its susceptibility to MNase
What is the core DNA?
- part of the nucleosomal DNA that has a fixed length of 146 bp aand is found on the core particles produced by prolonged digestion w/MNase
- The core DNA reflects the minimum length of DNA needed to form a stable nucleosome and is resistant to digestion by nucleases
What is the linker DNA?
-part of the nucleosomal DNA. It is a region of 8 to 114 base pairs and is susceptible to early cleavage by nucleases.
What is the structure of a nucleosome and what are the core histone subunits of a nucleosome?
SUBUNITS (core histones): H2A H2B H3 H4
a nucleosome contains ~200 bp of DNA and two copies of each core histone forming an octamer,
What is the histone octamer structure?
the histone structure has a H3(ii) - H4(ii) tetramer associated w/two H2A-H2B dimers
TRUE OR FALSE: The nucleosome consists of approximately equal masses of DNA and histones.
TRUE
Why are histones positively charged? What does this charge help them bind to?
b/c they are rich in arginine and lysine. b/c of this they bind to DNA w/high affinity and are responsible for DNA packaging in eukaryotes.
Which histones are the most conserved proteins known?
H3 and H4
Fill in the blanks: the histone proteins in the nucleosome form this _____ shaped ________ with the DNA wrapping around the disk, making about a ________ turn around the core. DNA enters and exits on the ________ side of the nucleosome
the histone proteins in the nucleosome form this DISC shaped CYLINDER with the DNA wrapping around the disk making about a 1 and 2/3 turn around the core. DNA enters and exits on the SAME side of the nucleosome
When looking at the crystal structure of a histone octamer, what is observed about how the histone subunits interact w/each other
Tracing the paths of the individual polypeptide back bones in the crystal structure shows that the histones are not organized as individual globular proteins, but that each is interdigitated with its partner: H3 with H4, and H2A with H2
What is a histone fold?
it is part of the core histone. It is a structural motif that consists of two short helices flanking a longer alpha helix.
from book:
All four core histones show a similar type of structure in which three a-helices are connected by two loops. This highly conserved structure is called the histone fold
What terminal histone tails do the core histones have?
H2A, H2B, H3, and H4 all have the N-terminal histone tails that extend out of the nucleosome
H2A and H2B also have the C-terminal histone tails
The non histone H1 is associated with what?
with linker DNA and may lie at the point where DNA enters or exits the nucleosome
Why are the N and C-terminal tails not seen in the crystal structure of the histone?
b/c the histone tails are flexible regions that cannot be visualized by crystallography. Hence, the position of the tail w/respect to the nucleosome is not known
How are histones modified?
they are covalently modified by:
Methylation Acetylation Phosphorylation Ubiquitylation Sumosylation ADP-ribosylation
and other modifications
Modifications of histones most often occur on what part of the histone? And which is the most common residue that gets modified?
Modifications most often occur at the histone tails.
The residue that is most commonly modified is lysine. The modification usually occurs on the epsilon amino group and can be acetylated, methylated, ubiquitylated and sumoylated.
Where do phosphorylation modifications occur on histones?
on serene and threonine residues.
Which histone modifications are reversible?
all modifications are reversible. Some of these can be transient while other are more stable.
What is a histone code?
a histone code is when a combination of specific histone modifications define the function of local regions of chromatin
-These modifications are not restricted to a single histone. the functional state of a region of the chromosome may be affected by modifications on a set of nucleosomes. some modifications of a particular histone can affect other histone modification events. a change in the charge caused by the modification can even alter the structure of the chromatin
What is one example of a protein domain that binds to specific modifications on histone tails?
Bromodomain, which is found in a variety of proteins that interact w/chromatic.
Bromodomains recognize what types of sequences?
It recognizes acetylated lysines on histones and a short four-amino acid sequence, one of which is the acetylated
Which protein motifs recognize methyl lysines or arginines? What is unique about them?
- Chromodomains
- PHD domains
- Tudor domains
-they can distinguish between mono, di, or tri-methylated lysine.
Fill in the blank: Nucleosomes are ______ modified
COVALENTLY
TRUE OR FALSE: Histone modification at specific sites can determine the functional state of the chromatin
TRUE:
different histone modifications play roles in replication, chromatin assembly, transcription, DNA repair, and splicing.
Give an example of how modifications at specific sites on specific histones can determine the functional state of chromatin.
1) core histones that are newly synthesized have specific patterns of acetylation, which are removed after the histone is incorporated in the nucleosome and this activates the gene.
2) other modifications are added or removed thus regulating transcription, replication, or DNA repair
FILL IN THE BLANK: Histone variants have been identified for all core histones except ____. Histone variants can be closely related or highly divergent from ________ histones. These variants have different function and their incorporation into the ___________ can alter the nature of the chromatin.
1) H4
2) canonical histone
3) nucleosome
How are the major groove and minor groove of DNA structured on a nucleosome?
DNA major groove on nucleosomes is smoothly bent while the minor groove has some abrupt kinks.
Why is the structure of DNA altered on nucleosomes/
the structure of DNA is altered so that it has an increased number of base pairs/turns in the middle but decreased number at the ends. This makes the central part of DNA a non-target for DNA binding regulatory proteins which generally bind into the histone tails or linker sequence.
What is the difference between the nucleosome 10nm fiber and the 30nm fiber?
- The 10 nanometer fiber is a continuous string of nucleosomes and represents the lowest level of chromatin organizational hierarchy. It looks like a string of beads w/the linker DNA between the beads are nucleosomes
- in the 30nm fiber, the histone tails are involved in inter-nucleosomal contact and form two start helix consisting of two rows of nucleosomes coiled into a solenoid.
Under what conditions is the 10nm and 30nm fiber seen in a microsope?
- 10nm is visible in low ionic strenght and does not require the linker histone H1
- if ionic strength is increased the 30nm fiber is obtained. This does require the linker histone H1
TRUE OR FALSE: replication of chromatin requires assembly of nucleosomes
TRUE
Why are nucleosomes structure disrupted when replication occurs?
b/c the double strand has to separate into single strand. this disruption is limited to the vicinity of the replication fork. Progression of the fork disrupts the nucleosomes for a short time b/c the nucleosomes form very quickly on the daughter strands as the fork moves forward
How do histones associate with DNA to generate nucleosomes? Do the histones pre form a protein octamer around which the
DNA is subsequently wrapped? Or, does the histone octamer assemble on DNA from free histones?
Either of these pathways can be used in vitro to assemble nucleosomes, depending on the conditions that are employed. In one
pathway, a preformed octamer binds to DNA. In the other pathway, a tetramer of H32-H42 binds first, and then two H2A-H2B dimers are added. This latter stepwise assembly i s the pathway that is used in replication
Why does the assembly of nucleosomes require accessory proteins or molecular chaperones?
-accessory proteins/molecular chaperones are involved in assisting histones to associate with DNA. accessory proteins bind to the histones to release either individual or complexes (H3-H4, or H2A-H2B) to the
DNA in a controlled manner. This is could be necessary as histones, which are basin proteins, have high affinity for DNA. Such interactions allow histones to form nucleosomes w/o becoming trapped in other kinetic intermediates
What are CAF–1 and ASF1?
They are histone assembly proteins that are linked to the replication machinery and function at the replication fork;
CAF-1 is a chromatin assembly factor and is recruited to the replication fork by the proliferating cell nuclear antigen or PCMA, which is the processivity factor for DNA polymerase.
ASF1 is an anti-silencing factor and interacts w/the helicase that unwinds the replication fork.
What are HIRA and histone H3.3
they are different assembly proteins variants from CAF1 and ASF1 that are used for replication-independent assembly
Describe the assembly of nucleosomes.
1) The replication machinery displaces the histone octamers, which then associate into the H3(ii)-H4(ii) tetramenrs and the H2A-H2B dimers.
2) these tetramers and dimers can be reused in the synthesis of new nucleosomes.
4) they join the pool of newly synthesized H3(ii)-H4(ii) tetramers and H2A-H2B dimers.
5) The nucleosomes assembles 600 bp behind the replication fork
6) The tetramers bind first to each daughter strand helped by the histone assembly proteins CAF-1 and ASF1. This is followed by the binding of the H2A-H2B dimers
TRUE OR FALSE: During replication histone octamers, tetramers, and dimers are conserved
FALSE: the entire histone octamer is NOT conserved during replication but the octamer dissociates into the H3H4 tetramer and H2AH2B dimers which are conserved.
What is the replication coupled pathway?
-the assembly of chromatin from a mix of old and new histones during the S phase.
What is the replication independent pathway?
- a way for nucleosomes to assemble that occurs during other phases of the cell cycle (excluding the S, since S phase is replication coupled pathway)
- nucleosomes may be needed following DNA damage or if they are displaced during transcription. Since the pathways cannot be linked to replication machinery, the replication independent pathway uses the histone variant H3.3 and HIRA
nucleosomes can be assembled in which two pathways?
1) replication coupled pathway
2) replication independent pathway
Do nucleosomes lie at specific positions?
nucleosome may form at specific positions as the result of either the local structure of DNA or proteins that interact w/specific sequences
What is indirect end labeling?
a technique for examining the organization of DNA in order to determine if each side of the DNA is always located at the same position on the nucleosome. Thiis is done by making a cut at a specific site and identifying all fragments containing the sequence adjacent to one side of the cut.
-it reveals the distance from the cut to the next break(s) in DNA
What is a common cause of nucleosome positioning ? How does this affect which DNA positioning?
when proteins bind to DNA and establish a boundary
-positioning may affect which regions of DNA are in the linker and which face of DNA is exposed on the nucleosome surface
DNA sequence determinants (exclusion or preferential binding may be responsible for what?
half off the in vivo nucleosome positions
The location of the DNA on the nucleosome can be described as what two positions?
1) rotational
or
2) translational
what is rotational positioning?
- describes the location of DNA on the nucleosome.
- the location of the histone octamer is relative to the turns of the double helix and this determines which face of the DNA is exposed on the nucleosome surface.
- This is important b/c the site of the DNA that must be recognized by regulatory protein can be accessible or unavailable based on its position
What is translational positioning ?
described the linear position of the DNA relative to the histone octamer.
- The location of a histone octamer at successive turns of the double helix determines which sequences are located in the linker regions.
- this does not affect which face of DNA is protected by the histone and which is exposed.
TRUE OR FALSE: nucleosome are displaced and reassembled during transcription
true: DNA completes one and 2/3 turns around the histone core. so as the polymerase moves along the template one would expect that the polymerase has to engage with the segment on the surface of the nucleosome. This would mean a structural change.
in the case of transcription, in vitro the RNA polymerase displaces the octamer s which re associate with the DNA as soon as the polymerase has passed
TRUE OR FALSE: most transcribed genes retained the nucleosomal structure although some heavily transcribed genes appeared to be devoid of nucleosomes
TRUE
What is the difference in nucleosome displacement during transcription when dealing with a large RNA polymerase as compared to a small RNA polymerase?
in eukaryotes where the RNA polymerase is much larger, transcription through the nucleosome is not as simple as in a smaller polymerase. It requires additional factors that act on the chromatin such as ATP dependent chromatin remodelers that use the energy of ATP hydrolysis to move or displace nucleosomes. another factor that has been identified is fact FACT or facilitates chromatin transcription, which behaves as a transcription elongation factor. It is well conserved in all eukaryotes and is associated with the chromatin of active genes. It is thought that FACT is involved in the displacement of the octamers during transcription and may also be involved in assembly of the nucleosomes after transcription by acting as a histone chaperone.
What are hypersensitive sites?
regions in the chromatin that are susceptible to digestion by endonucleases such as DNAse 1. They are also found at the promoters of expressed genes and origins of replication and centromeres.
-hypersensitive sites are mostly found in chromatin of active or poised genes but not in inactive genes
How are hypersensitive sites generated?
by the binding of factors that exclude histone octamers
Why is DNA much more exposed in hypersensitive sites?
b/c this site may not be organized into the nucleosomal structure. Hence on these sites, histone octamers are absent
A domain containing a transcribed gene is defined by?
increase sensitivity to degradation by DNAse I
What are insulators?
a class of elements that block passage of any activating or inactivating effects from enhancers, silencers, and other control elements.
- insulators are specialized chromatin structures that typically contain hypersensitive sites
- insulators can also provide barriers agains the spread of hetero-chromatin - increasing the precision of gene regulation
What happens when an insulator is located between the enhancer and promoter?
the insulator prevents the enhancer from activating the promoter. This might explain why an enhancer can activate only particular promoter despite the fact that these enhancers have long range interactions and can activate promoters from a distance.
What happens when an insulator is situated between an active gene and hetero-chromatin?
it will protect the gene from getting inactivated by the heterochromatin in what is called the barrier effect.
*heterochromatin may spread from a center and block any promoters that it covers
TRUE OR FALSE: Insulators can either have the block effect or the barrier effect but not both
FALSE: certain insulators may have both the block and barrier effects, while others might have either on
What is the locus control regions (LCRs)?
LCRs are located at the 5’ end of a chromosomal domain and consist of multiple DNAse hypersensitive sites.
- LCRs regulate gene clusters and behaves like a super enhancer and has the entire locus poised for transcription
- LCRs usually regulate loci that show complex developmental or cell-type specific patters of gene expressions
Genes that lie w/in cluster of other genes are usually regulated by what?
by a single regulatory element that regulate transcription and
What is an example of regulated gene clusters?
-The mammalian beta globin gene
How do LCRs interact with promoters?
through a physical contact between LCR and the promoter through loop structures.
Describe chromosome conformation capture (3C technique)
1) one starts out by using formaldehyde treatment to crosslink to fix DNA and protein that are in close contact.
2) Next, treatment with restriction enzymes, will result in digestion of the chromatin.
3) This is followed by ligation under dilute conditions to encourage intramolecular ligation, resulting in the ligation of DNA fragments that are held in close proximity to each other due to the crosslinking.
4) The cross links are then reversed and the new ligate junctions can be detected by PCR or even B sequence
Individual nucleosomes can be obtained by treating chromatin with what endonuclease?
-MNase, which cuts the DNA between nucleosomes (cuts linker DNA)
Using the 3C technique researchers found what about the beta globin locus and LCR?
researchers found that in the beta globin locus, the beta globin LCR sequentially interacts with each globin gene depending on the developmental stage at which that gene is active.
Example: interactions in the fetal stage show LCR interacts with the 3’ hypersensitive site and the gamma genes.
The TH2 locus and the LCR interacts with all three of its target genes simultaneously.
the interaction is seen in all T cells whether the genes are expressed or not. but the precise organization of the loop changes when a specific transcription factor such as SATB 1 binds and activates the genes. Thus, the TH2 LCR’s has all the genes poised in the T cells, ready to initiate transcription as soon as the genes are activated by specific transcription factors
