Epigenetics & miRNA

Question 1

Epigenetics

Answer 1

Study of heritable changes in gene expression or phenotype caused by mechanisms other than chanes in DNA sequences

Explains how individuals w/the same DNA sequences (e.g., identical twins) have different disease profiles

Question 2

3 major types of epigenetic modification

Answer 2

1. DNA methylation​​
2. Histone Modification
3. Microribonucleic Acids (miRNAs or miRs)

Question 3

DNA methylation

Answer 3

• ​​attachment of methyl group to a cytosine base is followed by a guanine base (CpG dinucleotide)
• silences/transcriptionally inactivates gene (heavily methylated = less likely to be transcribed to RNA)
• Aberrant methylation can lead to silencing of tumor-suppressor genes in development of cancer
• Same process in X inactivation

Question 4

Histone Modification

Answer 4

(e.g., histone acetylation and deacetylation, alterations in chromatin)

​Chromatin compaction & organization help to regulate gene expression

Question 5

Microribonucleic Acids (miRNAs or miRs)

Answer 5

• ​RNAs encoded by short DNA sequences (~22 nucleotides)
• located on introns of genes or transcribed from noncoding DNA located btwn genes
• untranslated

Question 6

Functions of noncoding RNA/miRNA?

Answer 6

• noncoding RNAs (ncRNAs) have been shown to regulate gene expressions: e.g., RNA interference, gene cosupression, gene silencing, imprinting, DNA methylation
• MiRNA perform critical functions during development & cell differentiation
• MiRNA regulate diverse signalling pathways - e.g., oncomirs stimulate cancer development and progression

Question 7

oncomirs

Answer 7

miRNA that stimulate cancer development and progression

Question 8

Why are miRNAs linked to carcinogenesis?

Answer 8

b/c can act as either oncogenes or tumor suppressor genes

Question 9

pluripotent

Answer 9

trait of embryonic stem cells - potential to become any type of cell in fetus or adult

Question 10

How is eukaryotic DNA compacted 10,000-fold into chromosomes?

Answer 10

Histones!

Question 11

What are histones

Answer 11

• The proteins that make up a nucleosome - there are four pairs
• histones are packers. They pack the protein. They are the beads – they are able to roll to the right or left, and unpack DNA as they have on them, expose it for whatever is needed – replication, transcription… Wind back in when the job is done.

Question 12

Types of histones

Answer 12

Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 and H5 are known as the linker histones.

Question 13

Histone octamer

Answer 13

The collective of the four pairs of histones that make up a spool for DNA to coil around (nucleosome)

Question 14

Histone Structure

Answer 14

• octomeric histone core (8 pieces). Come together to build bead structure.
• Histones contain an N-terminal tail which is subject to covalent modification
• Histones H2A and H2B form a dimer via a handshake
• Histones H3 and H4 form asimilar interaction
• N-terminal ends are different: They fold – when they pair and fold, their tails stick out in a certain way – very important

Question 15

nucleosome

Answer 15

a basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around eight histone protein cores.

Question 16

Chromatin

Answer 16

nucleosomes are organized into chromatin, the repeat building blocks of a chromosome

Question 17

Nature of movement of nucleosomes

Answer 17

Dynamic!

Note when a part of a nucleosome is exposed, a protein can bind to it

Question 18

What makes the nucleosome turn?

Answer 18

Chromatin remodeling complexes (ATP dependent!)

Question 19

How do chromatin remodeling complexes work?

Answer 19

Histone exchange and histone removal are catalyzed by chromatin remodeling complexes

Changes H2A part of H2A-H2B dimer, for a new H2A. These cannot be altered, so histone chaperone protects them during the process.

This uses ATP.

Whole nucleosome can be rebuilt over time – but needs to frequently replace H2A-H2B piece. Once H2A is replaced for different H2A, that nucleosome exposes gene for action.

Question 20

Who gets the replacement H2A?

Answer 20

Histone Tails!

The way they stick out, communicate message. Signals come from gene, and also from proteins attached to gene, and tails on proteins

Question 21

Histone Tails and the path of DNA

Answer 21

Tails may attach one nucleosome to another

A single histone H1 binds both the histone octomer and DNA and may change the path of the DNA as it exits from the nucleosome thus allowing binding of nucleosomes to each other

Question 22

Histone DNA contacts

Answer 22

primarily nonspecific

• hydrogen bonds (142) between basic histone Amino acids and the negatively charged DNA phosphodiester backbone
• hydrophobic and ionic interactions
• certain nucleotides favor staying inside, other favor outside. AA, TT, TA favor wrapping. G-C prefer outside.

Question 23

Why does DNA need packaging & how is it packaged?

Answer 23

DNA is a long molecule: needs packaging to fit in the tiny nucleus. Packaging involves coiling of DNA in a “left-handed” spiral around spools. These spools/nucleosomes are made of four pairs of proteins known as histones

Question 24

DNA methylation & tumor cells

Answer 24

Tumor cells usually exhibit hypomethylation, which can increase activity of oncogenes

Question 25

DNA methylation and promoter regions + examples

Answer 25

often hypermethylated --\> decrease in transcription rate & ability to inhibit tumor formation e.g., RB1 in retinoblastoma & BRCA1 in breast cancer

Question 26

DNA methylation & hereditary nonpolyposis colorectal cancer [HNPCC]

Answer 26

MLH1, whose protein product repairs damaged DNA, is methylated --\> inactive --\> damaged DNA accumulates --\> colon tumors

Question 27

mrRNAs and cancer

Answer 27

hypermethylation: miRNA encode small, 22 base pair RNA molecules that bind to mRNAs and degrade them/prevent translation hypermethylation of specific subgroups of miRNA assoc w/tumorigenesis methylation --\> overexpressed targets --\> metastasis

Question 28

DNA mutations vs epigenetic modifications

Answer 28

DNA sequences can't be directly altered, but epigenetic modifications can be reversed! e.g., demethylating agents like 5-azacytidine in Tx of leukemia & myelodysplastic syndrome

Question 29

treatment of histone modification problems

Answer 29

e.g. histone deacetylase (HDAC) inhibitors counteract removal of acetyl groups from histone proteins -- stop silencing tumor-suppressor genes T-cell lymphomas \*challenge is targeting only specific cancer-causing genes

Question 30

Imprinting

Answer 30

* process of gene silencing - in which genes are predictably silenced depending on which parent transmits them * typically via heavy methylation * clinical significance: if same group of genes are deleted, may cause different expression in child depending on whether inherited from mother or father - b/c different genes were active in that area based on gender * Role for miRNA

Question 31

location of miRNA

Answer 31

in the introns of genes (**Intronic**) or non-coding DNA between genes (**Intergenic**), or can code for multiple genes (**Polycistronic**)

Question 32

DICER

Answer 32

* also known as endoribonuclease Dicer or helicase with RNase motif (scissor) * An enzyme that in humans is encoded by the DICER1 gene. * Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into short double-stranded RNA fragments called small interfering RNA and microRNA respectively.

Question 33

process of miRNA

Answer 33

Drosha cleaves off hairpin piece of miRNA, which leaves nucleus and goes to cytosol. Dicer cuts pre-miRNA into miRNA and unused part, miRNA goes to target mRNA, e.g., to area of gene that should be silenced and blocks --\> gene not expressed

Question 34

Drosha

Answer 34

a Class 2 RNase III enzyme responsible for initiating the processing of microRNA (miRNA) A microRNA molecule is synthesized as a long RNA primary transcript known as a pri-miRNA ## Footnote *become short segments, like hairpins. As a result of cleaving enzyme (Drosha), primitive RNA is cleaved into a pre-miRNA. At that point, it exits the nucleus, goes to cytosol, where part is used and other is cut off (by dicer enzyme). It is now available – role in RNA interference, gene code suppression, gene silencing, imprinting*

Question 35

Clinical significance of miRNA

Answer 35

Don’t get translated but signals that help development, metabolism, fate of cell, most importantly cell death. cancer cells – we want that signal and why has cancer cell managed to escape it? Why have these cells altered miRNA, how can you restore the death signal in these cells?

Question 36

Where does methylation occur?

Answer 36

At the 5 position of cytosine

Question 37

when chromatin is open/active, genes are...

Answer 37

expressed

Question 38

when chromatin is condensed b/c of methylation or histone modification, genes are...

Answer 38

inactivated

Question 39

Where does histone acetylation happen?

Answer 39

At histone tails more acetylation (by HAT): open chromatin/gene expression deacetylation (by HDAC): closed (e.g., overdone in cancers) HAT & HDAC are chromatin remodeling complexes