Chapter 2

Question 1

Every somatic cell nucleus contains the same complete genome established in the fertilized egg. The DNA of all differentiated cells are identical. The unused genes aren’t destroyed or mutated. How does differential gene expression happen?

Answer 1

• Only a small percentage of the genome is expressed in each cell

Question 2

Regulation of gene expression: (4)

Answer 2

• Differential gene transcription - Pre-m RNA processing - Selective mRNA translation - Differential protein modification

Question 3

Cloning method: (Dolly) Somatic cell nuclear transfer

Answer 3

• Nuclear donor provides somatic cells. Ensure nuclei at G1 stage of cell cycle (diploid). Reprogrammed through demethylation. - Oocyte donor provides egg (oocyte). Important at the second meiotic metaphase (stage at which it’s normally fertilized). Nucleus removed - Donor cell and enucleated oocyte are brought together and electric pulses / chemicals destabilize the cell membranes allowing the cells to fuse - These same signals activates the egg to begin development - Implanted at blastocyst stage in surrogate mother

Question 4

Methylated DNA

Answer 4

Used to regulate transcription No gene expression (depending on lysine/cytosine methylated) Works by stabilizing nucleosome, preventing TFs from binding. Methylation gives negative charge => more attractive to positive nucleosome Methylation pattern of a cell changes during development Repressed states of chromatin will also attract proteins that facilitate DNA methylation.

Question 5

Acetylated DNA

Answer 5

gene expression Destabilizes nucleosome, pga makes DNA more positive => repulsive to positive nucleosome

Question 6

Differential gene expression through transcription factors, how?

Answer 6

• The enhancer sequences are the same in every cell type; different combinations of TF proteins though - The same gene can have several enhancers, enabling it to be expressed in different cell types. - Allows simultaneous activation of entire groups of genes (coordinated gene expression) - TFs act as bridge between DNA and histone-modifying enzymes (fx HATs – dissociates histones from DNA) - TFs stabilize the transcription pre-initiation complex that enables RNA pol II to bind to the promoter

Question 7

Trithorax and polycomb family of proteins affects gene transcription how?

Answer 7

They retain the memory of transcriptional state from generation to generation through mitosis. Trithorax proteins keep genes active Polycomb proteins keep the genes in a repressed state

Question 8

Silencers are ?

Answer 8

DNA regulatory elements that actively repress the transcription of a particular gene. They can silence spatially (in particular cell types) or temporally (at particular times)

Question 9

Insulator sequences limit ?

Answer 9

They limit the range in which an enhancer can activate gene expression. Insulating a promoter from being activated by another gene’s enhancers.

Question 10

Reprogramming cells? How? For?

Answer 10

By changing the expression of certain TFs, an entirely new network of gene expression can be effected => changing one adult cell type into another Fx been used to reprogram adult human fibroblasts into functional dopaminergic neurons (degenerates in Parkinson disease).

Question 11

Two general classes of promoters:

Answer 11

• High CpG-content promoters • Low CpG-content promoters

Question 12

Sodium bisulfite treatment of genomic DNA

Answer 12

leads to conversion of nonmethylated cytosine to uracil. • PCR amplifies uracil as thymine. • Methylated cytosines (5-methylcytosine) remain as cytosines. • Detection of ”C” in sequencing reaction indicates methylation at this CpG site (C can only be methylated if followed by G), detection of ”T” indicates no methylation.

Question 13

Promoters can exist in three major states

Answer 13

Active Repressed “Poised” - (intermediate, allowing for rapid response to developmental signals, characterizes high CpG promoters)

Question 14

How are promoters “poised”?

Answer 14

1. DNA is relatively unmethylated and nucleosomes enriched with “activating” H3K4me3. 2. RNA pol II is already present. 3. A small truncated transcript of nRNA is already initiated (but not completed). Rate limiting step is thus not initiation but elongation.

Question 15

To become an active protein, the nRNA must be

Answer 15

1. Processed into mRNA by removal of introns 2. Translocated from the nucleus to the cytoplasm 3. Translated by the protein-synthesizing apparatus 4. (Some cases) Posttranslationally modified to become active. Regulation during development can occur at any of these steps.

Question 16

Differential RNA processing is?

Answer 16

The splicing of mRNA precursors into messages that specify different proteins by using different combinations of potential exons (splicing isoforms), done by spliceosomes. About 92% of human genes are thought to produce multiple types of mRNA; genome around 20,000 genes => proteome far more complex.

Question 17

Dosage compensation is?

Answer 17

The equalization of X chromosome encoded gene products in males (one copy, XY) and females (two copies, XX), in drosophila, nematodes and mammals. Three possibilities: • Doubling of male X transcription rate (drosophila) • Partial repression of both X chromosomes so they in total provide the same amount as the single X (C.elegans) • Random irreversible X chromosome inactivation (mammals) => all tissues in female mammals are mosaics of two cell types (mom inactivated or dad) pga happens early in development. Inactivated = Barr body

Question 18

Heterochromatin

Answer 18

heavily condensed, inactive

Question 19

Euchromatin

Answer 19

less / not condensed, active

Question 20

Long nonconding RNAs (lncRNAs) are?

Answer 20

A group of transcriptional regulators often used to silence genes on one of the two chromosomes, fx X chromosome inactivation by Xist

Question 21

Control of gene expression at the level of translation Can occur by many means: (5)

Answer 21

• Longevity • Stored oocyte mRNAs • Ribosomal selectivity • microRNAs • Cytoplasmic localization

Question 22

Control of gene expression at the level of translation - Longevity

Answer 22

The longer an mRNA persists, the more protein can be translated from it. Selective stabilization of a message with an otherwise relatively short half-life at a specific time and place, fx often through length of polyA tail.

Question 23

Control of gene expression at the level of translation - Stored oocyte mRNAs

Answer 23

The oocyte often makes and stores mRNAs that will only be used after fertilization at certain times, most are needed during cleavage, but others encode proteins that determine cell fates. Most translational regulation is negative, pga prevent mRNA from being translated at inopportune times.

Question 24

Control of gene expression at the level of translation - Ribosomal selectivity

Answer 24

Selective activation of mRNA translation, ribosomal proteins are not the same in all cells, some are necessary for translating certain messages.

Question 25

Control of gene expression at the level of translation - MicroRNA

Answer 25

Specific regulation of mRNA, humans have more than 1000 miRNA loci and they modulate ca. 50 % of the protein-encoding genes in our bodies. Can regulate translation in two ways: o Binding can block initiation o Binding can recruit endonucleases to digest the mRNA, usually starting with the polyA tail.

Question 26

Control of gene expression at the level of translation - Cytoplasmic localization

Answer 26

A majority of mRNAs are localized to specific places in the cell, often accomplished through their 3’-UTRs. Three major mechanisms: o Diffusion and local anchoring – diffuse freely but is trapped by proteins that reside in particular regions, these also activate the mRNA allowing it to be translated o Diffusion and local protection – degraded everywhere, except where protected by local proteins o Active transport along cytoskeleton – most widely used, mRNAs are bound to “motor proteins” that travel along the cytoskeleton to their final destination, usually ATPases.

Question 27

Posttranslational regulation of gene expression

Answer 27

• Some newly synthesized proteins remain inactive until certain inhibitory sections are cleaved away. - Some must be “addressed” to their specific intracellular destinations in order to function - Some need to assemble with other proteins in order to form a functional unit. - Some are not active unless they bind an ion, fx Ca2+ or are modified by the covalent addition of a phosphate or acetate group.

Question 28

The Polycomb proteins fall into two categories that act sequentially in repression:

Answer 28

1. Methylates H3K27 and H3K9 2. Maintains methylation and methylate adjacent nucleosomes

Question 29

The Trithorax act to counter the effect of the Polycomb proteins, two ways:

Answer 29

• Some modify nucleosomes or alter their positions on chromatin - Others keep H3K4 trimethylated and activated

Question 30

Describe high CpG-content promoters.

Answer 30

Usually found in “developmental control genes”, regulating TFs and other proteins used in construction of organism Default state is “on”, active repression through histone methylation

Question 31

Describe low CpG-content promoters.

Answer 31

Usually found in genes whose products characterize mature cells Default state is “off”, can be activated by TFs, usually methylated (critical for preventing transcription)

Question 32

Is the CpG motif widespread in the genome?

Answer 32

The CpG motif is underrepresented in most of the genome (on average once every 100 bp). • Approximately 72% of the ~20,000 genes in the mammalian genome have a high CpG content in their promoter regions.

Question 33

Degree of cytosine methylation controls the level of the gene’s transcription. Works by:

Answer 33

• Blocking binding of TFs to enhancers - Can recruit additional proteins that facilitate methylation or deacetylation of histones (stabilizing the nucleosome)

Question 34

DNA methyltransferase 1 and 3 does what?

Answer 34

Dnmt3 (DNA methyltransferase-3) methylates DNA Dnmt1 transmits methylation pattern to next generation (recognizes methyl cytosines on one strand and places methyl groups on the newly synthesized strand)

Question 35

How is transcription paused in “poised” promoters?

Answer 35

Transcription is paused pga RNA pol II remains tethered to TFIID, (through Mediator complex). Which remains bound to the promoter sequence of the gene => elongation requires out-competing connection to TFIID by TEC (transcription elongation complex)