Lecture 10 - Eukaryotic Chromatin Conformation and its Regulation of Gene Expression

Question 1

What releases a uniform chromatin structure of a series of twisted loops containing supercoiled DNA and holds the loops in place?

Answer 1

• Lysis of the bacterial cells

- loops are held in place by proteins

Question 2

What are the most abundant chromosomal proteins in eukaryote cells?

Answer 2

the histones (H1, H2A, H2B, H3, and H4)

Question 3

What are the properties of the histones?

Answer 3

• tightly bound to DNA - requires high salt concentrations to extract the histones from DNA
• High content of basic (positively charged) amino acids
• Amino acid sequence of each of the histones is highly conserved from yeast to humans
• Most abundant chromosomal proteins: ~50% histones to 50% DNA therefore, not regulators of specific genes but structural proteins in chromatin. Histones in 1:1 ratio with DNA

Question 4

What are the properties of High Mobility Group (HMG-) proteins?

Answer 4

1) Abundant chromosomal proteins, > 10^6 molecules per nucleus - as such, not specific regulators of gene expression
2) Loosley bound to chromatin - extracted by low concentrations
3) High in basic (positively charged) and acidic (negatively charged) amino acids
4) More diverse in amino acid sequence of individual HMG-proteins from one species to another
5) HMG-proteins exhibit distinct tissue-specific patterns of distribution

Question 5

What are characteristics of other NHC proteins?

Answer 5

• low abundance

- specific transcription factors, e.g. steroid hormone receptor

Question 6

What are the two experimental approaches that defined the structure of chromatin in interphase nuclei?

Answer 6

• electron microscopy of chromatin released from nuclei

- biochemical studies using nucleases, such as DNase I or micrococcal nuclase, to digest DNA in chromatin

Question 7

What are the two types of chromatin in an interphase nuclei?

Answer 7

• beads-on-a-string (10nm-11nm fibre)

- “solenoid” (30nm fibre)

Question 8

How did the biochemical studies use nucleases (DNase I or micrococcal nuclease) to digest DNA in chromatin?

Answer 8

• purified nuclei were lysed to release the interphase chromatin
• chromatin was the subjected to digestion with nuclease for either short or long periods of time
• the particles were then sedimented by centrifugation
• protein and DNA constituents of the sedimented material were purified and assayed by gel electrophoresis

Question 9

What provided evidence that DNA is organized into fundamental units of 200bp in ALL eukaryotic chromatin?

Answer 9

the mild digestion of chromatin by nucleases

Question 10

What proteins are present after short nuclease digestion?

Answer 10

two of each of histones H2A, H2B, H3 and H4 and one copy of H1

Question 11

What proteins are present after longer nuclease digestion?

Answer 11

two each of histones H2A, H2B, H3 and H4, and no histone H1

Question 12

In short nuclease digestion, what is the single copy of histone H1 attached to?

Answer 12

a portion of undigested linker DNA

Question 13

What occurs during a longer period of nuclease digestion?

Answer 13

all of the linker DNA is destroyed and releases the attached histone H1 from the core nucleosome

Question 14

True or false, chromatin in the eukaryotic nucleus is free-floating.

Answer 14

False, it is not free-floating, chromatin loops are attached to an internal protein sub-structure called the nuclear matrix, scaffold, or cage

Question 15

Is there a relationship between chromatin conformation and gene expression in eukaryotes?

Answer 15

yes

Question 16

What is the background to the experiment that showed that there is a relationship between chromatin conformation and gene expression in eukaryotes?

Answer 16

• chicken erythrocytes (RBC) retain their nuclei with DNA unlike mammals
• the most abundant mRNA in RBCs code for the alpha and beta-globin polypeptides that constitute haemoglobin, the O2/CO2 carrier. In chicken RBCs, the alpha and beta-globin genes are actively transcribed

Question 17

What is the transcriptionally-active chromatin preferentially digested by over transcriptionally-inactive chromatin?

Answer 17

nucleases

Question 18

What form of chromatin is more resistant to nuclease digestion?

Answer 18

transcriptionally inactive chromatin in the condensed conformation of the 30 nm fibre (solenoid structure)

Question 19

What for of chromatin is more rapidly digested by nucleases?

Answer 19

transcriptivelly active or transcriptionally competent chromatin in the extended conformation of the 10 nm fibre (beads-on-a-string structure)

Question 20

What controls the transition of the chromatin from the condensed (30 nm fibre) to the extended (10 nm fibre) conformation and vice versa?

Answer 20

The acetylation and deacetylation of nucleosomal histones

Question 21

What adds acetyl groups to the histone tails?

Answer 21

Histone acetyltransferase (HAT)

Question 22

What removes the acetyl groups from histones?

Answer 22

Histone deacetylase (HDAT)

Question 23

What is the term used for the definition that patterns of DNA methylation can be inherited from one generation to the next. with the result that inheritance of traits independent of DNA sequence?

Answer 23

Epigenetics