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What are the 5 HAT families

HAT1

MYST

GNAT

P300

Rtt109

Are these markers cause repression or transcription?

H3K27me3

H3K4me3

H3K27me3 - repression

H3K4me3 - transcription

What are the writers, readers and erasers for:

• Lysine Acetylation

• Lysine Methylation

• Serine/Theroine (phosphate)

• Arginene (methylation)

1. Writer = HAT, Reader = Bromo, Eraser = HDAC

2. Writer = KMT, Reader = Chromo, Eraser = KMD

3. Writer = Kinase, Reader = SANT, Eraser = PPTase

4. Writer = PRMT, Reader = Tudor, Eraser = PAD

Which met markers cause silenceing

H1K26

H3K9

H3K27

H4K20

H4K59

What do H3K4/79 do

methylate aa residues in the histone core (not tail) -> activation

What is totipotency? Pluripotent?

state of cell when they have equal ability to become any tpe of cell as the embryo differentiates

ex. blastomeres

state of cells when they can become many cell types but not all

ex. Inner cell mass of trophoblast

Explain the process of first differentiation of totipotent cell

occurs between the morula and blastocyte stages

• blastomere is outside of morula → turns to trophoblast → will turn into placenta

• Inner Cell Mass cells of the blastocyte start development of embryo tissue

• trophoblast cant produce any of the cells derived from ICM and vice versa → both pluripotent

What are ESC derived from?

• ICM from day 5-8 of morula stage embryo

What are the 3 primordial germ layers differentiated from ICM?

• ectoderm - skin / neuronal/ pigement cells

• mesoderm - blood cells + bone + muscle

• endoderm - lung + thyroid + pancreatic cells

What are the markers for ESCs?

Oct4 and nanog Transcription factors

What is induced pluripotency + what genes/transcription factors are needed to induce pluripotency / how do you induce pluripotency

- overexpression of a small set of TFs so that somatic cells can resemble ESC without going through development

- Oct4, Sox2, Klf4, Cmyc

What does differentiation do to the epigenetic nature of chromatins

• decreases their dynamic nature → characteristics simillar to heterochromatin

• diheterochromatin markers go from disperse localization to concentrated foci in differentiated cells → increased global levels of H3K9me3 (heterchromatin marker linked to repression and decreased acetylation)

• acetylation of histones is linked to euchromatins

What is the epigenetic nature of ESC chromatin and genome

• ESC genome subjected to generalized histone acetylation and H3K4me → transcription activating modifications

• ESC chromatin nature is euchoromatic → high gene expression (makes sense due to its pluripotency) → becomes more heterochromatized when differentiated

What are bivalent domains? How are they relevant to ESCs?

large areas of repressive histone modification (H3K27me2/3) with smaller regions of permissive repression (H3K4me or H3K9me2 or H3K9ac) → markers usually mutually exclusive

• act to silence genes to maintain pluripotency in ESCs

During differentiaton, they “resolve” so that silent genes become concentrated for H3K27me2/3 only (the markers for no transcription)

Explain differentiation with bivalent domains

starts with bivalent domains where there are regions which repressive histone modifications (H3K27me3) and permissive epigenetic marks (h3k9ac or h3k9me2)

• during differentation they turn into a monovalent state where the genes must be either expressed (by losing the silencing marks/h3k27me3) or silenced by losing the permissive marks (h3k9ac or h3k9me2)

Explain relationship between ESCs and heterochromatin + LOCKS

Het is detected by presence of h3k9me2/3 which is found in esc but not in great amounts in comparison to the amounts found in differentiated cells

• LOCKS: large organized chromatin K9 modifications (large regions of heterochromatin) → increase in development/differentation of the esc → decreases the acetylation (which is required for the h3k9me2/3 modifications)

Explain the method that was used to create Dolly + why it was done

• Take donor nucleus from a sheep’s udder

• take egg cell from adult female sheep

Fuse the two together via electric shock

Fused cell → divides normally → develops into embryo

• Embryo placed into foster mother’s uterus

• Embryo develops normally

Questions:

Can an egg whose nucleus has been removed support development using genetic material from a different cell?

What is the nuclear characteristics of cilitates?

• nuclear dimorphism: small micronucleus and large macronucleus

Compare the characteristics of the micro and macro nucleus

Mic

• diploid (2n)

• germ genome

• transcriptionally silent

• for reproduction

• miotic growth

Mac

• polyploid

• somatic genome

• transcriptionally active

• for regular cell function

• amiotic growth

What are histone variants of the Mic and Mac ?

Mac: hv1 (H2A.Z) + h2v (h3.3) + Hholp (H1 linker)

• hyperacetylation of histones → H3K4me3 + H4ac (same histone tail)

• Associated sequences of H3K9me2/3 and H3k27me3 are deleted (markers = silencing)

Mic: Cnplp + linker micLH (centromere related histones)

• lack of histone acetylation

What is the general life cycle of the Mac + modifications

• Mic and Mac have diff genomes

• new Mac → original germline genome undergose modifications

• Remove repeitive elements

• unscramble sequence of protien coding genes

• increased ploidy

Explain the mechanism for the modification of removing repeitive elements (IES + MDS)

IES = internal eliminated sequences → sequences that are removed during Mac development

MDS = Macronucleus destined sequences → sequences that are retained and incorporated into new Mac

1. scnRNAs are produced during meiosis/sexual reproduction from the Mic DNA → used to identify sequences that should be eliminated

2. Info between the old and new Mac is compared (old provides info on what should be contained in the new Mac)

3. scnRNAs → homologous to Mic seq

4. scnRNAs that bind to the old Mac RNA sequence get deleted

5. scnRNAs that dont bind are tranported to the new Mac and target seq that need to be deleted

Explain the sexual cycle of paramecium cells

start with 2n Mic

1. Mics go through meiosis → haploid (n)

2. all except one haploid nucleus breaks down → which then goes through mitosis

3. the 2 cells exchange one haploid nucleus + fuses → cell contains zygotic nucleus

4. goes through 2 miotic divisions → four 2n nuclei

5. 2 of them differentiate into Mac

6. cell division occurs and each cell has 2 Mic 1 Mac

Why cant Ciliates reproduce asexually forever?

• the macronucleus contains an epigenetic state that can include the development of the new Mac

• the amitotic divisions dont allow for 50:50 chromosome segregation

• more miotic divisions → more imbalance of the Macs genome

• Mac becomes more homozygous and Mic becomes more heterozygous

what is ies elimination in paramecium?

ku70/80: recognizes + binds + stabilizes broken ends of dsdna

spt161: euk transcripton + dna replciation + repair

What is the mechanism for unscrambling modification?

Oxytrochia - some genes in Mic have DNA segments in the wrong order

• rearranged during Mac development

• Old macro produce long RNAs → sent to new Macro

• Long RNAs act as templates to guide and reorganize the segments

Epigenetic inheritence:

• The rearranged chromosome seq were found in the F2 and F3 generation of offsprings

What are the 4 HDAC families + significant sirutiins

HDAC - histone deaceyltases - enzymes that remove acetyl groups from histone tails

Zinc dependent:

Class 1 - for general function (nucleus + cytoplasm)

- Class 2 - restricted expression + for more specific functions (shuttles in and out nucleus)

- Class 4 - only member HDAC11 + structural similarities with class 1 and 3

NAD+ dependent:

- Class 3 - also called sirituins

SIRT4 and 5 are in the mitochondria -> dont acetylate histones in the nucleus and has different functions in the mitochondria

Explain the 3 types of blotting techniques

Southern: detects DNA seq

Northern: detects RNA molecules

Western: detects specific protiens

explain 3C method

• studies how different regions of DNA interact with each other DNAs 3d structure

1. crosslink DNA to associated protiens

2. use restriction enzymes to digest the DNA into smaller pieces

3. DNA fragments that were close together in the nucleus are ligated together

4. remove crosslink

5. use PCR to amplify

What is the fbx15 promoter?

• inactive in differentiated cells

• becomes active when cells are reprogrammed (by the 4 factors: oct4 sox2, cmyc,klf4 that induce pluripotency)

• act as a marker to identify which cells have been reprogrammed

In the Brownell study, what were they trying to awnser, what techniques did they use, why did they use them + the overall findings

Question:

• what is the gene or protien responsible for HAT A activity

• knew that there was a connection between histone acetylation and gene transcription but mechanism was unknown

Techniques:

1. protein purification - Why?: isolate the p55 gene from the macronuclei and

2. peptide sequencing - Why?: identify the amino acid seq of p55

3. PCR - Why?: used the aa-seq as primers to amplify fragments of the gene

4. antibody prcduction - Why? create antibodies against the p55 peptides to identify whether the protien they identified WAS present in HAT A activity

5. clone Gcn5p and introduce it into bacteria

Why?: to see if it had HAT activity

Findings

• p55 gene is responsible for HAT A activity

• p55 gene is homologous to Gcn5p

• Test HAT activity in Gcn5p

In the Takahashi study, what were they trying to awnser, what techniques did they use, why did they use them + the overall findings

Question:

- what genes are responsible for reprogramming differentiated fibroblasts in iPS cells and do they behave like ES cells?

Techniques:

1. G418 antibiotic selection to identify cells activating fbx15 (promoter associated w esc identity)

2. ChIP to examine histone modifications at gene promoters

3. Bisulfite sequencing to examine dna methylation

4. Western blotting to identify protein expression

5. Blastocyte injection to introduce iPS cells into developing embryos

Findings

- identified four factors: Oct4, SOx2, Klf4, cmyc (Nanog was not required) -> needed to reprogram a differentiated cell back to pluripotent state

- normal: embryonic pluripotent stem cell -> differentiated fibroblast

- reprogram: differentiated fibroblast -> induced pluripotent stem cell

what is ChIP used for ?

used to determine if a specific protien is associated with a specific DNA sequence

• crosslink DNA w protien → fragment the dna → add antibody that binds to the protien → reverse cross link → PCR