L3 Chromatin Structure and Assembly in Eukaryotes Learning Objectives

Define histone variants, give one example, and say where in the genome the variants are found

• The four core histones are the most common

• Other histone variants can be incorporated into nucleosomes, and are found at special chromatin locations

• For example, H2A.X is phosphorylated at sites of DNA double-stranded breaks and is thought to recruit repair machinery

Predict where along a gene nucleosomes are absent, and explain why promoters and terminators are relatively histone-free

Explain what a DNase I hypersensitive site indicates about the local chromatin state

They’re present in chromatin regions that lack nucleosomes

Describe how a nucleosome is built: which dimers associate with DNA first, how much DNA wraps the octamer, and the numbers that go with it

• DNA wraps around a histone complex in a left handed manner to form a nucleosome - the histone complex at the center is the histone octamer

• The histone octamer has two each of the four core histones H2A, H2B, H3, and H4 (histone variants exist)

• The two H3-H4 dimers associate with DNA

• The two H2A-H2B dimers then associate to form the octamer

• About 146bp of DNA wrap around the octamer to form the nucleosome (1.76 turns)

Describe what H1 contributes to compaction, and what happens to chromatin when H1 is removed

• H1 is a linker histone that binds to the linker DNA in between successive nucleosomes, helping compaction. Formation of the 30 nm fiber involves histone H1. The core histone tails are also involved in the formation of the 30 nm fiber, but it is not fully understood how. The 30 nm fiber is then compacted further into compact chromosomes in which large loops of chromatin are anchored to a central scaffold

• Knocking out H1 leads to loss of chromatin structure and embryonic lethality

Explain how micrococcal nuclease digestion produced the ~200 bp ladder, and what that ladder established about how DNA is organized

• Micrococcal nuclease is an extracellular enzyme (from Staphylococcus aureus) that digests both DNA and RNA by targeting the linker protein. Used on DNA it produced fragments of various lengths between 170-205 bp long. The 205 bp fragments were generated by one cut about half-way between the histone octamers, and the shorter 170 bo fragments resulted from “nibbling” up to the histone octamer. Longer fragments resulted from “multiples” - where the nuclease did not cut between one or more histone octamers

• This showed that DNA is organized in a regular, repeating subunit structure and is not a random complex of DNA and protein

Explain why the left-handed wrap leaves negative supercoiling when the octamer is removed, and why that helps transcription and replication

• The wrapping of the DNA is done in a left-handed position where, if the histone is removed, it will result in a linking number deficit that will create negative supercoiling making it slightly untwisted, and this is helpful because through this loop created the two loose strands can be used for DNA transcription and replication

Distinguish the histone-fold domain from the N-terminal tail by location and function, and say which one is required for 30 nm fiber formation

• Histone-fold domain:

1. Structurally conserved motif near the C-terminus in every core histone

2. 70 amino acids - 3 alpha helices separated by two loops

3. Responsible for the binding of histones into heterodimers

• N-terminal tail:

1. Extends outwards of the core histone between the DNA coils

2. Tails are up to 25 amino acids, and have an undefined structure

3. Tails interact with other nucleosomes to help compact DNA further

4. Can also be chemically modified which are important for chromatin structure and function

• The N-terminal tail is required for the 30 nm fiber formation

Compare the solenoid (one-start) and zig-zag (two-start) models, and explain why the question is still open

• Similarities:

1. Both supported by experimental evidence

2. Both may exist in different areas of chromatin

• Solenoid (one-start):

1. Nucleosome in a spiral shape

2. Flat slides of nucleosome disks next to each other

3. H1 linker bent inside center of the spiral to provide constant thickness of the spiral

• Zig-Zag (two-start):

1. Presence of H1 linker favors this model (due to its binding geometry, physical interaction with linker DNA, and the energetic costs)

2. Zigzag histone pairs stack on each other

• The question is still open because the in vitro results yielding a uniform 30 nm structure was different than the observed 10 nm disorganized structure in vivo

Distinguish euchromatin from heterochromatin, and give one consequence each for transcription, replication timing, and recombination

• Euchromatin: Chromatin in relatively decondensed regions and stains lightly.

• Heterochromatin: More compacted regions and stains more darkly. Some regions of chromosomes are particularly rich in heterochromatin. Telomeres (special DNA at the ends of chromosomes) and centromeres are heterochromatic. Regions with highly repetitive DNA

• Chromatin structure affects transcription, but also replication, recombination, and chromosome transmission:

1. Rearrangements that place an origin of replication into heterochromatin result in late replication, possibly delaying cell division

2. Recombination involves breaking DNA and joining it to a different molecule. Heterochromatic regions experience less recombination, which can protect some parts of the genome, like the ribosomal RNA synthesis genes

3. The special histone CENP-A is needed to form a functional centromere, which is needed for proper chromosome separation

4. Some transcription does occur in heterochromatic regions, but translocation of a gene from a euchromatic to heterochromatic region can actively prevent transcription