Gene Expression II: Mechanisms of Regulation

Question 1

Explain the concept of combinatorial gene control

Answer 1

It is the norm for eukaryotes. Multiple different regulatory proteins can bind to the identical regulatory element in a gene. The particular combination of proteins that bind to an element is dependent on the cell type or the physiological state of the cell. Some combinations will activate and some will repress gene expression. It is likely a “competition” of activators and repressors. The overall outcome is the net effect of these.

Question 2

Describe how the combinatorial regulation of Myc, Max, and Mad binding to the E-box element can regulate the switch between cell division and cell differentiation

Answer 2

Myc, Max and Mad are bHLH (basic HLH zip) transcription factors that are involved in promoting the expression of genes associated with cell division.

Max is constitutively expressed, meaning it is always expressed and max will dimerize with itself to form Max-Max. When it dimerizes it binds to the E-box element but it will not turn on transcription in this case, transcription will simply occur at the basal level of ongoing expression of this gene but we aren’t doing anything to promote or inhibit it.

If the cell wants to DIVIDE, it will upregulate Myc. When Myc is expressed in high enough levels, it will displace Max from the Max-homodimer and form a Myc-Max heterodimer. This gives a strong signal to produce proteins that are involved in cell division. My binds to the E-box and we turn on or activate transcription.

If the cell wants to DIFFERENTIATE, we can’t have genes involved in cell division expressed. Differentiation is an opposing force of division, they are opposite. In this case, Mad will be produced and replace Max from the Max-Max homodimer to form a Mad-Max heterodimer. It will then bind to the E-box element but now we get transcription turned OFF or silenced.

Repressing a gene is leaky, or it can still produce some mRNA and thus protein so we need to completely silence it! When we want activation, we get acetylation because if causes the TFIID to bind to the DNA because it is less tightly packed.

Question 3

Max-Max homodimer

Answer 3

Max is constitutively expressed, meaning it is always expressed and max will dimerize with itself to form Max-Max. When it dimerizes it binds to the E-box element but it will not turn on transcription in this case, transcription will simply occur at the basal level of ongoing expression of this gene but we aren’t doing anything to promote or inhibit it.

Question 4

Myc-Max Heterodimer

Answer 4

If the cell wants to DIVIDE, it will upregulate Myc. When Myc is expressed in high enough levels, it will displace Max from the Max-homodimer and form a Myc-Max heterodimer. This gives a strong signal to produce proteins that are involved in cell division. My binds to the E-box and we turn on or activate transcription

Question 5

Mad-Max Heterodimer

Answer 5

If the cell wants to DIFFERENTIATE, we can’t have genes involved in cell division expressed. Differentiation is an opposing force of division, they are opposite. In this case, Mad will be produced and replace Max from the Max-Max homodimer to form a Mad-Max heterodimer. It will then bind to the E-box element but now we get transcription turned OFF or silenced.

Question 6

Myc, Max, and Mad Domains Characteristics

Answer 6

Myc heterodimerizes with other bHLH-ZIP proteins
Myc-Max dimers induce cell proliferation
Mad-Max dimers inhibit proliferation or cell division and initiate cell differentiation.
Myc-Max and Mad-Max complexes have opposing functions in transcription and Max plays the central role.
Max is constitutively expressed, meaning it is always expressed.
Myc is ONLY expressed in the G1 and S transition of the cell cycle because this is when we need cell division after replication.
When Max is bound, it doesn’t really have an activation domain so it won’t really be doing anything! This is where the leakiness comes from, we get the basal level of expression. Myc, however, has a very large activation domain, so we get this large activation signal when it binds to the E-box.
Mad is in-between, it has an activation domain but it is not “activating” it has repression activity. Its function is to repress. This is so we don’t get any expression

Question 7

Transcription factors can be activated in 7 ways

Answer 7

1) Protein Synthesis
2) Ligand Binding
3) Covalent Modifications
4) Addition of second subunit
5) Unmasking
6) Stimulation of nuclear energy
7) Release from membrane

Question 8

Protein Synthesis

Answer 8

The transcription factor didn’t previously exist so it will be synthesized as an active protein that can signal

Question 9

Ligand Binding

Answer 9

There is a ligand that will interact with the transcription factor and activate it

Question 10

Covalent Modifications

Answer 10

The transcription factor is expressed but it only becomes active when it is phosphorylated by a KINASE

Question 11

Addition of second subunit

Answer 11

There is an addition of a second subunit that is necessary for the activation of this transcription factor

Question 12

Unmasking

Answer 12

There is an inhibitor bound to the transcription factor and when its phosphorylated (the inhibitor) it will leave.

Question 13

Stimulation of nuclear energy

Answer 13

The translocation of the transcription factor into the nucleus causes an inhibitor to dissociate and the transcription factor to be active.

Question 14

Release from membrane

Answer 14

A membrane-bound transcription factor is cleaved releasing the active portion into the cytoplasm.

Question 15

Autocrine Pathway

Answer 15

The cell releases signals that will act on itself

Question 16

Paracrine Pathway

Answer 16

Neighboring cells release a signal to act on a neighboring cell or cells

Question 17

Kinase Cascade

Answer 17

A “cascade” in which one kinase phosphorylates another kinase which then phosphorylates another and then another and it is repeated as a cascade to get an end result like a kinase entering the nucleus and activating transcription via phosphorylating a transcription factor. Jun and Fos are an example of a kinase cascade.

Question 18

Describe how different signal transduction pathways activate kinases that regulate the AP-1 transcription factors Jun and Fos

Answer 18

It is a kinase cascade that eventually leads to JNK being phosphorylated which will then phosphorylate Jun causing it to be able to bind to the AP-1 site now. ERK is also part of a kinase cascade that will phosphorylate Fos that will then be able to bind to the AP-1 element. Jun and Fos are bZIP heterodimers connected via a leucine zipper and bind with a scissor motif to the AP-1 element.

Question 19

JNK

Answer 19

JNK is the kinase that phosphorylates and thus activates the Jun dimer

Question 20

ERK

Answer 20

ERK is the kinase that phosphorylates and thus activates the Fos dimer

Question 21

Describe models for how the position of a gene in the heterochromatin or euchromatin of a chromatid influences its expression

Answer 21

Positioning a gene in condensed or heterochromatin inhibits gene expression where placing it in euchromatin or less condensed regions increases gene expression. The chromatin structure can regulate cell phenotype. If the gene is positioned in heterochromatin it will not be expressed. If it is in euchromatin it will be expressed. An example is the inactivation of one X chromosome to form the Barr Body.

Question 22

Describe how the X chromosome is inactivated in females

Answer 22

An example is the inactivation of one X chromosome to form the Barr Body. If both gene pairs in the X chromosomes were expressed in females, we would have a dosage problem. Basically, the chromosome undergoes extensive deacetylation to allow it to interact with the DNA and adjacent nucleosomes more and it also undergoes methylation that tells the cell to condense the DNA in a compact manner there. The cells are thus mosaic in females because this is done randomly. Some cells have the X from mom expressed, some the X from dad. The progeny cells will express the same X chromosome being condensed as was determined randomly by the parental cell. But, the condensing of one of the X chromosomes is done randomly.

Question 23

Explain the role of the locus control region in regulating Beta-Globin gene expression in red blood cells

Answer 23

In this case, we are again looking at how heterochromatin can regulate gene expression. The hemoglobin gene is only expressed in young RBCs and it is not expressed in any other cell type. So the other cell types must have the region of chromosome 11 present as heterochromatin so that it will not be expressed. However, in early RBCs this region of chromosome 11 must be exposed in order to express the hemoglobin gene. Thus, it is present as euchromatin. The key is this locus control region because transcription factors will be produced by early RBCs and will bind to the locus control region and cause expression. In an embryo, the cells know to make the appropriate transcription factors to bind to the locus control region to express epsilon-globin. Then, when it reaches the fetal stage, it will produce gamma-globin instead of epsilon now. Then when the child is born it will switch to beta-globin. The locus control region is critical because it will bind all of these different transcription factors and say which globin to express. Heterochromatin structure regulates the hemoglobin gene expression.

Question 24

Define the term epigenetic

Answer 24

Epigenetic refers to the inheritance or passage of information from parental cells to progeny cells by a mechanism other than from the “instructions” within the DNA sequence. Due to epigenetics, two alleles can have the same nucleotide sequence but give different inheritable genetic information. Epigenetic mechanisms occur through modifications of either DNA or gene regulatory proteins.

Question 25

Explain how positive feedback transcription factor loops can generate cellular memory

Answer 25

Positive feedback transcription factor loops cab generate cellular memory because there is some gene A that is turned on in a parent cell and makes protein A. Then, if protein A is a regulatory element that promotes expression of gene A, it will feedback and continue to make itself. Then, when the parent cell divides, it will inevitably pass on some of the protein A to the progeny cells. Then, the protein A will continue to regulate itself and express itself in the progeny as well. This is seen in the placenta which is asymmetrical. In other words, in different regions of the placenta, different signals can be produced that will cause the cells to produce different factors, including different transcription factors. We start with an embryonic cell and on one side is told to increase transcription of gene 1 but the other side is not. When the cell divides, it will pass on this memory to the side where it was turned on but not to the side that was not. So one set of progeny will express protein 1 and one will not. This can continue to occur to get this memory and asymmetry

Question 26

Explain how DNA methylation affects gene expression and how a pattern of DNA methylation can be passed from parental cell to to progeny cell

Answer 26

DNA methylation is involved in epigenetic mechanisms. It is NOT the same methylation involved in histone tails! We are looking at DNA now, not protein. Methylation in DNA occurs on “Cs” or cytosines but ONLY Cs followed by Gs (guanine). Methylation sequence is palindromic! The methylation of a promoter or enhancer region of a gene inhibits the gene expression. If a C on one strand is methylated, the C on the other strand will also be methylated, remember it is palindromic. It is termed dimethylation. The methylation will affect and prevent the transcriptions factors from binding to their regulatory element, they can inhibit transcription. After replication, only one DNA strand comes from the parent cell and has the methylation marks. So, the methylation needs to be reestablished to the newly synthesized strand.
Maintenance Methylase recongnizes these sites and methylates them. If a particular CG was not methylated, it will not methylate it. The methylation also promotes the binding of inhibitory proteins!

Question 27

Maintenance Methylase

Answer 27

Maintenance Methylase recongnizes these sites and methylates them. If a particular CG was not methylated, it will not methylate it. This is how sites of methylation are inherited by progeny cells. It will methylate and HEMIMETHYLATED sites or those where only one strand is methylated.

Question 28

Understand the function of CG Islands in maintaining the expression of housekeeping genes.

Answer 28

There are some genes that we never want to turn off because they are essential or “housekeeping” genes. If we did turn them off, it would be a toxic condition. For these gene we don’t want methylation because if we did have it, it would turn off these essential genes. CpG islands are regions upstream of housekeeping genes that contain a high concentration of CGs and they tend to be very compact. It seems counter-intuitive but these actually RESIST methylation and keep the gene on.

Question 29

Explain how histone acetylated regions of the genome can be passed from parental cell to progeny cell.

Answer 29

Just like there are maintenance methylases, there are maintenance acetylates that act to acetylate histone proteins of nucleosomes that are semi-acetylated in newly formed progeny cells. Thus, the same acetylation regions within the chromatin of a parental cell may be maintained in the progeny cell. The maintenance acetylase simply comes in and makes the same marks the parental cells had.

Question 30

Maintenance Acetylase

Answer 30

The maintenance acetylase simply comes in and makes the same marks the parental cells had.

Question 31

Understand how most genes in the DNA within the egg and sperm are recycled (stripped of methyl groups) except for a small number of genes where methylation may be maintained.

Answer 31

Imprinting is sex-dependent. In other words, a particular gene retains its methylation in either the sperm or egg, but not in both. Genomic imprinting, or the retention of the methylation groups is known to occur in about 50 human genes. The others are simply stripped of their methylations.

Question 32

Explain how genomic imprinting may occur in a sex dependent manner

Answer 32

Imprinting is sex-dependent. In other words, a particular gene retains its methylation in either the sperm or egg, but not in both. It leads to sex-dependent non-mendelian genetic expression of the imprinted gene. Normally, we inherit 2 genes from our parents, 1 from mom and 1 from dad. If one is mutated, it usually isn’t a big deal because there is still a good gene. Generally speaking, methylation marks are erased when making sperm or egg. There are some genes that remain “imprinted” however, and these genes are methylated or turned off in a parent specific manner. Certain genes are always methylated or turned off in mom and others in dad. After fertilization, these genes retain their methylation and the gene is not expressed. Imprinted genes though can lead to pathology because if there is a mutation in the expressed copy, the other is turned off!