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BIOC352 > Lynette's Brownfield > Flashcards

Lynette's Brownfield Flashcards

1
Q

what is transcription and what are the stages of transcription?

A

making an RNA copy of a DNA sequence; catalysed by DNA-dependent RNA polymerase (RNAPII)

stages: Pol II recruitment, initiation and early elongation, productive elongation, termination

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2
Q

discuss inducible/ developmental genes?

A

genes that have diff expression levels in diff cell types during development/in response to environmental stimuli

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3
Q

what are some examples of how inducible development of gene expression is influenced by the environment?

A

growers spraying crops with HiCane to cause fruit trees to break dormancy; naturally triggered by cold period over winter

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4
Q

outline the ‘machinery’ involved in the early stages of transcription?

basically just like how does it start

A

an activator binds a cis-regulatory element

co-activators recruited

chromatin at core promoter changed to open conformation

first general TFs recruited to core promoter

RNAPII recruited to core promoter

pre-initiation complex complete

helicase activity of general TFs opens up transcription bubble

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5
Q

how is the early stages of transcription controlled?

A

activator activity

chromatin modifications around core promoter (polycomb and trithorax proteins)

early elongation (promoter proximal pausing)

events at the core promoter/PIC formation

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6
Q

what are some experimental techniques used to study the control of gene expression?

A

early studies performed on a gene-by-gene basis (mutants, overexpression)

new technologies now enable genome-wide or global analysis e.g. RNA-seq, Chip-seq, bioinformatics

this has lead to increased understanding but also lots more questions

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7
Q

what are the two types of chromatin and key differences between them?

A

heterochromatin - tightly packaged, low transcription, repetitive sequences, few protein-coding genes

euchromatin - not tightly packaged, high transcription, rich in protein-coding genes, dynamic (changes to control transcription)

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8
Q

what is the difference between closed and open chromatin?

A

when chromatin is closed (e.g. heterochromatin) activators cannot access cis-regulatory elements (CREs) so is not transcribed

when chromatin is open (e.g. euchromatin) activators can access CREs so can be transcribed

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9
Q

discuss how chromatin is dynamic i.e. how is structure made more open or closed?

A

DNA methylation - methyl group added to/removed from cytosine/ repressed

histone tail post-translational modifications (PTMs) - acetylation, methylation; these added by things called writers (e.g. methyltransferase); marks on histone tails dont do much to chromatin but can recruit reader proteins which recognise mark, bind, impact whats happening

nucleosome remodelling complexes - nucleosome/chromatin remodelers use ATP to eject or slide nucleosomes

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10
Q

what is the nomenclature of the mark/PTM H3K27me3?

A

H3 is the histone, K is the amino acid (lysine), 27 is the position, me3 is the modification (methylation by adding 3 methyl groups)

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11
Q

what are nucleosomes?

A

DNA (or chromatin; DNA w proteins) is wrapped around histones

eight histones form a nucleosome

nucleosomes can be spread apart making DNA accessible

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12
Q

how were polycomb group (PcG) and trithorax group (TrxG) proteins discovered?

A

mutant screens in drosophila - seeing if mutations affected phenotype implying a gene is being disturbed

mutations altered expression of Hox genes (control specification fo cell fate) causing incorrect specification

PcG mutants had increase in Hox gene expression (so PcG proteins repress transcription)

TrxG mutants had decrease in Hox gene expression (TrxG proteins aid transcription (not activators cause dont activate on their own), and are antagonistic to PcG proteins (anti-repressors))

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13
Q

outline the importance of PcG and TrxG proteins in development of diverse eukaryotes?

A

animals/mammals: important for specification of cell fate, X chromosome inactivation i.e. regulate cell identity and cell fate genes, mutations often embryo lethal and often perturbed in cancers (cause involved in controlling cell fate/decisions)

plants: important for cell specification, phase transitions (e.g. dormancy breaking), organ development

fungi: less characterised but also have functions

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14
Q

what are PcG and TrxG proteins?

A

lots of proteins often with multiple homologs in an organism; these proteins interact to form multi-subunit complexes; subunits of these complexes can vary between cell types and between organisms

PcG: polycomb-repressive complex 1 (PRC1) and polycomb-repressive complex 2 (PRC2)

antagonistic interactions between PcG and TrxG proteins allow controlling of transcription; PcG and TrxG complex activity is regulated at inducible genes

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15
Q

outline polycomb repressive complex 1 (PRC1) core complex?

A

catalytic site has ubiquitin ligase activity; ubiquitinates H2AK119 which is a histone tail PTM

this done by RINGA/B proteins and inhibits RNAPII

core complex usually interacts with many other proteins and is also identified in animals and plants

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16
Q

outline polycomb repressive complex 1 canonical complex (cPRC1)?

A

inclues CBX protein which has reader domain allowing it to bind the H3K27me3 mark, also has charged region

also has other proteins that help w oligomerisation and other protein-protein interactions

main role of this complex is chromatin compaction; has low H2AK119 ubiquitination

identified in animals

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17
Q

how does cPRC1 cause chromatin compaction?

A

has the positively charged region and H3K27me3 binding reader domain on CBK protein

DNA negatively charged; chromatin compaction involves positively charged region interacting with negative regions allowing cPRC1 to bind multiple histones

other cPRC1 protein cause oligomerisation and compacts chromatin

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18
Q

what is variant/non-canonical PRC1 (vPRC1/ncPRC1)?

A

includes a range of diff complexes which enhance ubiquitin ligase activity (mostly of H2AK119ub)

other roles include histone deacetylation, demethylation

identified in mammals and maybe plants

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19
Q

what are the three main types of PRC1 complex we need to know?

A

PRC1 (core complex)

cPRC1 (canonical complex)

ncPRC1 (aka variant complex)

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20
Q

what are the three main types of PRC2 complex we need to know?

A

PRC2 (core complex)

PRC2.1

PRC2.2

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21
Q

what is the polycomb repressive complex 2 (PRC2) core complex?

A

catalytic site (SET) has histone methyl-transferase activity; H3K27 methylation (me2 and me3)

has reader domains which bind H3K27me3 and H2AK119uq

also binds nucleosomes, RNA/DNA

identified in animals, plants, fungi

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22
Q

what is PRC2.1 and PRC2.2?

A

alters methyltransferase activity

also can demethylate histones

involved in a variety of protein interactions (these only thing different between 2.1 and 2.2 i.e. what proteins)

identified in animals

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23
Q

what are TrxG complexes?

A

heterogenous (linked by function) i.e. chromatin modification and acts antagonistically to PcG proteins; includes a number of complexes involved in general transcription mechanisms

functions include: ATP-dependent nucleosome remodellers and accessory proteins, histone methylation (di/tri; H3K4me2/3, H3K36me2/3), histone demethylation (H3K27me3; removes mark the PcG add; antagonistic), histone acetylation (H3/H4), reader domains to recognise the marks they put down

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24
Q

outline the tug of war battle going on between PcG and TrxG?

A

PcG and TrxG proteins act antagonistically; gene expression controlled by a balance between PRC repression and TrxG relieving this (anti-repressors kinda)

TrxG complexes causing chromatin remodelling via histone acetylation/methylation antagonise PcG complexes trying to compact chromatin

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25
Q

how do PcG complexes antagonise TrxG complexes?

A

PRC complexes antagonise TrxG complexes by:

  • reinforcing themselves
  • inhibiting histone acetylation
  • H3K27me prevents H3K27ac
  • demeth of H3K4, H3K36
  • H2AK119uq inhibits chromatin remodelling/RNAPII (early elongation)
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26
Q

how do TrxG complexes antagonise PcG complexes?

A

reinforce themselves

H3K27ac prevents H3K27me (reducing PRC reinforcement)

H3K36 and H3K4 methylation inhibit PRC2 activity (access to histone tails)

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27
Q

outline how genes controlled by PcG/TrxG proteins can be active, inactive or poised?

A

inactive genes - PcG winning

active genes being transcribed - TrxG winning

Poised genes - have bivalent marks i.e. activating (TrxG) and repressive (PcG) marks

28
Q

what are the different theories of how PcG and TrxG activity is regulated?

A

multiple homologs/complexes

post-translational modifications

recruitment to target genes

29
Q

outline regulation of PcG/TrxG activity by mutliple homologs/complexes?

A

multiple homologs expressed in different cells in response to different signals

complexes with differing activities/substrate specificities depending on composition

e.g. plant development regulated by diff PcG and TrxG protein complexes which change in composition over time; modifying composition allows them to modify what genes they target

30
Q

outline regulation of PcG and TrxG activity by post-translational modifications?

A

phosphorylation

e.g. EZH protein in PRC2; phos thr487 disrupts binding to other subunits, phos ser21 suppresses methyltransferase activity

31
Q

outline regulation of PcG/TrxG proteins by recruitment to target genes?

A

no universal mechanism, differs between organisms

possible mechanisms (prob a combo of these) include:

  • DNA binding protein interactions
  • RNA-mediated recruitment
  • chromatin-mediated recruitment
  • chromatin sampling
32
Q

discuss recruitment of PcG and TrxG proteins by DNA binding protein interactions?

A

in drosophila polycomb repressive elements (PREs) have motifs bound by various DNA-binding proteins that interact w PcG proteins

in mammals its thought non-core subunits of PcG proteins might recognise hypomethylated CG rich regions

recent research suggesting PcG get recruited by a repressor and TrxG recruited by an activator e.g. myogenin gene; working out stimulates certain DNA-binding proteins to bind muscle genes which recruit TrxG leading to RNAPII recruitment and more muscle

33
Q

discuss recruitment of PcG and TrxG proteins by RNA-mediated recruitment?

A

evidence that RNAs can influence recruitment of these proteins:

some examples of lnc RNAs recruiting TrxG/PcG complexes to regions of DNA they work on

short RNAs often found around promoter region where PcG proteins acting; potentially influencing recruitment?

34
Q

discuss chromatin-mediated recruitment of PcG/TrxG proteins?

A

PRC2: certain histidines bound by PRC1 and PRC2 in certain methylation (or ubiquitinated) states

TrxG: reinforced by H3K4 methylation status

unclear if marks just reinforcing these proteins or if may be influencing recruitment

35
Q

discuss recruitment of PcG and TrxG proteins by chromatin sampling?

A

this is the idea that all those theories of recruitment are correct

PRC2 weakly associates all over the genome and is constantly sampling chromatin and what things are there (activator or a repressor (TF), marks, RNAs) will influence whether it sticks around leading to self reinforcement and silencing

recruitment influenced by the environment at that chromatin at that time

36
Q

what is the core promoter?

A

core promoter is the minimum promoter region required to initiate transcription and is where RNAPII recruited to (with help) and overlaps transcription start site (TSS)

37
Q

what is the traditional view of the core promoter?

A

core promoter has low basal activity which can be further suppressed (chromatin modification) or activated (activators binding cis-reg elements)

so trad view basically that core promoter is part of the machinery

38
Q

how/what experiments suggest a regulatory role of the core promoter?

A

a range of RNA-seq approaches indicate pervasive transcription (throughout genome in many places) due to lots of RNAs found (e.g. eRNA, uaRNA); led to question; why have a core promoter if transcription can initiate without a core promoter?

further experiments combined genome sequencing, RNA-seq and variants, ChIP-seq and showed three types of core promoter; led to question; why have different core promoter types if its machinery?

prob cause regulatory function

39
Q

outline the role of the core promoter?

A

recruit RNAPII which it does by building pre-initiation complex (PIC) which consists of general TFs (GTFs) called TFII(letter)

formation of PIC can occur in diff order but main thing is recruits RNAPII to TSS - this is required for transcription initiation

40
Q

discuss how the core promoter can act as a regulator via RNAPII recruitment?

A

RNAPII can be recruited transiently (on/off) or more stably; the stronger (longer) RNAPII recruitment the more transcription

41
Q

discuss how the core promoter may act as a regulator through stability?

A

core promoter maybe more important in early transcription with roles of ensuring RNAPII is stable on DNA so it makes long transcripts and also ensuring stability of those transcripts (e.g. adding 5’ cap)

not necessarily mutually exclusive with fc 40

42
Q

what is TFIID and how is it recruited to the PIC?

A

the first general transcription factor (GTF) recruited to PIC

is a complex and can have diff combinations of subunits

these subunits bind diff motifs in the core promoter with varying strengths, can interact with diff coactivators and activators which vary at diff genes, can also be influenced by marks (some subunits have reader domains)

TFIID recruitment regulated by changes in these factors

43
Q

outline formation of the pre-initiation complex (PIC)?

A

PIC consists of general transcription factors (GTFs) named TFII and then a letter

formation can occur in different order; ultimately recruits RNAPII to TSS

required for transcription initiation

44
Q

what are mediators and what suggests they are part of the machinery?

A

mediate RNAPII transcription and found in all eukaryotes

is a co-activator linking activators w GTFs so promoting PIC assembly

initially thought to be machinery cause it found at any gene where PIC formed; if regulatory would (in theory) be at some genes and not others

45
Q

what is the structure of the mediator and functions of its components?

A

flexible multiprotein complex w four parts/modules

tail - interacts w many diff activators, influenced by PTMs

head and middle - interacts w PIC components, histone tails, mRNA export machinery

CDK8 kinase - transiently associates; inactivates RNAPII

has core conserved subunits and non-essential subunits that can vary between cells/ organisms/ conditions

46
Q

what is an example of the mediator as a regulator?

A

doing RNA-seq in yeast showed mutating tail module resulted in similar gene expression to wildtype under stress

mutating the same tail module subunit so it can’t be phos showed this important for its function

conclusion from this paper was tail subunit MED15 involved in regulation of expression and phos state important for its function

47
Q

outline how events at the core promoter control transcription?

A
  • stability of PIC/RNAPII recruitment
  • stability of RNAPII during initiation

influenced by TFIID subunits, core promoter structure, activators, histone tail mods

  • mRNA transcript stability

influenced by PIC

all three influenced by mediator subunits

48
Q

how can we think of the core promoter as a control panel?

A

cause it not necessarily the primary thing controlling transcription but helping to regulate it by integrating and modifying signals

49
Q

how was promoter proximal pausing identified?

A

ChIP-chip study using RNAPII antibodies discovered RNAPII pauses after initiation about 20-120nt from TSS

they called this promoter proximal pausing (PPP)

this was confirmed in many other studies using other methods and found to happen in all metazoans and prob plants

50
Q

outline establishment of PPP?

A

many diff theories

related to events during early elongation e.g.

  • PIC components (GTFs/TFIIH)
  • RNAPII CTD has ser5 phos
  • CTD ser2 not phos (elongation factors not recruited but needed for full stability)
51
Q

discuss the theory of establishment of PPP due to DNA sequence?

A

based on: genes showing pausing in mammals and dros often G/C rich just after TSS

drosophila genes often A/T rich region just after the G/C rich region i.e. pause button; RNAPII slips on A/T rich bit cause RNA/DNA hybrid helix less stable and backtracks and pauses on G/C rich region

high G/C content favours RNA/DNA hybrid helix between nascent RNA transcript and template strand forming R-loop

52
Q

discuss the theory of establishment of PPP due to nucleosome position?

A

not all paused genes have G/C rich region after TSS

core promoter nucleosome free but still some in gene body; first one usually right next to pausing region in paused genes

idea is that RNAPII starts transcribing but hits first nucleosome and gets stuck cause no CTD ser2 phos

53
Q

discuss the theory of establishment of PPP due to it just happens?

A

it just happens and isnt regulated or anyhting

54
Q

discuss maintenance/stabilisation of PPP?

A

paused RNAPII stabilised by interacting with proteins called pausing factors

examples of these include negative elongation factor (NELF) and DRB sensitivity-inducing factor (DSIF)

if these recruited RNAPII will sit in paused state

apparently want to remember these names

55
Q

what causes release of PPP into productive elongation?

A

relies on recruitment of PTEF-b complex

PTEF-b has kinase activity; phos ser2 of CTD allowing recruitment of elongation factors, removal of nucleosome and increased stability

phos NELF making it dissociate and DSIF making it an elongation factor

56
Q

if PPP is regulated, which stages would be regulated?

A

establishment - maybe

maintenance - prob not cause just paused if pausing factors present

release - likely by control of P-TEFb recruitment and activity

57
Q

what evidence is there for regulation of the release stage of PPP?

A

drosophila heat shock protein:

pause usually lasts >20 min but after heat shock it last about 4 seconds

suggests regulation by control of P-TEFb recruitment

mayb pausing used in stress response genes and when u wanna turn on quick everything already there (activator bound, core promoter stuff, transcription initiated)

58
Q

what three complexes can P-TEFb exist in in the cell?

A

inactive - in 7SK complex

active 1 - in association w BRD4

active 2 - in association w super elongation complex (SEC)

59
Q

outline P-TEFb in the 7SK complex?

A

inactive

7SK complex a mix of proteins and RNA which sequesters most of cellular P-TEFb and inhibits its kinase activity

90% P-TEFb in this state so prob default state

60
Q

outline P-TEFb in association w BRD4?

A

active

BRD4 has reader domain and can recognise histone marks and get recruited to RNAPII

it can then recruit PTEF-b which will then have kinase activity active

BRD4 is actually a TrxG protein

61
Q

outline P-TEFb in association with SEC?

A

active

bunch of proteins in this complex some with reader domains, some which interact w co-activators/mediator

complex gets recruited to RNAPII and increases kinase activity

62
Q

what causes P-TEFb to alter which complex it’s associated with?

A

we don’t know but would be cool to

63
Q

outline the hypothesis that PPP is a checkpoint?

A

mayb PPP happens all the time and acts as checkpoint for transcription

we know productive elongation doesn’t start unless PTEF-b recruited and CTD ser2 phos

mayb PPP ensures 5’ cap added and ensures important elongation factors and splicing factors recruited and that RNAPII is stable and intron recognised

64
Q

how might PPP be both regulation and machinery?

A

probably both regulation and machinery

machinery as it acts like checkpoint but also provides opportunity for regulation

65
Q
A

BIOC352 (4 decks)

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