Chromatin structure and organization

•What would be the consequence of replicating DNA and assembling all new nucleosomes? What would be the consequence of one daughter molecule inheriting all the parental molecule's histones?

"Without having some of those old histones incorporated into the new nucleosomes, you will lose the epigenetic marks that recruit modifying enzymes. You lose all those patterns of gene expression at the epigenetic level." *** epigenetics involves passing on chromatin modification and DNA modification A daughter molecule inheriting all parental molecule histones would 100% inherit the epigenetic marks. Modified old histones that are passed onto the daughter strand recruit enzymes that will modify neighboring new histones in the same way.

______ and _______changes charge

- Acetylation - Phosphorylation

Chromatin remodeling complexes include a subunit that _______ ATP into ADP+P. Each time this happens, the DNA is moves by how many base pairs?

- Hydrolyzes - One bp

Chromatin Remodeling complexes are _______ (small/large) complexes with _______ (little/many) subunits. They require an energy input of ________

- Large - Many - ATP

Histone modification is a _____ _______ modification. It involves adding functional groups to

- Posttranslational - N-terminus tails sticking out of nucleosomes. Tails are accessible to be modified.

What two big factors influence inherited patterns of gene expression (epigenetics)?

- histones and their modifications - DNA modifications (adding functional groups to nitrogenous bases)

Why is DNA packed into chromosomes? (3)

- space - Management - protection

What are the two types of histones?

1. Core histones 2. Linker histones

•Describe different levels of chromatin compactions and the protein and factors that are important for each level.

1. Double stranded DNA. 2. 10 nm fiber. 2(H3+H4) + DNA + (H2A +H2B). DNA wraps around the H3/H4 tetramer. Then the H2A/H2B dimer joins. You get nucleosomes with linkage DNA in between. 3. 30 nm fiber. Histone tails and H1 protein aid in compacting the chromatin fiber further. 4. 30nm fiber gets wrapped up in coils. Compaction more than 30 nm requires scaffold proteins. Structural maintenance chromosomes (nonhistone proteins) allow chromatin loops to build into more complex structure. Topoisomerase II relaxes and rearranges topology. 4. Loops coil even further, forming a high order chromosome (metaphase chromosome)

What specialized sites are required in DNA to produce a Eukaryotic chromosome that can be replicated and segregated properly during mitosis?

1. Origin of replication 2. Telomere 3. Centromere

Chromatin remodeling complexes: What are three mechanisms?

1. They can slide the nucleosome more apart from each other with the unput of ATP. It is hydrolyzed to ADP + P 2. Nucleosome gets ejected out to make the sequence available. 3. Variants of a dimer histone (like H2A H2B variants) replace a normal histone protein to change how loose the DNA is.

1. No centromere would lead to.... 2. One centromere would lead to... 3. Two centromeres would lead to...

1. none = random segregation of chromosome. 2. One chromosome for each cell. 3. Chromosome breakage

Each nucleosome core particle is found to consist of how many histone proteins?

8 histone proteins with two molecules each

Chromatin is made of

A complex of histone and nonhistone proteins with nuclear DNA of the eukaryotic cells

What is CENP-A?

A modified H3 variant that designates where the centromere is and allows for the centromere to attach to the kinetochore proteins required for sister chromatid separation

Centromere

A specialized DNA sequence that allows one copy of each duplicated and condensed chromosome to be pulled into each daughter cell as the cell divides.

_________ results in loose packing of nucleotides by removing positive charges. Creates neutral charge

Acetylation

•Explain why acetylated histone tails would make DNA associated with those nucleosomes more accessible.

Acylation neutralizes a positive charge between the histone (+) and DNA (-), making the DNA more accessible.

Chromatin remodeling complex

An ATP dependent protein that catalyzes nucleosome sliding (makes the DNA less tightly bound to the histone core).

Heterochromatin condensation through HP1 protein can spread along the chromosome until it encounters a ________. It allows distinct areas of euchromatin and heterochromatin next to each other.

Boundary element - Where nonhistone proteins are bound to the DNA.

______ delivers H3H4 tetramers to newly synthesized DNA.

CAF-I

•What would happen if CENP-A was incorporated into nucleosomes at the wrong places chromosomes?

CENP-A binds to sequence specific regions that are designated as a "centromere region". CENP-A forms a tetramer with H4 (it is similar to H3 but structurally different.) Then a kinetochore protein will bind to the tail of CENP-A to form a complex. If CENP-A is incorporated in the wrong place of the chromosome, the kinetochore complex will form at the wrong portion of the chromosome. That means that the centromere will be at the wrong place. The spindle fibers will connect to sister chromatids at the misplaced centromere and pull them apart during anaphase in a way that cells have unequal distribution of chromosomes.

Direct formation of the kinetochore complex, which is the attachment point for spindle microtubules to separate duplicated chromosomes.

Centromeres

Epigenetics

Control mechanisms beyond the gene's sequence. Inherited patterns of gene expression. -Differentiation of cell types and maintaining through CDC (cell division cycle)

____ DNA is tightly associated with the histone protein

Core

Nucleosome assembly: H2A and H2B form a ________. Two these dimers are recruited to the complex to complete the nucleosome.

Dimer

How many N-terminal tails should you expect to find in the final histone octamer? What are their functions?

Eight. They are highly flexible and serve as binding sites for other proteins. They get different post-translational modification which can regulate assembly.

How are eukaryotic chromosomes different from prokaryotic chromosomes?

Eukaryotic chromosomes are linear. They are 50% DNA, 50% proteins. Prokaryotic chromosomes are circular. They have proteins too but they are different in structure.

Which histones are the linker histone? (not associated with the spool DNA)

H1

Which histones are core histones?

H2A H2B H3 H4

a class of enzyme that add acetyl groups

Histone Acetyltransferase

All four histones that make up the core of the nucleosome (H2A, H2B, H3,H4) share a structural motif called the ________ _______, formed from three alpha helices connected by two loops

Histone Fold

In assembling a nucleosome, the histone folds first bind to each other to form H3-H4 and H2A-H2B dimers. The H3-H4 dimers combine to form tetramer. An H3-H4 tetramer then further combines with two H2A-H2B dimers to form a compact _______ _______, the core around which the DNA is wound.

Histone Octamer

A class of enzyme that adds a negatively charged phosphate group to Ser or Thr

Histone Phosphorylase

A class of enzyme that removes acetyl groups

Histone deacetylase

•You want to study the potential interaction between nucleosome-bound DNA and a specific histone deacetylase. You decide to perform an electrophoretic mobility shift assay (EMSA). You use a 32P end-labeled, linear template DNA that contains two nucleosome positioning sites. You assemble two nucleosomes on the DNA template before incubation with and without the histone deacetylase. For some reactions, you use unmodified nucleosomes. For other reactions, you use nucleosomes that are methylated at lysine 36 of the histone protein H3. Based on the data below, propose a model for the interaction between the histone deacetylase and nucleosome-bound DNA. What type of protein domain do you predict allows the histone deacetylase to interact with the nucleosomes?

Histone deacetylase removes acyl groups, which silences transcription. Methylation is also associated with silencing transcription. In this case, you want to methylate the chromatin and then deacetylate it to make it even more compact. In this electrophoresis, DNA is still associated with its protein (Normally you strip all proteins and end up with negative DNA). Bands that are less associated with proteins will migrate further. We are essentially separating DNA protein complexes in the gel.

A class of enzyme that removes methyl groups from lysine and arginine from H3/H4

Histone demethylase

A class of enzyme that adds methyl groups to lysine and arginine from H3/H4

Histone methyltransferase

•Describe why histones are basic proteins.

Histones have a positive charge and DNA has a negative charge. The histones are able to neutralize some of the negative charge. It prevents the two DNA strands wrapped around the histone protein from repulsing each other. Lysine and Arginine (basic amino acids are abundant in histone proteins.

•Stem cells can be induced from differentiated cells. What do you think has to happen to to chromatin modifications in the process of de-differentiation?

Imagine a stem cell that can differentiate into cell type "a" or cell type "b". They both express a different set of proteins that allows them to become specialized. Epigenetic modifications allow this to occur. A differentiated cell can revert back into a stem cell only if epigenetic moderations are reversed. Epigenetic markers are removed. Maybe a gene that allowed the cell to identify as a stem cell was silenced and needs to be turned back on. Maybe methylation take place.

•Name the types of bonds that occur between histone proteins and DNA and the region of DNA where these bonds form. Are these interactions sequence-specific? Explain why or why not.

Interactions are not sequence specific. Hydrogen bonding occurs between the minor groove of DNA and histone octamer. DNA is (-) due to phosphate backbone while histone proteins are (+) due to basic amino acids like lysine and arginine.

A protein called the _______ forms at the centromere and attaches the duplicated chromosomes to the mitotic spindle in a matter that causes the two sister chromatids to be pulled apart.

Kinetochore

DNA between nucleosomes

Linker DNA

______, _______, and _______ groups are examples of functional groups that get added to or taken off of the N-terminus tails that stick out from nucleosomes during post-translational modification.

Methyl, Acetyl, Phosphate

_________ induces tighter wrapping of DNA around the histone core, making genes inaccessible.

Methylation

Why do histone modifications and DNA modifications matter for gene expression/ which genes are turned on or off?

Modifications on histone and DNA change how accessible the promoter is to transcriptional machinery. Nucleosomes are repositioned to allow accessibility.

Witan a histone octamer, each of the core histones contain an ______ _______ tail, which is subject to several formal of covalent modification and a histone fold region.

N-terminal tail

________ delivers H2aH2B dimers to a newly synthesized daughter strand.

NAP-I

Can nucleosomes self assemble?

No

Are parental histones evenly distributed in daughter DNA?

No, some are all new, some are all old. Some can be 1/4 old or 3/4 old.

The nucleosome core particles can be isolated by digesting chromatin with an enzyme called _______ that cut DNA. After digestion, the exposed linker DNA is degraded.

Nucleases

________ rings serve as markers for newly synthesized DNA

PCNA

Histones have a _____ charge while DNA has a ______ charge.

Positive Negative

What facilitates interaction of DNA with histone?

Sequence independent contacts. There are 14 different sites of contact. One exists for every time a minor groove faces the octamer. - Interactions between the DNA backbone and the minor groove.

DNA binding proteins can lay down a pattern for where nucleosomes are located. They can ensure that a space is accessible at all times (preventing nucleosome from binding) or the DNA binding proteins can be locked into place to prevent nucleosome sliding, which would do what to a gene?

Silence a gene

Origin of replication

Site where the replication of a DNA molecule begins, consisting of a specific sequence of nucleotides. - Eukaryotic cells can have many origins of replication

Repeated sequences at the end of linear chromosomes that is the solution to a replication issue with linear chromosomes.

Telomeres

______ are at the ends of each chromosome. They contain repeated nucleotide sequences that enable the ends of the chromosomes to be efficiently replicated.

Telomeres

Nucleosome assembly: H3 and H4 form a dimer. The dimers combine to form _______

Tetramer

What are chromosomes?

The genetic material that gets passed down from one generation to the next via mitosis and meiosis.

Telomere function

The repeated telomere DNA sequences form structures that protect the end of the chromosome from being mistaken by the cell for a broken DNA molecule in need of repair.

Why do histone proteins have a high percentage of basic amino acid residues like Lysine and Arginine (two amino acids with basic side chains)

Their positive charge neutralizes the negatively charged DNA backbone. - It allows virtually any part of DNA to be bound on a histone octamer core.

•Name the types of bonds that occur between histones and DNA.

There are 14 contact sites (every time the minor groove faces the histone octamer.) The negatively charged backbone of the DNA hydrogen bonds with positively charged/basic histones. This is not an ionic association. The main takeaway is that hydrogen bonding is occurring between the backbone and nitrogenous bases in DNA.

What are RNA genes?

They generate an RNA molecule instead of a protein.

Quiz: in assembling a nucleosome, _____ first form a tetramer which binds and bends the DNA, after which two ______ dimers join to complete the octamer of core histones a) H3 and H4 ; H2A-H2B b) H2A and H2B; H3-H4 c) H2 and H3 ; H1-H4 d) H1 and H2; H3-H4

a) H3 and H4; H2A-H2B

Which of the following statement(s) is/are true regarding histones? a) post-translational modification of histones occurs on their N-terminal tails. b) Histone N-terminal tails are unstructured regions of the protein. c) Histone variants incorporated into nucleosomes mark specialized regions on a chromosome. d) Histones are considered basic proteins because they have a simple globular structure. e) Histone H1 is part of the nucleosome core structure.

a,b,c are correct d - They have positively charged amino acid residues like Lysine and Arginine. e - Histone H1 is located outside of the core. Required for the 10 nm fiber to condense into the 30 nm fiber. Note: H3 variant CENP-A is included in nucleosomes around centromeric DNA.

•Address the general properties of the chromatin within the following types of DNA: -a. DNA undergoing active transcription -b. DNA within a mitotic chromosome -c. DNA immediately after passage of the replication fork -d. centromeric DNA

a. Chromatin is loose either through phosphorylation or acetylation of histone. b. Mitotic chromatin is highly condensed (heterochromatin). Limited gene expression. Telomers and centromeres present. c. Helicase unwinds segments of DNA and breaks hydrogen bonds so that synthesis of a new strand can occur. DNA will repackage afterwards. Nucleosomes begin to form with the help of histone octamer and H1 histone protein. The chromatin is somewhat loose (Euchromatin). d. Heterochromatin is found. Centromeric (and telomere) DNA is difficult to access. It is very compacted. Contains sequences where CENP-A (H3 variant) binds, allowing assembly of kinetochore.

Image you isolate a batch of 10nm fiber chromatin from nuclei and then digest that chromatin to completion with a non-specific DNase. You then remove ALL proteins from the DNA and separate out the digestion product by agarose gel electrophoresis. What kind of banding pattern would be expected on the gel? a) There would be a ladder banding pattern, with bands increasing by ~200bp per band up the gel. b) There would be one band, migrating at ~147bp. c) There would be no bands as the DNA was completely digested into nucleotides by the DNase. d) There would be one band, migrating at ~50bp.

b) There would be one band, migrating at ~147bp. The only DNA that survives full digestion is the core DNA that is wrapped around and protected by its association with the nucleosome. a - There would be a ladder pattern if a partial digestion occurred. Some linker DNA would still be included c - Implies that core DNA is also digested. d - implied that the linker DNA survives, and the core DNA is digested

H3 lysine 9 acetylation ______ a) adds a negative charge to histone tails b) removes a positive charge on histone tails c) adds a positive charge to histone tails d) is found in compact chromatin

b) removes a positive charge on histone tails Acetylation will loosen chromatin. Nothing adds a positive charge. Methylation is found in compact chromatin Phosphorylation adds a negative charge

A gene actively undergoing transcription _____. a) would have chromatin with H3K27 methylation. b) would have chromatin with H3K27 acetylation. c) would have the transcription start site located in the DNA wrapped around a nucleosome. d) would have chromatin compacted into the 30nm fiber.

b) would have chromatin with H3K27 acetylation - Transcription activation occurs when chromatin is loose. Acetylation is associated with euchromatin and active transcription. c- transcription start site is not accessible when wrapped around the nucleosome. d- 10nm state required for transcription machinery to pass through protein complex. Technically some parts of the 30nm fiber can be unraveled into 10 nm. a- methylation contributes to heterochromatin and silenced gene.

Hp1 is a ______ protein that contributes to heterochromatin condensation. It binds to histone H3 N-terminal tails trimethylated at lysine 9. (Methylation recruits the HP1 proteins).

heterochromatin

Methylation contributes to ___________ (heterochromatin/euchromatin) and ________ (silenced/activated) genes

heterochromatin; silenced

Histones are responsible for the most basic level of chromosome packaging called

nucleosome

A spool of protein around which DNA is wrapped in an ordered manner. It is wrapped around histone protein.

nucleosomes

______ are the basic unit of chromatin structure

nucleosomes

A histone ______ forms a protein core around which the double stranded DNA is wound

octamer

_______ confers a negative charge to the histone, resulting in a more open chromatin conformation because the negatively charged DNA backbones are no longer neutralized (causes repulsion)

phosphorylation

DNA binding proteins can help to ______ nucleosomes

position

Methylenation is often associated with ________ (repression/activation).

repression

Simply wrapping around the spool allows for ______ fold compactions

six fold

There are about _____ origins of replication per humane genome

~10,000

What type of bonds occur between DNA and the histone Octamer?

~40 hydrogen bonds; They are the driving force behind the bending.