Altklausur Flashcards
In a light microscopy image of a cell with stained DNA, you can see two states of chromatin. What are they? Describe their properties.
The two states are euchromatin and heterochromatin. Euchromatin is less densely packed, often transcriptionally active, and stains lightly. Heterochromatin is densely packed, transcriptionally inactive, and stains darkly.
What is Xist?
Xist (X-inactive specific transcript) is a long non-coding RNA that plays a crucial role in X chromosome inactivation in female mammals. It coats the X chromosome to be inactivated, recruiting proteins that modify chromatin to a silenced state.
Describe and explain the results of some ChIP experiments that are shown in the figure below.
ChIP (Chromatin Immunoprecipitation) experiments show the binding of specific proteins or histone modifications to regions of DNA.
For example, a stretch of DNA with coding regions and transposons might show binding of LHP1 to regions with histone modifications like H3K27me3, indicating regions of gene silencing.
Bld von ChIP einfügen als bespiel
Develop and explain three different experimental approaches to study if the gene AG is a target of PRC2.
- ChIP-Seq: Immunoprecipitate chromatin with antibodies against PRC2 components (like EZH2) and sequence the DNA to see if AG is enriched.
- Reporter Assays: Fuse AG promoter to a reporter gene and measure reporter activity in the presence or absence of PRC2.
- RNAi/CRISPR Knockdown: Knock down PRC2 components and measure AG expression levels through qPCR or RNA-Seq.
Describe three different types of histone modifications and name the residues on which they occur.
- Acetylation: Typically on lysines (e.g., H3K27ac).
- Methylation: On lysines or arginines (e.g., H3K4me3, H3K27me3).
- Phosphorylation: On serines or threonines (e.g., H3S10ph).
Explain why in the inheritance tree below, Mendel’s rule of inheritance is violated and explain the underlying mechanism responsible for it.
paramutation
What is a Barr body and what does it have to do with dosage compensation?
A Barr body is an inactivated X chromosome in female cells, visible as a dense spot in the nucleus.
It is a mechanism for dosage compensation to ensure that females (XX) do not have twice the number of X-linked gene products as males (XY).
Explain the effect of silencing miR156 on the SPL gene and explain what happens if the miR156 target sites in SPL are mutated.
miR156 silences SPL genes by binding to their mRNA, preventing translation.
If the miR156 target sites in SPL are mutated, miR156 can no longer bind, leading to increased SPL gene expression.
SQUAMOSA promoter binding protein-like (SPL) genes make up a unique transcription factor family mostly in green plats
Explain the underlying mechanism of vernalization.
Vernalization is the process by which prolonged cold exposure induces flowering in plants.
It involves the epigenetic silencing of the floral repressor FLC by histone modifications such as H3K27me3, mediated by the PRC2 complex.
- Prolonged Cold Exposure
- VIN3 Induction
- PRC2 Recruitment
- H3K27me3 at FLC
- FLC Silencing
- Flowering Promoter Activation
- Flowering
Define epigenetics and explain why the following example is an epigenetics phenomenon: a state change induced environmentally and then passed on to the next generation.
Epigenetics is the study of heritable changes in gene expression that do not involve changes to the underlying DNA sequence.
Environmental factors can induce epigenetic changes, such as DNA methylation or histone modification, which can be passed to offspring, affecting gene expression in the next generation.
The ribosome has three tRNA binding sites. Please name the three sites & briefly describe their function.
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a) Describe the action/function of chromatin remodeling complexes
b)Where does the energy for their activities come from?
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How is the gene dosage of X Chromosomes maintained in male & female mammals? Name
one factor or process that is involved.
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Explain how siRNAs silence transposons in the genome.
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Name four histone modifications
1.Methylation
2. Acetylation
3. Phosphorylation
4. Ubiquitination
Where in the genome are siRNA genes mostly located?
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How is DNA methylation in the CG context inherited across cell divisions (DNA replication)?
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How do histone posttranslational modifications affect
Mechanisms of Epigenetic inheritance: DNA Methylation
- DNA methylation→ inactivate gene replication → hemimethylated state
- DNA methylase methylates other Cytosine
How does the genome defend itself against transposons?
- RNAi
- DNA methylation
RNAi - How does it work?
RNAi:
gene expression silencing due to action of short RNA molecules
How does RNAi work?
1. required: long dsRNA
2. dsRNA processed by DICER → siRNA (20-25 nt)
3. siRNA associate with ARGONAUTE effector complex (RISC)
4. complementary mRNA (sequence specificity!) bound by RISC and inhibited
Where does dsRNA originate?
* Transgene (hairpin constructs)
* Complex transposon integration
* natural cis-antisense transcripts
* RNA-dependent RNA Polymerase (RDR)
Small RNAs, siRNAs and miRNAs
- Small RNAs contribute to the regulation and defense of the genome, and confer silencing specificity through base-pairing
- siRNA targets include repetitive-rich heterochromatin, transposons, viruses or other pathogens
- miRNAs and tasiRNAs targets include regulatory genes affecting developmental timing or patterning, nutrient homeostasis and stress responses
How are nucleosomes structured?
- Nucleosomes consist of 8 Histones (Octamer). H3, H4, H2B and H2A
- DNA is wrapped around Histones (ca. 147bp/Octamer) –> “beads on a string”
H3 and H4: form a tetramer (H3-H4)2, which is the core of the nucleosome. The H3 and H4 histones are centrally located within the nucleosome structure.
H2A and H2B: form dimers (H2A-H2B) and are positioned on the outer surface of the H3-H4 tetramer, making contact with the DNA as it wraps around the nucleosome.
Which methods are there to analyze histone modification? Describe the process
MNase assay:
* MN (Micrococcal Nuclease) Enzyme that digest DNA
* MNase can only digest linker DNA (Located between nucleosomes, forming the “link” or spacer between the tightly wrapped nucleosomal DNA.Located in euchromatin and heterochromatin).
1. Isolate Nuclei: Obtain intact nuclei from cells.
2. Isolate Chromatin: Extract chromatin from nuclei.
3. Add MNase: Introduce enzyme to chromatin.
4. Inkubate: Incubate to allow digestion.
5. Stop Inkubation: At different times with stop solution.
6. DNA Extraction: Separate DNA from histones.
7. Gel Electrophoresis: Visualize DNA fragments. Nucleosome Ladder: Mononucleosomes (~147 bp), dinucleosomes (~300 bp), etc.
Note To identify which gene is on linker DNA and which on Histones use different gels. Mark DNA extraction with probe for youre gene.
8. Sequencing DNA
ChIP with Histone specific Antibodys
* ChIP is a technique used to analyze protein-DNA interactions.
1. Use cross-linking proteins to cross-link DNA to Histones
2. shearing the DNA into smaller pieces
3. add specific antibodies to modified histones
4. affinity purify histone-DNA-complexes
5. Remove cross-link
6. Examine by PCR, NGS or microarrays
DNase I hypersensitivity assay
* DNaseI can only access open chromatin (Refers to broader regions of the genome where the chromatin is in a relaxed, accessible state, typically associated with active regulatory elements.Located only in euchromatin)
solate Nuclei: Obtain intact nuclei from cells.
1. Isolate Chromatin: Extract chromatin from nuclei.
2. Add DNase I: Introduce enzyme to chromatin.
3. Inkubate: Incubate to allow digestion.
4. Stop Inkubation: At different times with stop solution.
5. DNA Extraction: Separate DNA from proteins.
6. Gel Electrophoresis: Visualize DNA fragments. –> Hypersensitive Sites: Smaller fragments indicating open chromatin regions.
