Zygotic Genome Activation

Question 1

Define “transcription factors.”

Answer 1

Proteins required to initiate OR regulate transcription.

Question 2

Define “cis-acting locus.”

Answer 2

An area on a DNA molecule that affects the sequencing of DNA on the SAME molecule (and doesn’t code for proteins).

Question 3

Define “promoter.”

Answer 3

Nucleotide sequence in DNA to which RNA polymerase binds to to begin transcription.

Question 4

Define “enhancer.”

Answer 4

Regulatory DNA sequence to which gene regulatory proteins bind in order to alter the rate of transcription on structural genes that could be many thousands base pairs away.

Question 5

Define “TATA box.”

Answer 5

Sequence in promoter region of many eukaryotic genes to which general transcription factors bind in order to initiate transcription at that position or of that gene.

Question 6

What does the “threshold nucleo-cytoplasmic ratio” idea refer to?

Answer 6

In xenopus embryos, zygotic genome activation occurs after DNA replication and cell division occurs very quickly and repressive factors are titrated out. During this time, interphase becomes long enough to synthesize transcripts of zygotic DNA.

Question 7

Does the “threshold nucleo-cytoplasmic ratio” model hold true for mammals?

Answer 7

No (doesn’t seem to)

Question 8

Converting two parental haploid genomes into a single diploid genome involves what two major steps?

Answer 8

1. Remodeling: Dramatic reprogramming to alter mature oocyte/sperm into new TOTIPOTENT genome.
2. Reprograming: Epigenetic programing to make sure that alleles are expressed properly and especially at imprinting regions in the genome.
3. NOTE: Zygotic genome transcription MUST take place AFTER remodeling and reprogramming is complete to ensure safe and healthy child!

Question 9

Zygotic genome activation is a multi-step processing consisting in what four main changes?

Answer 9

1. Ooplasm (also note histones made will be translated from inherited maternal mRNA)
2. Nuclear structure - non-permeable sperm membrane replaced with permeable one to help form pro-nucleus.
3. Chromatin structure - protamine-histone exchange in male pronucleus for chromatin decondensation (maternal chromatin also decondenses).
4. Methylated haploid parental genomes undergo demethylation (more in next lecture).

Question 10

Zygotic genome activation is also called what?

Answer 10

Embryonic genome activation

Question 11

Define “zygotic genome activation.”

Answer 11

The transformation of highly differentiated gametes into the totipotent blastomeres which will begin their own differentiation later.

Question 12

Contrast between maternal and paternal chromatin before the formation of the zygotic genome.

Answer 12

1. Sperm chromatin condensed around protamines with S=S bonds replacing histones.
2. Maternal chromatin also very condensed until female pronucleus is formed.
3. Note: both maternal and paternal genome appear to be transcriptionally inactive at this stage.

Question 13

What three reasons make maternally-derived histones essential for zygotic genome activation?

Answer 13

1. Hyperacetylated histone enriches zygotic chromatin.
2. Histone acetylation patterns change folding of nucleosomal fiber to make chromosomes more accessible.
3. RNA polymerase II is also enriched temporarily at the nuclear periphery (to begin transcription of zygotic genome?).

Question 14

Onset of zygotic genome activation depends on what three things?

Answer 14

1. Maternally-derived inherited proteins.
2. Post-translational modification of these maternally-derived proteins.
3. Protein phosphorylation which is catalyzed by cAMP-dependent protein kinase.
4. Note: protein synthesis is NOT required for onset of zygotic genome activation.

Question 15

What are three purposes of zygotic genome activation?

Answer 15

1. To destroy oocyte-specific transcripts.
2. To replace maternal transcripts that are only common to oocyte and early embryonic development.
3. To promote reprogramming of gene expression that is not coupled in oocyte.

Question 16

On an absolute scale, when does zygotic genome activation truly begin and why?

Answer 16

After 1-cell embryo becomes transcriptionally active with functional RNA polymerase I, II, and III (only a few hours after fertilization).

Question 17

When are various times of zygotic genome activation in mouse, human, and cow embryos?

Answer 17

1. Mouse = 2-cell stage (minor activation because of acetylation of histones)
2. Human = 4-8 cell stage
3. Cow = 2-cell (recent studies; older studies say 4 or 8)

Question 18

What are three possible mechanisms for the first round of embryonic DNA replication leading to the initiation of transcription?

Answer 18

1. DNA replication may be involved in chromatin remodeling.
2. DNA replication may disrupt nucleosomes which inhibit translation.
3. DNA replication may provide an opportunity for transcription factors to have access.

Question 19

What happens when 1-cell zygotes are treated with DNA transcription inhibitors?

Answer 19

No zygotic activation occurs and zygote remains at 1-cell stage.

Question 20

What are five maternally inherited translational factors found in oocytes and early stage embryos?

Answer 20

1. Sp1
2. TBP
3. CBP
4. m-TEAD-2
5. (in mice) eIF-1A

Question 21

Describe the abundance of maternally inherited transcription factors in oocytes vs. early stage embryos and explain a reasoning for this change.

Answer 21

1. Decrease during oocyte maturation.
2. Increase at 2-cell stage.
3. Change reflects degradation of maternal mRNA & replacement with zygotic factors following zygotic genome activation.

Question 22

Maternal mRNA and maternally inherited proteins stored in oocytes is sufficient to support blastocyst development up until what stage?

Answer 22

8-cell stage.

Question 23

Describe the three stages of maternally inherited mRNA in a developing embryo.

Answer 23

1. It becomes polyadenylated (the polyA tail needed for RNA maturation and function).
2. It becomes translated.
3. It begins to degrade within a defined period after fertilization.

Question 24

What are two possible ways in which maternal mRNA activity/recruitment may be involved in zygotic genome activation?

Answer 24

1. Involving its core components of transcriptional machinery.
2. Changing polymerase II into an activated form for transcription.

Question 25

What is the difference between paternal pronuclei transcription levels and maternal pronuclei transcription levels after fertilization and why?

Answer 25

Paternal pronuclei have higher levels of transcription and it is possibly due to the difference in chromatin structure particularly regarding the protamine-histone exchange.

Question 26

How does the protamine-histone exchange facilitate transcription?

Answer 26

By providing access to the cis-cognate DNA-binding sequence which codes for initiation of RNA synthesis.

Question 27

What are two specific biochemical differences between paternal and maternal DNA that may account for the different levels of transcription between the two?

Answer 27

1. Higher paternal acetylation on histone H4

2. Lower paternal methylation of DNA

Question 28

Why is 1-cell embryo DNA transcribed but not translated?

Answer 28

There exists an uncoupling of the transcription and translation during this stage (mechanism unknown) which may prevent premature gene expression before chromosome remodeling and formation of single zygotic pronucleus.

Question 29

In the absence of translation, what are two possible reasons why a 1-cell embryo is transcribed?

Answer 29

1. Involvement in chromosome remodeling.

2. May mark promoters for following cleavage.

Question 30

When is coupling established between transcription and translation in early embryos?

Answer 30

Rapidly established following first cleavage.

Question 31

Studies on mice show what protein as the first produce of zygotic genome activation and what is one of its roles?

Answer 31

Heat shock protein 70; chaperone protein assists other proteins in molding into correct configurations and positions.

Question 32

What cell stage marks the transition from maternal to zygotic gene dependence?

Answer 32

2-cell stage.

Question 33

At what cell stage is maternal mRNA ~90% degraded?

Answer 33

2-cell stage.

Question 34

Zygotic gene activation is sensitive to the protein synthesis of what molecule and what are four reasons why?

Answer 34

Alpha-amanitin, because:

1. Inhibits RNA polymerase II and III.
2. (obviously) increases alpha-amanitin.
3. Decreases protein synthesis.
4. Embryo is arrested.

Question 35

How many proteins are needed for zygotic genome activation and when?

Answer 35

1. 40

2. After completion of first mitosis.

Question 36

What may happen in vitro to indicate incompetence of zygotic genome activation?

Answer 36

If embryo cleavage is arrested in vitro culture.

Question 37

At what stage during embryo development does transcriptional repression occur?

Answer 37

Starts sometime between S-phase in 1-cell embryo or the formation of the 2-cell embryo and increases as developing embryo becomes 4-cells.

Question 38

What are four things to note about chromatin’s involvement in the repression of transcription during early embryonic development?

Answer 38

1. Histone-1 and 4 (scaffolding proteins) change structure after first cleavage.
2. Histone-1 leads to chromosome condensation (repression of transcription).
3. Although Histone-4 is hyperacetylated at the 1-cell stage, it becomes deacetylated at 2-cell (proceeding to 4-cell stage) and this represses transcription.
4. Note: transcription repression also reduces promoter activation.

Question 39

What are two ways in which the second round of DNA replication may be involved in transcriptional repression of early embryos?

Answer 39

1. Transiently allowing expression of endogenous genes.

2. Preventing transcription complex from assembling on their respective promoters.

Question 40

How could one argue that gene expression is an opportunistic process during the early stage of embryo development?

Answer 40

By pointing out that only genes with strong promotors and/or enhancers added to the correct transcriptional factors present will be expressed.

Question 41

What is the significance of a TATA-box in transcription initiation?

Answer 41

It can stimulate promoter activity to begin DNA transcription.

Question 42

What is the difference between a DNA promoter and DNA enhancer?

Answer 42

Promoters include local transcription binding sites but enhancers include transcription sites distal to their position which impose specificity on promoter activity.

Question 43

What enables an embryo to overcome transcriptional repression (eventually) and how?

Answer 43

Enhancers (which require co-activators) by adhering to DNA chromatin areas with DEacetylated histone-4 and stimulate “weak” promoter activity.

Question 44

What three points prove that TATA-box’s are not needed to relieve transcriptional repression in early embryos?

Answer 44

1. ~70% of oocyte transcription derived from TATA promoters.
2. In contrast, 25% of 2-cell and <10% of 8-cell embryo transcription is initiated from TATA promoters.
3. By the time the embryo reaches the blastocyst stage, only ~5% of transcription is derived from TATA promoters.

Question 45

What are three reasons why TATA-box isn’t needed for transcription repression relief in embryos?

Answer 45

1. Many housekeeping genes are regulated by promoters without TATA-boxes.
2. Preimplantation development may need TATA-box promoters more to help support the large requirement of housekeeping genes.
3. In early development, TATA-box promoters also enhance expression of other critical early-development genes like Oct4 (marker of totipotency).

Question 46

What are two possible reasons why transcription is repressed immediately following zygotic genome activation?

Answer 46

1. To prevent expression of inappropriate genes before appropriate gene reprogramming occurs.
2. To sculpt new gene expression profile in a way where it is more compatible for further development.