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What do intrinsically disordered regions allow for on TFs?
allow for weak mutivalent interactions with other TFs and cofactors
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What happens when IDRs (intrinsically disordered regions) promote protein-protein interactions?
nuclear condensates
These biomolecular condensates enable liquid-liquid phase separation and the establishment of “transcription factories” specializing in specific stages of transcription
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When do transcriptional condensates form?
Transcriptional condensates have also been described to form transiently in response to stress signals (e.g., heat shock)
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How do transcriptional condensates act as regulatory hubs?
Those condensates are thought to serve as regulatory hubs that rapidly turn on/off the expression of target stress-responsive genes
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Epigenetics
Epigenetics is the study of heritable genetic information that does not involve changes in the nucleotide sequence of DNA, but is manifested as chemical marks that are added to either DNA or chromatin proteins following replication
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Epigenome
The sum of the epigenetic marks in a genome is called the epigenome and the patterns of these marks vary between different cell types, tissues, and individuals of the same species
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What do epigenetic differences in chromatin structure result in?
differences in transcription and lead to different cell fates and morphologies
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Example of epigenetic DNA modification in eukaryotes
methylation of cytosine
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What are the ways that histones can be modified?
- Histone acetylation
- Histone methylation
- Histone phosphorylation
- Histone ubiquitination
- Variant versions of histones
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Nucleosomes
Nucleosomes, the basic unit of DNA packaging, consist of ~200bp of dsDNA wrapped nearly twice around a histone core made of 8 subunits: two H2A–H2B dimers and one H3–H4 tetramer
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What are the basic unit of chromatin?
Nucleosomes
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What feature of histones determines the affinity the nucleosomal particles have?
the positive charge of histones
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What is the "histone fold"
a highly conserved structure (the so-called histone fold) made of three a-helices connected by two loops
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What formations do the four core histones form?
All four core histones form a highly conserved structure (the so-called histone fold) made of three a-helices connected by two loops
These interact to make heterodimers
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globular core of the nucleosome
The histone fold domains of the eight core histones come together to form the globular core of the nucleosome
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Name the partners that histones are interdigitated with:
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What feature of histones allow for post-translational modifications?
- flexible N- and C- terminal tails on core histones
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Which histone plays a key role in the formation of higher order chromatin structures?
- H1
- binds at point of entry and exit of DNA from the core particles
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Chromatin formation with and without histone H1
- with: zigzag pattern
- without: beads on a string
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What supplys the energy for chromatin remodeling?
ATP hydrolysis
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Three ways that nucleosomes can rearrange to make DNA accessible
- sliding
- adjust spacing
- displace the nucleosome
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Why is DNase hypersensitivity an effective assay for detecting chromatin rearrangements?
Naked DNA regions or gaps in the nucleosome-covered DNA are no longer “protected” by the histones and are hypersensitive to endonuclease digestion
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Describe the process of DNase-seq
Cells are lysed to release nuclei and DNase I treatment is performed on isolated, permeabilized nuclei
After the digestion, nuclei are lysed, and the chromatin digestion products are fractionated (e.g., size-separated on a gel)
50-100bp DNA fragments are purified, and sequencing adaptors are ligated
Next-gen sequencing is performed, and the reads obtained are mapped to the genome to identify DNase-hypersensitive sites
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Describe the process of MNase-seq
Nuclei are isolated and chromatin is crosslinked and digested with Micrococcal Nuclease that in the presence of Ca2+ digests all exposed DNA
Nucleosomes and TFs shield DNA “footprints” from digestion and protected DNA fragments are purified, adapted, next-gen sequenced and the reads are mapped to the genome
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Describe the process for ATAC-seq
ATAC = Assay for Transposase-Accessible Chromatin
Nuclei are isolated and treated with Tn5 transposase that cuts and tags accessible DNA with adapters (“tagmentation”)
Tagged DNA fragments are purified, amplified by PCR, next-gen sequenced and the reads are mapped to the genome
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Describe the process of FAIRE-seq
FAIRE = formaldehyde-assisted isolation of regulatory elements
Cells are treated with a crosslinking agent (formaldehyde), chromatin is purified and sheared by sonication
Phenol/chloroform extraction is performed leading to the partitioning of protein-DNA complexes into the organic phenol phase and leaving naked DNA in the aqueous phase
DNA from the aqueous phase is purified, adapted, next-gen sequenced and the reads are mapped to the genome
Alternatively, qPCR can also be performed on select targets of interest
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What process is a prerequisite of transcription, replication and DNA repair in all eukaryotes?
Chromatin remodelling
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What enzyme is a key component of all chromatin remodeling complexes?
ATPase
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Name the four major classes of chromatin remodeling machineries:
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