Ch. 4 DNA Packaging in Eukaryotes

Human genome is about 3 giga-bp (approx. 2 meters). A typical human cell is ~20 micrometers and a nucleus is ~5-10 micrometers. How does our genomic DNA fit into such a tiny nucleus?

DNA interacts with specific proteins to form a nucleoprotein complex called chromatin

Can cells develop into something else?

Despite having the same genome, cells not only develop into distinct types but also maintain their identity across cell divisions. For example, blood cells stay blood cells, they can’t turn into a neuron.

True or False: DNA exists only transiently for a brief phase in mitosis, when chromosomes must be accurately separated into daughter cells, in the highly condensed structures (X shaped).

True

Cells spend most of their time in what phase?

interphase (G1, S, and G2), when DNA, RNA, and proteins are actively synthesized on demand and depending on the needs of the cell

During interphase, chromatin exists in 2 different states of condensation. What are they?

1. Euchromatin

2. Heterochromatin

Euchromatin

• Less condensed and more accessible to transcription factor

• Generally associated with active gene transcription

• It is more open and spread. It is more accessible.

Heterochromatin

• More condensed and less accessible

• Generally associated with transcriptionally inactive genes

• More closely packed

Can chromatin transition between euchromatin and heterochromatin by changing the proteins associated with DNA?

yes, these changes help regulate whether genes are turned on or off

What are the 3 levels of chromatin organization?

1. DNA wrapped around histone proteins (nucleosomes) like “beads on a string”

2. Multiple nucleosomes coiled (condensed) into 30 nm fiber (solenoids) structures

3. Higher-order packing of the 30 nm fiber into the eventual familiar metaphase chromosome

Histones

a type of protein that plays a central role in DNA packaging

What are the 5 major classes of histones?

H1, H2A, H2B, H3, and H4

Are histones basic or acidic? Positively or negatively charged?

basic, positively charged proteins, rich in lysine and arginine

What enables the amino acids in histone to bind to the acidic, negatively charged phosphodiester backbone of double-helical DNA

their positively charged side chains

True or False: BOTH eukaryotes and prokaryotes have histones.

False, only eukaryotes do.

What do the “beads on a string” mean?

• Beads: DNA-wrapped nucleosomes

• String: linker DNA of uniform length connecting adjacent nucleosomes

What is the basic unit of chromatin?

nucleosome

What does the nucleosome core particle contain?

• ~1.47 bp of DNA wrapped around a histone core ~1.7 turns

• a histone octamer containing 2 copies each of H2A, H2B, H3, and H4 histones

linker histone

Histone H1. H1 binds near the sites where DNA enters and exits the nucleosome, stabilizing DNA around the nucleosome and promoting higher-order chromatin compaction.

histone fold

consists of 3 alpha-helices connected by 2 short loops

What terminus of the protein does not participate in the fold and plays a crucial role in the regulation of chromatin structure?

The N-terminus

What 2 sub complexes are first formed in the assembly of the nucleosome core?

• H3 interacting with H4 to form a H3.H4 heterodimer

• H2A interacting with the H2B to form H2A.H2B heterodimer

Histones use their histone folds to fit together in what kind of interaction?

a “handshake” interaction

2 H3.H4 heterodimers interact to form a tetramer, and DNA begins to wrap around it. 2 H2A.H2B heterodimers then do what?

caps the complex at the top and bottom to complete the histone octamer

Electrostatic interactions

negatively charged DNA phosphate backbone interacts with positively charged amino acids in histones

Hydrogen bonds form where in histone-DNA interactions?

between histones and DNA bases

True or False: Most histone-DNA interactions are not sequence-specific, allowing nucleosome to package almost any DNA sequence.

True

What areas of the genome are where nucleosomes position themselves preferentially?

Regions with A-T rich sequences which can bend more easily as DNA wraps around the histone octamer

H1 binds DNA where…

Where the DNA joins and leaves the histone octamer and helps lock the DNA into place, acting as a clamp around the nucleosome

H1 changes the path of the linker DNA, which helps what?

orient neighboring nucleosomes so they can interact and pack more closely together

Non-histone proteins are used for?

higher order structures that help organize and compact chromatin to form the highly condensed metaphase chromosome

What are some non-histone proteins?

condensins, cohesions, and scaffold proteins

Can a highly condensed chromatin metaphase chromosome be transcribed into any RNA or protein?

No

DNA compaction allows the genome to fit inside the nucleus, but it also limits DNA accessibility to the proteins required for gene expression. What must occur for gene expression to occur?

chromatin must be able to switch between condensed and decondensed states

Changes to chromatin structure can be brought about in what 2 ways that are not mutually exclusive adding layers of complexity to regulation?

• via histone modifications

• via chromatin remodeling complexes

histone tails are rich in what?

amino acids that can be chemically modified (lysine, arginine, serine, and threonine) and can undergo various post transitions modifications (PTMs)

epigenome

the set of chemical modifications or marks that influence gene expression and are transferred across cell divisions and, in some limited cases, across generations of organisms

epigenetic marks

histone modifications that help regulate chromatin structure and gene expression without changing the DNA sequence

What are the 3 common histone modifications?

1. methylation

2. acetylation

3. phosphorylation

Methylation of lysine and arginine

• adding methyl groups by methyltransferase

• can activate or repress gene expression, depending on which amino acid residue is modified and the degree of methylation

• often creates binding sites for reader proteins

Acetylation of lysine

• adding acetyl groups by acetyltransferase

• neutralizes the positive charge of lysine, weakening histone-DNA interactions

• Generally promotes more open chromatin and active gene expression

Phosphorylation of serine and threonine

• adding negatively charged phosphate groups by protein kinases

• Phosphorylation changes the electrical charge of the histone and can alter histone-DNA or protein-protein interactions

• Acts as regulatory signals involved in several processes, including chromosome condensation, DNA damage responses, and gene expression

Writers

enzymes that add chemical modifications to histones

Erasers

enzymes that remove histone modifications

What enables dynamic regulation of gene expression?

the opposing activities of writers and erases allow histone modifications to be dynamically added or removed at specific genomic regions

Modifications not only regulate chromatin structure by merely being there, but they also…

recruit proteins that recognize particular modified amino acids residues

Proteins with bromodomains recognize…

acetylated lysine residues

Proteins with chromodomains recognize…

methylated residues

Protein and histone-modifying enzymes often function within a larger multi protein complex and can recruit what?

chromatin-remodeling complexes that can further alter nucleosome positioning

Chromatin remodeling complexes

multiprotein complexes that alter chromatin structure by repositioning nucleosomes

Chromatin remodeling complexes uses what mechanisms?

• sliding nucleosomes along DNA

• evicting nucleosome components (such as H2A-H2B dimers)

• ejecting full nucleosomes (creating nucleosome-free regions)

• replacing with variant histone subunits

Different chromatin remodeling complexes all have at least one enzymatic ATPase subunit. What is the function of ATPase subunit?

It allows them to utilize the energy released from ATP hydrolysis to reposition nucleosomes

Chromatin-remodeling complexes are often recruited to chromatin how?

by recognizing histone modifications or by interacting with proteins that modify or recognize histones

Because chromatin structure plays a key role in gene expression regulation, changes in acetylation signaling resulting from dysregulated histone acetyl transferases (HATs) or histone deacetylases (HDACs) can cause…

abnormal gene expression patterns and have been identified in numerous cancers

**There are a wide variety of small-molecule inhibitors targeting acetylation signaling pathways in development for use as anti-cancer drugs.