lecture 4

Question 1

what are the post translational modifications of histones?

Answer 1

acetylation, methylation, ubiquitylation, phosphorylation

Question 2

what are the post translational stages of modification?

Answer 2

1. writing - add modifications
2. erasing - remove modifications
3. reading - proteins with these domains that regulate these modulated histones

Question 3

what is the histone code?

Answer 3

different modifications constitue a code which dictates a transcriptional state of underlying DNA

Question 4

what occurs in ATP-dependent chromatin remodelling?

Answer 4

-nucleosome is repositioned
-uses energy from ATP hydrolysis to promote different reactions with chromatin

Question 5

what different reactions with chromatin are promoted?

Answer 5

sliding with nucleosome, unwrapping of DNA, space chromosomes neatly, promote histone variant exchange

Question 6

what is responsible for spacing out nucleosomes?

Answer 6

ISWI

Question 7

what are the properties of SWI/SNF?

Answer 7

-hydrolyses 1000 ATP molecules per minute
-related to DNA helicases
-molecular motor as uses energy from ATP hydrolysis to track along DNA and induce torsion
-disrupts histone

Question 8

how doesSWI/SNF remodel chromatin?

Answer 8

-uses energy from ATP hydrolysis as molecular motor tracks along DNA and introduces torsion and twists
-causes disruption of DNA with the nucleosome
-effectively wraps the DNA around nucleosome
-location of binding site is moved

Question 9

how do ATP-dependent and HAT complexes co-operate?

Answer 9

-commonly recruited to the same promoters
-remodel function together
-transcription factor binds to DNA and recruits acetyl histone transferase
-this acetylates nearby nucleosomes and brings about recruitment of SWI/SNF

Question 10

what do bromodomains in Snf2 allow?

Answer 10

to tether it to acetylated nucleosomes

Question 11

what are the functions of the SWI/SNF complex in yeast?

Answer 11

-general transcription

Question 12

what are the 3 complexes in the SWI/SNF in humans?

Answer 12

PBAF, cBAF, ncBAF

Question 13

what are the roles of these complexes?

Answer 13

-general transcription (needed for txn factors)
-cell control via interaction with Rb and cyclin E
-development
-role in tumour suppressor pathways

Question 14

how is SWI/SNF linked with cancer?

Answer 14

-at least 9 different genes encoding SWI/SNF are recurrently mutated in cancer

Question 15

what is the tumour suppressor activity of SWI/SNF likely to be because of?

Answer 15

-roles in facilitating transcription factor function

Question 16

how is transcription repressed?

Answer 16

-mediated by recruitment of chromatin modifying factors
eg. histone deacetylases, ATP dependent remodellers, histone methylases (formation of heterochromatin)

Question 17

which region is hyperacetylated?

Answer 17

active regions

Question 18

which regions are hypoacetylated?

Answer 18

repressed regions (low transcriptional activity, low acetylation)

Question 19

what is deacetylation mediated by?

Answer 19

histone deacetylases (HDACs)

Question 20

what are the 4 major groups of histone deacetylases?

Answer 20

class I, class II, class IV (all zinc dependent enzymes), class III (require NAD as cofactor)

Question 21

how do HDAC co-repressor complexes work????

Answer 21

-recruited to promoters by interaction with site specific DNA binding proteins (repressor proteins)
-deacetylation of N terminal tails
-UME6 repressor binds to upstream repressor sequence through binding domain
-regulatory domain interacts with sim3
-nucelosome is deacetylated to prevent proteins helping the PIC formation

Question 22

what commonly mediates transcriptional repression?

Answer 22

ATP-dependent remodellers

Question 23

what does closed chromatin mean?

Answer 23

transcription off

Question 24

what does open chromatin mean?

Answer 24

transcription on

Question 25

what are the two basic types of chromatin?

Answer 25

euchromatin and heterochromatin

Question 26

what are the properties of euchromatin?

Answer 26

-gene rich -potential to be transcribed -viewed as light stained through microscope

Question 27

what are the properties of heterochromatin?

Answer 27

-gene poor -repetetive regions -transcriptional silencing -usually dark stained through microscope

Question 28

what are the biochemical features of heterochromatin?

Answer 28

1. hypoacetylation - very low levels of acetylation 2. specific histone H3 methylation 3. association of specific silencing factors

Question 29

how is heterochromatin assembled?

Answer 29

1. lysine 9 cant be acetylated (to form heterochromatin) so histone deacetylase needed to remove any acetyl groups associated with the molecule 2. a histone methyltransferase called suvar39 methylates lysine 9 on histone H3 3. this forms a docking site/assembly site for a component of heterochromatin

Question 30

what are the properties of heterochromatin protein 1?

Answer 30

-chromodomain -can recognise methylated lysine -forms pocket where lysine 9 fits (docking site)

Question 31

what does binding of HP1 cause?

Answer 31

-compaction of nucleosomal arrays -acts as platform for recruitment of further activities that prevent recruitment of RNA pol II

Question 32

how does binding HP1 prevent recruitment of RNA pol II?

Answer 32

-HP1 has region that attracts other enzymes like histone deacetylases to ensure all histone tails are deacetylated

Question 33

how are reporter silencing assays used to analyse heterochromatin?

Answer 33

-reporter gene is located -ade6 repressed

Question 34

what occurs in the 'normal' wild type?

Answer 34

-ade6 gene expressed -white colonies

Question 35

what occurs in silencing reporter stain?

Answer 35

-ade6 silenced -red colonies

Question 36

what occurs when the ade6 silencing is alleviated?

Answer 36

white/pink colonies

Question 37

what is controls Barr body formation?

Answer 37

-non coding RNAs Xist and Tsix -regulate X chromosome inactivation

Question 38

what area drives X chromosome inactivation?

Answer 38

Xic

Question 38

what leads to the X chromosome being inactivated?

Answer 38

1. in early development, both X chromosomes are making the tsix transcript 2. later, one X chromosome represses tsix and instead upregulates the xist 3. the other X chromosome continues to express tsix and becomes the active chromosome (lots of genes expressed) 4. xist binds to x inactivation centre and coats the entire chromosome 5. indirectly, xist recruits binding of xist and recruitment of a methylase which methylates h3k27 leading to chromodomain protein polychrome that silences expression from X chromosome

Question 39

what is the function of the tsix transcript?

Answer 39

prevents transcription initiation to drive formation of xist (an antisense transcript)