MBB 267 Week 9: Mitchel 5

Question 1

What is heterochromatin and euchromatin?

Answer 1

densely packaged chromatin is described as heterochromatin while loosely packaged chromatin is described as euchromatin. These states were originally distinguished cytologically but are now empirically defined through the presence of epigenetic markers (histone modifications, DNA methylation) at the DNA sequence level. Euchromatin can be transcriptionally active. Transcriptional activation is often coupled to chromatin decondensation.

Question 2

What are the types of heterochromatin?

Answer 2

Constitutive heterochromatin (DNA that is maintained in a heterochromatic state) comprises repetitive DNA sequences (satellite DNA, centromeric and telomeric regions) and is stably inherited upon cell division. Facultative heterochromatin is readily able to become decondensed.

Question 3

What are the effects of the post-translational modified histones?

Answer 3

Specific modifications have both direct and indirect effects that drive chromatin condensation or decondensation and so act as markers for either transcriptionally silent heterochromatin or transcriptionally active euchromatin. Since their introduction or removal serves to modulate gene expression, they serve as markers for distinct epigenetic states of activated or repressed transcription for a given gene.

Question 4

What do H3K9Ac and H3K4me modifications show?

Answer 4

Acetylation of lysine 9 in histone H3 (H3K9Ac) and methylation of lysine 4 in histone H3 (H3K4me) are markers of actively transcribed DNA

Question 5

What do H3K9me and H3K27me modifications show?

Answer 5

methylation of lysine 9 or lysine 27 in histone H3 (H3K9me, H3K27me) are markers of heterochromatin

Question 6

What do histone modifications effects?

Answer 6

Histone modifications directly affect interactions between nucleosomes and DNA, and modulate interactions with proteins that promote chromatin condensation or decondensation.

Question 7

What is the histone code?

Answer 7

The correlation between histone post-translational modifications and chromatin structure is referred to as the histone code.

Question 8

What is a chromodomain?

Answer 8

The Chromodomain (CD) of proteins bind to histone tails that are methylated at specific lysine residues. Heterochromatin protein 1 (HP1) is the most abundant protein found in heterochromatin and binds to nucleosomes containing H3K9me

Question 9

What is a chromoshadow domain?

Answer 9

Some proteins containing a chromodomain also contain a structurally related domain called the chromoshadow domain. Chromoshadow domains mediate protein/protein interactions. Thus, by interacting with nucleosomes and itself, HP1 can draw together adjacent nucleosomes carrying the H3M9me3 mark

Question 10

What are methyltransferases?

Answer 10

Methyltransferases are a large group of enzymes that all methylate their substrates.

Question 11

What is a SET domain?

Answer 11

The SET domain is a protein domain that typically has methyltransferase activity.

Question 12

How is heterochromatin formed?

Answer 12

The histone methyltransferase Suv3-9 has 2 domains; a CD and a catalytic SET domain. Binding of the Suv3-9 CD to H3K9me3 stimulates SET domain activity on H3K9 in the neighbouring nucleosome. Adjacent methylated nucleosomes are then linked by HP1. Heterochromatin spreading occurs until it is limited by boundary elements.

Question 13

How is heterochromatin formed in yeast?

Answer 13

Heterochromatin formation in yeast occurs by the action of the histone deacetylase Sir2.

Question 14

What is a chromatin boundary?

Answer 14

Chromatin boundary elements define the edges of heterochromatic regions

Question 15

What are insulator elements?

Answer 15

Insulators, consisting of clusters of binding sites for specific DNA-binding proteins. Insulators (i) prevent heterochromatin spreading into actively transcribed genes, and (ii) restrict cross-activation or repression of enhancer or silencer elements. They are equivalent to TAD boundaries.

Question 16

What was the method used that showed the transcription starts at the 5’ end?

Answer 16

Digestion of chromatin with micrococcal nuclease releases mono-, di- and tri-nucleosomes etc, which can be resolved through agarose gels.Gene-specific probes can reveal whether a gene is in nuclease- resistant or –sensitive chromatin.This approach showed that the 5’ end of the globin gene is nuclease sensitive in cells where it is actively transcribed.

Question 17

What does Chromatin immunoprecipitation (ChIP) do?

-how does it work?

Answer 17

allows the analysis of protein/DNA interactions in vivo.
Interactions are stabilised by chemical crosslinking. The DNA is fragmented and protein/DNA complexes are purified by immunoprecipitation. The DNA is then analysed by PCR or microarray (ChIP/Chip). This approach has enabled the genome-wide mapping of protein binding sites including RNA polymerase II and specific histone markers.

Question 18

Where are nucleosome free regions usually seen?

Answer 18

ChIP assays reveal that nucleosome free regions (NFRs) are found in promoter and terminator regions. Nucleosomes adjacent to NFRs are enriched in euchromatin markers, e.g. H3K9-Ac, H3K4me3

Question 19

How can genes be activated?

Answer 19

by recruitment of histone acetyltransferase complexes, e.g. the SAGA complex

Question 20

What is the function of SAGA complexes?

-How do they apply their function?

Answer 20

function as transcriptional coactivators. SAGA triggers acetylation of multiple sites within core histones, leading to nucleosome decondensation. Acetylation removes the positive charge of lysine side chains and blocks ionic interactions with the DNA of neighbouring histones. Moreover, proteins with bromodomains are able to interact with acetylated histones and can promote transcription initiation
-SAGA is recruited to UAS elements through interaction with transcriptional activators such as Gcn4 and Gal4.

Question 21

What are Chromatin remodelling complexes (CRMs)?

Answer 21

Such as the SWI/SNF, complexes are able to “push” DNA sequences into nucleosomes using ATP-dependent helicase activity, causing the nucleosome to slide along the DNA. CRMs are recruited to promoter regions through interactions with transcriptional activators or repressors

Question 22

How do transcriptional repressors work?

Answer 22

transcriptional repressors can direct the condensation of chromatin by recruiting histone deacetylase complexes (HDACs)It is recruited to upstream regulatory sequences (URS) of many genes through interaction with the repression domain of Ume6. Other corepressor complexes contain histone methyltransferases.