MCB 252: Handout 7

What is a nucleosome? Describe its structure.

A nucleosome is the basic unit of chromatin structure. It is made up of an octameric core histone that has 2 nm DNA wrapped around it approximately twice. A nucleosome also includes the H1 linker histone that interacts with the DNA wrapped around the core histone to further compact it. It also includes the histone tails that extend from the core histone, and serve a role in the regulation of the level of chromatin condensation.

What are the two (general) domains of a transcription factor?

A transcription factor will have two major domains: a DNA binding domain and an activation domain. The DNA binding domain of a transcription factor will bind to the DNA at the binding site for that transcription factor. The activation domain of a transcription factor will bind to other factors to initiate/activate other functions (i.e. recruitment of other factors, complexes, etc.)

What role does acetylation play in modification of chromatin architecture?

Acetylation is one of the only epigenetic modifications that has a concrete correlation between extent of modification and level of chromatin condensation. Acetylation of lysine residues in histone tails is correlated with promoting chromatin decondensation. Deacetylation of lysine residues in histone tails is correlated with promoting chromatin condensation.

Describe how histone tail acetylation promotes chromatin decondensation.

Acetylation of histone tails promotes chromatin decondensation because: 1) it sterically inhibits interactions between histone tails and DNA; 2) it removes the positive charge in the histone tails that makes them attracted to the negatively charged DNA backbone, promotes an interaction; 3) it recruits bromodomain proteins that promote chromatin decondensation.

What is an enhancer? Function?

An enhancer is a region on a gene that helps to "fine-tune" transcriptional activity of that gene. Since the general transcriptional machinery at the promoter just provides very basic transcriptional control, it is the role of the enhancers to serve as binding sites for very specific transcription factors which will appropriately modify transcription of the associated gene.

What occurs when SWI/SNF or another chromatin remodeling complex has successfully unmasked the sequence of the regulatory promoter?

As SWI/SNF or another chromatin remodeling complex rearranges the chromatin to move nucleosomes away from the regulatory promoter, it allows specific transcription factors to bind to the regulatory promoter and to mediate assembly of the core transcriptional machinery by stabilizing the pre-initiation complex.

What are chromatin remodeling complexes dependent on?

Chromatin remodeling complexes are dependent on ATP hydrolysis to provide the necessary energy to rearrange nucleosomes. Because of this, every chromatin remodeling complex will have at least one ATPase subunit [In SWI/SNF complex, the ATPase is Swi2/Snf2]

What interaction do chromatin remodeling complexes change?

Chromatin remodeling complexes have to break the interaction between DNA and the core histone/histone tails, that is creating a strong nucleosome. By interfering with this interaction, chromatin remodeling complexes are able to move the nucleosomes.

Describe how histone tail deacetylation promotes chromatin condensation.

Deacetylation of histone tails promotes chromatin condensation because: 1) it returns the positive charge to lysine residues in the histone tails, promoting their interaction with the negatively charged DNA backbone.

Describe the structural differences between heterochromatin and euchromatin? How does this contribute to their functions?

Euchromatin is the much less condensed form of chromatin. It exists in a 10 nm fiber state, in which DNA is wrapped around core histone complexes to form nucleosomes. The less condensed state of euchromatin contributes to its role in containing genes that are being actively transcribed. Heterochromatin is the more condensed form of chromatin. It exists in a 30 nm fiber state and then in more complex, highly condensed states that we do not understand. Heterochromatin can be so tightly condensed because of the fact that the genes it is encoding are being stored/not actively transcribed.

What is Swi5? Its significance?

Every gene will have a different TF that recruits a chromatin remodeling complex to its transcriptional start site. In the case of the HO gene, the transcription factor Swi5 recruits SWI/SNF complex to the transcriptional start site.

Describe the general locations of heterochromatin versus euchromatin.

Heterochromatin is typically localized to the nuclear periphery, where it interacts with the nuclear lamina to anchor it and keep it highly organized. Since euchromatin is being actively transcribed, it is typically located more central within the nucleus.

What class of enzymes are responsible for histone tail acetylation?

Histone Acetyl Transferases (HATs) are responsible for acetylation of lysine residues in histone tails.

What class of enzymes are responsible for histone tail deacetylation?

Histone Deacetylases (HDACs) are responsible for deacetylation of lysine residues in histone tails.

Describe how histone tail methylation promotes chromatin condensation.

Histone tail methylation promotes chromatin condensation, as the methylated histone tails recruit chromodomain proteins. The recruited chromodomain proteins bind the methylated histone tails and interact with associated DNA, promoting chromatin condensation.

In general, how is the level of chromatin condensation controlled via histone tails?

Histone tails extending from the core histone (and the nucleosome) are subject to epigenetic modifications (i.e. phosphorylation, acetylation, methylation, ubiquitylation). Depending on the location(s) and type(s) of epigenetic modification(s), can promote chromatin condensation or decondensation.

What has to be assumed if a transcription factor binds to its DNA target sequence and initiates transcription?

If we are told that a transcription factor can bind its DNA target sequence and initiate transcription, we have to assume that it was able to access its target sequence. There are certainly ways to access a target sequence without the help of a chromatin remodeling complex, ie if the sequence is partially exposed at the top of a nucleosome or in the linker DNA sequence.

What do chromatin remodeling complexes do to chromatin?

It is really important to recognize that chromatin remodeling complexes (i.e. SWI/SNF complex) DO NOT condense or decondense chromatin. Chromatin remodeling complexes only rearrange the nucleosomes within chromatin to temporarily change the accessibility of certain areas. They are not sufficient to decondense chromatin.

How can TFs regulate transcription by interacting with HATs and HDACs?

Not all genes rely on chromatin remodeling complexes to initiate transcription by repositioning nucleosomes. In some situations, TFs can interact with HATs or HDACs to bring them directly to the regulatory promoter, where they can act on chromatin to promote decondensation or condensation, respectively.

How do nucleosomes pose a challenge to transcription?

Nucleosomes must be moved in or removed from the DNA in order to make sequences in the DNA available to be recognized by the pre-initiation complex and/or specific transcription factors. And then, even after transcription has been initiated, the nucleosomes have to be actively moved/removed as RNAPII processes down the DNA.

What maintains SWI/SNF at the chromatin of the gene it was initially recruited to?

SWI/SNF remains associated with the chromatin, because as it begins to remodel chromatin, it recruits a histone acetyltransferase (HAT), which hyperacetylates histone tails in that region. The hyperacetylated histone tails strongly attract SWI/SNF to the chromatin at that region, because it contains a bromodomain protein component (Swi2/Snf2).

What is the primary function of Swi2/Snf2?

Swi2/Snf2 encodes a protein that is a part of a major chromatin remodeling complex, the SWI/SNF complex. SWI2/SNF2 act as ATPases within the SWI/SNF complex, which allows them to hydrolyze ATP to produce energy for the remodeling of chromatin.

What is another characteristic of Swi2/Snf2 that results in its facilitation of chromatin decondensation?

Swi2/Snf2 is a bromodomain protein, meaning that it is recruited to hyperacetylated histone tails, where it carries out its decondensation actions on the chromatin.

Describe the mechanism of Swi5-mediated SWI/SNF recruitment.

Swi5 binds to its partially exposed DNA binding site upstream of the HO gene via its DNA binding domain. Binding of Swi5 to its binding site then causes recruitment of the SWI/SNF complex to the HO gene. Once at the HO gene, SWI/SNF begins to remodel chromatin by shifting nucleosome positioning in an ATP-dependent manner. As the chromatin is remodeled by SWI/SNF complex, a histone acetyltransferase (HAT) and SAGA complex get recruited to the gene, where they promote chromatin decondensation.

What is responsible for activating the ATPase function of Swi2/Snf2 (of the SWI/SNF complex)? Why?

The ATPase activity of Swi2/Snf2 is activated upon binding of the SWI/SNF complex to the DNA/chromatin. This makes sense, because the energy generated by the ATPase activity of Swi2/Snf2 is only needed once the SWI/SNF complex begins to remodel chromatin.

What class of protein complexes does the SWI/SNF complex belong to? What do they all have in common?

The SWI/SNF complex belongs to a class of protein complexes known as the "ATP-dependent chromatin remodeling complexes". The major conserved feature of protein complexes in this class of proteins is their use of an ATPase in their complex to provide energy for chromatin remodeling.

Describe the structure of the core histone.

The core histone is an octameric complex that is composed of 2 H3-H4 dimers and 2 H2A-H2B dimers. (4 sets of dimers, total). Additionally, the core histone has histone tails extending from each histone that are epigenetically modified to control levels of chromatin condensation.

What is a core promoter? Function?

The core promoter is the region on virtually all genes where the general transcriptional machinery assembles and then recruits RNAPII to. It does not provide specific control to transcription, and rather a much more general transcriptional control that can be modified by the functions of specific transcription factors.

What is the holoenzyme?

The holoenzyme is a way of referring to the assembled mediator and RNAPII complexes.

Describe the assembly of the pre-initiation complex. What is its role in transcriptional regulation of a particular gene?

The pre-initiation complex is formed when the mediator complex associates with the RNAPII complex to form the "holoenzyme". It is only once a holoenzyme has been assembled that the general transcription factors which have assembled at the gene's core promoter can recruit the holoenzyme to the core promoter to initiate transcription.

How do the roles of the pre-initiation complex and the transcription factors bound to enhancers overlap?

The transcription factors bound to enhancers play the role of interacting with the pre-initiation complex to assure that it was correctly assembled, and to stabilize the formation of the pre-initiation complex. TRANSCRIPTION FACTORS INTERACT WITH THE PRE-INITIATION COMPLEX AND INCREASE THE RATE OF PRE-INITIATION COMPLEX FORMATION.

What are the mechanisms of chromatin remodeling? Why is degradation of nucleosomes not a viable option?

There are two mechanisms of chromatin remodeling that both target nucleosomes. One mechanism is nucleosome sliding, in which the nucleosome is simply pushed away from the region that needs to be accessed. Another mechanism is nucleosome transfer, in which the nucleosome is removed from the chromatin, and is replaced into another region of chromatin. Degradation of nucleosomes is not a potential mechanism of chromatin remodeling, because there is not a constant source of nucleosomes. Nucleosomes are only produced by the cell in S phase, and therefore, degradation would deplete the cell's supply rapidly (think about how much chromatin remodeling gets done at all times in a cell)

Describe the elements that control transcription of a generic gene.

Transcription of a generic gene is controlled by a core promoter, which is where the general transcription machinery assembles, and enhancers, which are bound by more specific transcription factors to enhance the transcriptional activity of a gene.

Is there a relationship between nuclear location of chromatin and its transcriptional activity?

Typically, at the nuclear periphery, the chromatin is not as transcriptionally active. Chromatin more centrally located within the nucleus is more transcriptionally active.

Typically, what role does methylation of histone tails play in modification of chromatin architecture?

[DISCLAIMER: THIS IS NOT ALWAYS, BUT MOST OF THE TIME!] Methylation of histone tails leads to condensation of chromatin. Demethylation of histone tails leads to decondensation of chromatin.