Lecture 5 Flashcards

Question

What is Luciferase Reporter Assay ?

Answer 1

Tests whether a transcription factor can activate a specific promoter Cells are transfected with: - plasmid encoding the transcription factor - plasmid with a promoter of interest driving the expression of Luciferase enzyme - Luciferase is expressed if the transcription factor activates the promoter *Could mutate specific sites, in a cell but still not very physiological because all on plasmids (no chromatin structure)

Answer 2

ChIP-Seq to Determine Genome-Wide Binding of Chromatin-Interacting Proteins In Vivo - Tests for in vivo binding of the protein to chromatin in a specific genomic region - In this case the binding can be indirect, for example mediated by other proteins. - Protein can be TF, a histone, a histone with specific post-translational modifications  1. Cross-link proteins to DNA (with paraformaldehyde) 2. Sheer DNA into one-nucleosome fragments by sonication or treatment with an enzyme such as micrococcal nuclease (fragments ~1 nucleosome in size, 150-200nts?) 3. Add an antibody specific for your protein of interest, such as a transcription factor, or certain form of modified histone, and perform immunoprecipitation 4. Reverse the crosslinks, isolate the DNA and perform sequencing 5. Perform bioinformatics analysis – this involves aligning the sequences against the genomic sequence to determine what regions of the genome have been pulled- down... These are the regions bound by your protein!   Plot nucleotide positions in the genome (X) vs Fold enrichment of the sequence over control in the ChIP- Seq experiment (Y) —> get peaks at sites that where your protein of interest interacts with DNA

Answer 3

Both answer the same question Related Techniques to ChIP-seq to Map Protein Binding Across the Genome CUT&RUN —> ProteinA-MNase CUT&Tag —> Protein A-Tn5 Instead of using an antibody to precipitate the protein-DNA complexes (as in ChIP-seq), you use the antibody to deliver an enzyme to the DNA, either b) micrococcal nuclease (MNase) to cut the DNA or c) transposase (Tn5) to insert adapter sequences into the DNA. - Can tell by site where DNA is modified where the protein was binding - No cross linking - Have Ab against protein of interest but instead of using it to precipitate, you use it to deliver enzymatic activity to the site of interest - Can work with lower cell numbers that ChIP-Seq

Answer 4

Assay for Transposase-Accessible Chromatin with sequencing - no Ab involved, just expose the nucleus content - Technique to measure chromatin accessibility on a genome-wide scale - Open chromatin is tagged by Tn5 transposase, amplified, and sequenced Requires very low cell numbers and single-cell ATAC-seq is feasible (nucleus is exposed to Tn5 transposase) - No antibody is used – data independent on any assumptions about antibody specificity (which is 100% the case in ChIP-Seq) - Unbiased, non-hypothesis driven - Can know about changes in DNA chromatin accessibility at different cell stages of development for ex The more Tn5 transposase modifications are found in a specific DNA site, the more the chromatin is accessible at this site

Answer 5

Technique to detect interaction between distant parts of genomic DNA: 1. Chemical cross-linking of interacting DNA sequences 2. Enzymatic fragmentation of DNA and ligation into continuous DNA fragments 3. Sequence and alignment against the genomic sequence (Purify + PCR template?) Application: measure interactions of enhancers with promoters

Answer 6

It is at the molecular basis of pluripotency Genes not expressed in stem cells but poised and ready to be expressed when lineage committment Bivalent means the presence of both activating and repressing histone marks at a promotor or enhancer Defined primarily by ChIP-Seq studies for H3K4me3 and H3K27me3 histone modifications Not property unique to stem cells, bivalent chromatin is not 100% lost as soon as lineage committment - These bivalent marks are also actively maintained by enzymes (transcriptional regulators) - These loci have binding of RNA pol II (ready for expression - Bound by key writers of histone modifications, including Polycomb and COMPASS protein complexes Potential conformations of bivalent promoters: A) H3K4me3 and H3K27me3 occupy neighboring nucleosomes  B) H3K4me3 and H3K27me3 co- occupy the same nucleosome, localizing either on different histone tails or on the same histone tail.

Answer 7

ChIP-seq analyzes bulk cell populations – not individual cells. Hence, two scenarios may explain the co-occurrence of H3K4me3 and H3K27me3 observed by ChIP-seq on bivalent promoters: 1. In the case of ‘true bivalency’ most cells in the population carry both histone marks simultaneously. 2. Alternatively, the analyzed cells may contain a mixture of cells that either express (green) or do not express (red) the gene in focus. This will also present as ‘occurrence of both marks’ when the overall cell population is analyzed by ChIP-seq.

Answer 8

ChIP-seq analyzes bulk cell populations – not individual cells. Hence, two scenarios may explain the co-occurrence of H3K4me3 and H3K27me3 observed by ChIP-seq on bivalent promoters: 1. In the case of ‘true bivalency’ most cells in the population carry both histone marks simultaneously. 2. Alternatively, the analyzed cells may contain a mixture of cells that either express (green) or do not express (red) the gene in focus. This will also present as ‘occurrence of both marks’ when the overall cell population is analyzed by ChIP-seq.

Answer 9

2-step ChIP protocol: Tests for the presence of both histone modifications on the same DNA-molecule 1. ChIP for H3K4me3 (Ab specific for) 2. ChIP for H3K27me3 (Ab specific for) 3. Analyze to identify the DNA carrying bivalent histones ChIP followed by Mass Spectrometry analysis of the Histones Combining ChIP with mass spectrometry analyses of the histone modifications. So, if we precipitate the H3K4me3-marked histones, can we detect H3K27me3, and vice versa? * ∼15% of all H3K4me3 nucleosomes are also marked with H3K27me3 in ESCs. * For fibroblasts the co-occurrence of H3K4me3 and H3K27me3 is much lower.

Answer 10

A) H3K4me3 and H3K27me3 occupy neighboring nucleosomes  B) H3K4me3 and H3K27me3 co- occupy the same nucleosome, localizing either on different histone tails or on the same histone tail.

Answer 11

The discovery that just four transcription factors—Oct4, Sox2, Klf4, c-Myc (the “Yamanaka factors”)—can convert a differentiated fibroblast back into a pluripotent stem cell tells us something profound: A small, tightly interconnected transcriptional network is both necessary and sufficient to establish and maintain pluripotency. This means the ES-cell state is not dependent on irreversible epigenetic settings or permanent embryonic context, but rather on a self-sustaining transcriptional circuit.

Answer 12

Oct4, Sox2, Klf4, cMyc, Nanog (Yamanaka + NANOG) - could identify where they bind on a genome-wide level by ChIP-seq - Oct4, Sox2 and Nanog regulate each others’ expression through a series of complex regulatory loops. - Can act both as transcriptional activators and repressors - Genes co-regulated by Oct4, Sox2 and Nanog include both the genes required to maintain ES-cell identity and the genes repressed in ES- cells involved in ES-cell differentiation and lineage specification pathways.

Answer 13

Pluripotency transcription factors (OCT4, SOX2 and NANOG) and epigenetic regulators, including the many types of histone modifying enzymes & chromatin remodeling factors, work together to maintain ESC/iPSC pluripotency. Pluripotency factors OCT4, SOX2 and NANOG interact with histone modifying enzymes and chromatin remodeling complexes.

Answer 14

1. Microarrays (before) 2. Single cell RNAseq (more recent)

Answer 15

1. By Using Knockout Studies in Animal Models We gain knowledge about the molecular mechanisms controlling hematopoietic stem cell function by studying:  - transgenic & knockout mouse lines in which manipulation of specific genes causes changes in hematopoietic functions. * human congenital diseases of hematopoiesis (very rare) * human hematologic malignancies. Examples of Transcription Factors needed for normal Hematopoietic Stem Cell function : RUNX1 ** 2. Co-localization of the HSC transcription factors at many gene promoters (Direct protein interaction between RUNX1 and several other core hematopoietic stem cell transcription factors)

Answer 16

Example: RUNx1 KO HSCs fail to develop, the mouse is not viable (critical for hematopoiesis, but not much more information) To study the effect of the different TFs at different stages of development: Conditional Knockout Mouse Models: selectively inactivate a gene in a specific cell type (Cre/LoxP System) - Cre recombinase – an enzyme that catalyzes recombination between two loxP sites; - LoxP sites – specific 34-base pair (bp) sequences that Cre acts on. *Organ specific due to tissue-specific promotor activation

Answer 17

1. Primitive hematopoiesis 2. Hemtopoietic Stem Cell Formation 3. Fetal HSCs and Hematopoiesis 4. Adult HSCs and Hematopoiesis

Answer 18

Mouse model: VE-cadherin-Cre – Runx1 deletion in vascular endothelium VAV1-Cre – Runx1 → fine HSCs Conditional deletion of RUNX1 in VE-cadherin-positive endothelial cells demonstrates that it is indeed essential for HSC formation in mice. In contrast, Runx1 is not required in VAV1 expressing hematopoietic cells . Collectively these data show that RUNX1 function is essential in endothelial cells for hematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

Answer 19

Protein complexes responsible for transcriptional activation by histone METHYLATIONS - SET1 and MLL1 are the catalytic subunits of the COMPASS and COMPASS-like complex - Translocations of MLL1 locus are very common in acute lymphoblastic leukemia (ALL, >50%)

Lecture 5 Flashcards

(43 cards)