BIOL4417 Epigenetic Regulation and Cancer Cells

co-factor proteins

Help transcription factors in binding

Where are most CpG islands found?

In gene promoters

What methylates CpG islands?

Methyltransferase adds methyl groups to Cytosines, inactivating the promoter or the gene

When does de novo methylation take place?

Following implantation in the uterus

5 DNMTs

1. DNMT3a

2. DNMT3b

3. DNMT1

4. DNMT2

5. DNMT31

Which DNMTs are responsible for de-novo methylation

DNMT3a and DNMT3b

2 mechanisms through which DNA methylation silences genes

1. Methylation restricts access of transcription cofactors

2. Methylated nucleotides recruit MBD proteins, restricting access to other TFs or cofactors

How can DNA methylation be lost?

1. Passively during DNA replication

2. Actively via enzymatic action

DNA bisulfite conversion

Treating DNA with bisulfite salt can cause methylated Cs to be converted into Uracil, allowing them to be detected and allow for tracking of methylation patterns

Which DNMT is responsible for maintenance methylation?

DNMT1, following DNA replication

Formation of closed chromatin steps

1. CpG island is methylated

2. Methyl binding proteins bind

3. H3K9 gets deacetylated, then methylated

4. Recruits HP1, which closes the chromatin

2 waves of demethylation of the genome

1. First occurs in primordial germ cells, which will differentiate into gametes and undergo de-novo methylation

2. Second occurs upon fertilization, followed by de-novo methylation upon implantation

What is special about imprinted genes?

They do not get demethylated for the 2nd time, allowing for parent specific epigenetic markers to be passed down

5 types of modifications which can be done to histones

1. Acetylation

2. Methylation

3. Ubiquitination

4. SUMOylating

5. Phosphorylation

Nucleosome structure

147bp wrapped via supethelical turns around histone octamer, itself consisting of H2A, H2B, H3, H4

Which terminal are histones modified at?

N-terminal tails

Histone structure of an actively transcribed promoter

H3K4me4

Histone structure of an active enhancer

H3K4me1

What histone modification is generally linked to activation

Acetylation

Mechanism through which acetylation allows for active transcription of genes/ making them open.

1. Histone tails are positively charged, and acetyls are negatively charged, thus neutralizing the charge, and ensuring positively charged histone does not interact with negatively charged DNA

How does acetyl get onto Lysine on histone tails

Histone acetyltransferases use acetyl-CoA as a donor

Epigenetic changes can either ensure hematopoietic stem cells differentiate into healthy or tumor lineages

Epigenetic changes can either ensure hematopoietic stem cells differentiate into healthy or tumor lineages

ES cells chromatin structure

It is bivalent.

1. Activating H3K4me3

2. Repressive H3K27me3

both of these tails exist, allowing for rapid demethylation of whichever tail is fated for demethylation

Which 2 enzymes are needed for poising of genes in ES cells? How do they work?

1. Trithorax trimethylated H3K4 recruits chromatic remodeling complexes and histone acetylases, preparing gene for activating

2. Polycomb complex negatively regulates activation of genes by trimethylating H3K27, promoting closed chromatin structure

How does PRC repression work?

1. PRC2 recognizes and trimethylated H3K27

2. This mark is recognized by PRC1, which brings other histones closer, repressing chromatin

Trithorax G actions on chromatin

1. Demethylate H3K27me3

2. Methylate H3K4

3. Methylate H3K36

What does miR-145 do? Where is it expressed?

1. Blocks translation of Klf4, Oct3/4, Sox2

2. Expressed in differentiated cells

What does miR17/92 do?

Regulates TGF-beta signaling on cellular differentiation

How are iPS cells created

Induce specialized cells to produce Oct3/4, Sox2, Klf4, c-Myc

Order in which pluripotency genes are added to specialized cells, and why it matters.

1. c-Myc induces immortality and opens chromatin

2. Klf4 represses apoptosis and senescence

3. Oct3/4 changes cell fate from tumor cells to stem cells

4. Sox2 established pluripotency

How does retroviral pluripotency gene transduction work

Use a retrovirus to insert the necessary genes into the cell, which will then be transcribed. As time passes, viral promoter is repressed, but by then the cell should be differentiated back into a stem cell

Problem with using retroviruses to insert new genes

They can cause cancers

Polycistronic lentiviral vectors

Contain all necessary genes in a single lentivirus, ensuring lesser chance of cancer development

2 iPS cell problems

1. Low efficiency

2. Variation in gene expression profiling between ESCs and iPSCs

How was pluripotent mouse derived

Form a chimera mouse, and that descendants of one original iPSCs will become primordial germ cells and then a new generation of mice

What is more efficient at being transformed into iPSCs than fibroblasts? Why?

Keratinocytes, because they express higher levels of Klf4 and c-Myc, as well as being in epithelial state

What needs to happen to somatic cells before they are introduced to pluripotency factors

They must undergo MET

1. Higher expression of E-cadherin and Epcam

2. Downregulation of Snail and N-cadherin

Which of the 4 factors is an oncogene

c-Myc

How to ensure that inserted pluripotency genes do not cause cancer

1. Do not use retroviruses or lentiviruses, you can use episomes

2. Makes sure that the genes are transient, and are not fully integrated

3. Small molecules which can trigger transcription of necessary factors

Epigenetic memory

Inability of iPSCs to completely demethylate their genome, causing dissimilarities between them and ESCs

3 cellular origins of cancer stem cells

1. Mutation in a stem cell

2. Mutation in a somatic cell, resulting in transformation into a cancer stem cell

3. Fusion of somatic and stem cell

Risk factors for formation of cancer cell

1. Mutagen

2. Environmental stress

3. Viral infection

4. Chronic inflammation

Cancer stem cell hypothesis

Suggests that there is a subset of cancer stem cells which are responsible for tumor growth. Just like in regular stem cells, they can undergo symmetric or asymmetric division

Implications of CSCs (2 of them)

1. Strategy for tumor killing would have to focus on eliminating all cancer stem cells, and majority of differentiated cells

2. This complicates the search for a definitive cure, as new therapies are needed, and CSCs seem to be more resistant to radiation treatment compared to other cancer cells

Progression of colon cancer, from healthy to carcinoma

1. Loss of APC leading to hyper plasticity

2. DNA hypomethylation, leading to loss of cancer suppressants, such as p53

3. Adenoma turns into carcinoma by loss of p53

Multi-hit model of cancer

Since cells have multiple safeguards against cancer development, a combination of LOF and GOF mutations are needed to cause cancer

3 layers or the colon crypt

1. Differentiated upper layer

2. Middle area with proliferating and differentiating cells

3. Bottom layer with stem cells which replenish the stem cells

How does colon cancer start? What gene is affected?

APC gene is turned off on both alleles, and thus the cells on top can no longer undergo apoptosis

How does APC gene work?

It codes for APC protein which is antagonistic to Wnt signaling pathway. It does so by binding Beta-catenin. It’s inactivation leads to beta-catenin entering nucleus

Leukemia stem cells

They represent a small subset of total leukemic population, less than 1%, but they produce all the leukemia cells. If you transfer them into mice, mice will get AML

Leukemia stem cell markers

Thy1-

CD34+

CD38-

3 ways to target cancer stem cells

1. Target CSC specific receptors

2. Target CSC molecules, like nanog

3. Take advantage of differential signaling required for CSC

Label retaining experiment

1. Pulse the cells with a marker, like BrdU

2. Fast proliferating cells will dilute the marker faster than slow proliferating cells

Clonogenic assay

Tests the ability of a cell to form colonies.

The colonies are grown on Agar, Matrigel, HEMA-fibroblasts, methylcellulose

Side population assay

1. Identify stem cells using flow cytometry

2. Hoechst DNA dye is passively absorbed by all cells

3. Stem cells and progenitor cells have a pump which allows them to remove the dye, done via ATP-binding cassette (ABC)

4. These cells low i Hoechst dye are called side-population, and are the cancer causing cells

What drugs inhibit ABC protein pump

Verapamil