VL 25 (Isabel Bäurle) Flashcards
The conundrum
Nucleosome as basic unit of chromatin
- Human DNA: 2m
- Human nucleus: 10 μm→10,000x compaction
- Chromatin organizes and compacts DNA
Definitions:
* Nucleosome: basic structural chromatin SU; 200 bp DNA; histone octamer
- Histone tails: flexible amino-/carboxy-terminal regions of core histones that extend beyond nucleosome surface; sites of extensive PTM
- Linker histones: e.g. H1; histone family; not components of nucleosome core; bind nucleosomes and/or linker DNA; promote 30 nm fiber formation
- Nonhistone: any structural protein found in chromosome except one of the histones
DNA Is Organized in Arrays of Nucleosomes
-
core DNA:
145-147 bp; found on core particles produced by prolonged Micrococcal nuclease (MNase) digestion -
Linker DNA:
7-115 bp; susceptible to early cleavage by nucleases
Chromosomes consist of heterochromatin
Originally this distinction was based on cytological experiments.
Heterochromatin and euchromatin form distinct domains in nuclei:
A) „holes“ where heterochromatin is located
B) dots = heterochromatin; negative picture of A
C) each dots represents 1 chromosome position
Chromatin regulates accessibility and compacts
Nucleosomes are covalently modified:
- methylation, acetylation, phosphorylation, ubiquitylation, sumoylation
- modifications combined→defined function of local chromatin region (“histone code hypothesis”)
How do histone posttranslational modifications affect
How to study chromatin? – Chromatin-Immunoprecipitation (ChIP)
goal:
–> detect which sequences are associated; chromosome location
1. covalent cross-linking between DNA-lamins by means of aldehydes (e.g. glutaraldehyde)
2. lysing cells
3. sonicate cells mech.
→DNA separation
→protein-associating DNA-segments, alternative: RE (DPN1, MBO1)
4. Ab against lamin; latex beads with coupled protein A
→ bind Ab
→precipitation of lamin-DNA (Immunoprecipitation)
5. wash→remove non-lamin-bound DNA-fragments
6. DNA-detachment from immunoprecipitated
7. analysis
How to analyze histone modifications?
Chromatin immunoprecipitation (ChIP) with antibody that recognizes specific modification
followed by analysis of precipitated DNA
- Quantitative PCR
- Microarray hybridization (ChIP-chip)
- Next generation sequencing (ChIP-seq)
Writing, erasing, reading of histone modifications
Histone modifying enzymes are recruited to promoters via DNA-binding transcription factors
Histone Variants Produce Alternative Nucleosomes
- All core histones except H4 are members of families
of related variants - Histone variants can be closely related to or highly divergent from canonical histones
- Different variants serve –> different functions in the cell
Three H3 variants: H3.1, H3.3, CENH3
- H3.1 incorporated onto chromatin at sites of DNA repair
- Non-nucleosomal H3.1/3 with set of modifications before deposition
→determine their final PTMs in nucleosomes - Histone variants can be closely related to or highly divergent from canonical histone
When is chromatin assembly required?
during:
* replication
* transcription
* DNA repair
Replication requires assembly of nucleosomes:
- accessory proteins assist nucleosome assembly
- CAF-1, ASF1 = histone assembly proteins; linked to replication machinery
- HIRA (assembly protein) + H3.3 for replication-independent assembly
Picture:
* chromatin structure duplication involves two concerted reactions during DNA replication forks passage
* 1st reaction: transfer of parental/pre-existing histones (blue) behind replication forks
* nucleosome array gaps (created by DNA duplication) filled-in by deposition of newly synthesised histones (orange; acetylated at distinct Lys residues
Method: How to determine nucleosome occupancy and positioning?
- DNAse I, MNase hypersensitivity
- ChIP with histone-Ab
- process: polynucleosome
→mononucleosome via MNase digestion
→separation via gel electrophoresis
The nucleosome free region (NFR):
- at 5 ́/3 ́ gene ends
- sites of transcription initiation for mRNA, noncoding RNA (ncRNA)
- NFR regulation affects transcription initiation
- multiple factors known to facilitate transcription initiation by positive regulation of NFR-formation/-size
- 4 classes
Chromatin Remodeling Is an Active Process
Numerous ATP-dependent chromatin remodeling complexes use energy provided by hydrolysis of ATP.
- All remodeling complexes contain a related ATPase catalytic subunit, and are grouped into subfamilies containing more closely related ATPase subunits.
- Remodeling complexes can alter, slide, or displace nucleosomes.
- Some remodeling complexes can exchange one histone for another in a nucleosome
An Locus control regions (LCR) can control a domain:
- LCR at 5 ́ end of chromosomal domain
- multiple DNAse hypersensitive sites
- regulate gene clusters
- usually regulate loci that show complex developmental or cell-type specific patterns of gene expression
- control transcription of target genes by direct interactions, forming looped structures
Picture
* LCR makes physical contact to gene which is supposed to be activated
* 3C goal: with which regions is this particular region in contact with?
Insulators define transcriptionally independent domains:
- boundary regions between TADs contain insulator elements → block passage of (in)activating effects from enhancers/silencers…
- barriers against spread of heterochromatin
- specialized chromatin structures with hypersensitive sites
Heterochromatin blocked by insulator;
euchromatin doesn ́t change into heterochromatin
