Lynette's Brownfield Flashcards

Question

how do PcG complexes antagonise TrxG complexes?

Answer 1

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)

Answer 2

reinforce themselves H3K27ac prevents H3K27me (reducing PRC reinforcement) H3K36 and H3K4 methylation inhibit PRC2 activity (access to histone tails)

Answer 3

inactive genes - PcG winning active genes being transcribed - TrxG winning Poised genes - have bivalent marks i.e. activating (TrxG) and repressive (PcG) marks

Answer 4

multiple homologs/complexes post-translational modifications recruitment to target genes

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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

Answer 9

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?

Answer 10

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

Answer 11

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

Answer 12

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)

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

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

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

- 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

Answer 24

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

Answer 25

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

Answer 26

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)

Answer 27

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

Answer 28

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

Answer 29

it just happens and isnt regulated or anyhting

Answer 30

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

Answer 31

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

Answer 32

establishment - maybe maintenance - prob not cause just paused if pausing factors present release - likely by control of P-TEFb recruitment and activity

Answer 33

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)

Answer 34

inactive - in 7SK complex active 1 - in association w BRD4 active 2 - in association w super elongation complex (SEC)

Answer 35

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

Answer 36

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

Answer 37

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

Answer 38

we don't know but would be cool to

Answer 39

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

Answer 40

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