Lecture 8: Chromatin, histones, and the nucleosome

on or basal, off, nucleosomes block transcription factors from binding

The ground state of bacterial promotors is _____ while in eukaryotes it’s _____ because _______

formation of the 30nm fibre

Histone H1 plays a critical role in…

6, solenoid

The 30nm fibre has ____ nucleosomes per turn and they are organized into a _____

remodelling into the 10nm fibre

Chromatin _______ is a prerequisite for gene activation

the angle of the exiting DNA

Histone H1 allows for tighter DNA wrapping by changing _____

constant, variable

The histone core DNA is _____ while the linker DNA is ____ between species

1.66

DNA wraps around the nucleosome ~____x

histone fold, 3 alpha helices, carboxy helices, an amino helix

The ______ domain of the all histone tails is well conserved and is composed of _____. Histones 2A and 2B have _____ and H3 has _____

two H3 and two H4 forming a tetramer that binds DNA, H2A and H2B dimers incorporated one at a time

Nucleosome assembly starts with ______ followed by _____

central 60bp, outer 13bp, central-flanking 30bp

The H3-H4 tetramer binds _____ and _____ while the H2A and H2B dimers bind _____

lysine, arginine, nucleosomes are positively charged

The histones contain many _____ and ____ amino acids residue therefore ______

Micrococcal Nuclease

Nuclease that can digest the linker DNA between nucleosomes

DHS

DNAse I hyper-sensitive site

identify regulatory regions of genes, nucleosome-free, promoters of expressed genes or enhancers, binding of TFs that displace the nucleosomes

DHS refers to a technique used to ______. DNAse I hyper-sensitive sites represent regions that are ______ which are often found at ______ and are generated by ______

40, 33, 7, sequence-specific binding, is related to DNA sequence

The histone octamer makes ~____ contacts with DNA; of these, ~___ are to the phosphate backbone and ~____ contact the ____. Nucleosomes do not _____ but their positioning _____

proteins binding to DNA lock the nucleosomes into a specific location

The most common cause of nucleosome positioning is when…

which regions of the DNA are in the linker and which face of DNA is exposed on the nucleosome surface

Nucleosome positioning may affect:

poly A/T, transcriptional regulation, nucleosome-free, gene activation

_____ tracts are highly enriched in eukaryotes but not prokaryotes— they are important for ______. They are stiffer regions of DNA which keeps them _______ to facilitate ______

free region, positioning sequence

The nucleosome ______ is flanked by nucleosome _____

NPS

Nucleosome positioning sequence

NFR

Nucleosome-free region

induce dsDNA breaks in the linker DNA with micrococcal nuclease, isolate cleaved DNA and cut with REs, separate on agarose gel and run on Southern blot

To test nucleosome positioning at a define locus:

cross-link chromatin with formaldehyde, digest with micrococcal nuclease, deep-sequence all 147bp fragments

To test nucleosome positioning genome-wide:

Type I Promoter

Found in adult tissue-specific genes or terminally differentiated cell-specific genes— can contain TATA boxes and/or Inr

Type II Promoter

Found in housekeeping genes that contain CGI in mammals

Type III Promoter

Found in developmental transcription factor genes and contain CGI in mammals

focussed and sharp, dispersed and broad, dispersed and broah

Type I promoters are _____, type II are _____, and type III are _______