Lynette's Brownfield Flashcards
what is transcription and what are the stages of transcription?
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
discuss inducible/ developmental genes?
genes that have diff expression levels in diff cell types during development/in response to environmental stimuli
what are some examples of how inducible development of gene expression is influenced by the environment?
growers spraying crops with HiCane to cause fruit trees to break dormancy; naturally triggered by cold period over winter
outline the ‘machinery’ involved in the early stages of transcription?
basically just like how does it start
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
how is the early stages of transcription controlled?
activator activity
chromatin modifications around core promoter (polycomb and trithorax proteins)
early elongation (promoter proximal pausing)
events at the core promoter/PIC formation
what are some experimental techniques used to study the control of gene expression?
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
what are the two types of chromatin and key differences between them?
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)
what is the difference between closed and open chromatin?
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
discuss how chromatin is dynamic i.e. how is structure made more open or closed?
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
what is the nomenclature of the mark/PTM H3K27me3?
H3 is the histone, K is the amino acid (lysine), 27 is the position, me3 is the modification (methylation by adding 3 methyl groups)
what are nucleosomes?
DNA (or chromatin; DNA w proteins) is wrapped around histones
eight histones form a nucleosome
nucleosomes can be spread apart making DNA accessible
how were polycomb group (PcG) and trithorax group (TrxG) proteins discovered?
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))
outline the importance of PcG and TrxG proteins in development of diverse eukaryotes?
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
what are PcG and TrxG proteins?
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
outline polycomb repressive complex 1 (PRC1) core complex?
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
outline polycomb repressive complex 1 canonical complex (cPRC1)?
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
how does cPRC1 cause chromatin compaction?
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
what is variant/non-canonical PRC1 (vPRC1/ncPRC1)?
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
what are the three main types of PRC1 complex we need to know?
PRC1 (core complex)
cPRC1 (canonical complex)
ncPRC1 (aka variant complex)
what are the three main types of PRC2 complex we need to know?
PRC2 (core complex)
PRC2.1
PRC2.2
what is the polycomb repressive complex 2 (PRC2) core complex?
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
what is PRC2.1 and PRC2.2?
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
what are TrxG complexes?
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
outline the tug of war battle going on between PcG and TrxG?
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
how do PcG complexes antagonise TrxG complexes?
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)
how do TrxG complexes antagonise PcG complexes?
reinforce themselves
H3K27ac prevents H3K27me (reducing PRC reinforcement)
H3K36 and H3K4 methylation inhibit PRC2 activity (access to histone tails)
outline how genes controlled by PcG/TrxG proteins can be active, inactive or poised?
inactive genes - PcG winning
active genes being transcribed - TrxG winning
Poised genes - have bivalent marks i.e. activating (TrxG) and repressive (PcG) marks
what are the different theories of how PcG and TrxG activity is regulated?
multiple homologs/complexes
post-translational modifications
recruitment to target genes
outline regulation of PcG/TrxG activity by mutliple homologs/complexes?
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
outline regulation of PcG and TrxG activity by post-translational modifications?
phosphorylation
e.g. EZH protein in PRC2; phos thr487 disrupts binding to other subunits, phos ser21 suppresses methyltransferase activity
outline regulation of PcG/TrxG proteins by recruitment to target genes?
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
discuss recruitment of PcG and TrxG proteins by DNA binding protein interactions?
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
discuss recruitment of PcG and TrxG proteins by RNA-mediated recruitment?
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?
discuss chromatin-mediated recruitment of PcG/TrxG proteins?
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
discuss recruitment of PcG and TrxG proteins by chromatin sampling?
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
what is the core promoter?
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)
what is the traditional view of the core promoter?
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
how/what experiments suggest a regulatory role of the core promoter?
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
outline the role of the core promoter?
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
discuss how the core promoter can act as a regulator via RNAPII recruitment?
RNAPII can be recruited transiently (on/off) or more stably; the stronger (longer) RNAPII recruitment the more transcription
discuss how the core promoter may act as a regulator through stability?
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
what is TFIID and how is it recruited to the PIC?
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
outline formation of the pre-initiation complex (PIC)?
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
what are mediators and what suggests they are part of the machinery?
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
what is the structure of the mediator and functions of its components?
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
what is an example of the mediator as a regulator?
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
outline how events at the core promoter control transcription?
- 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
how can we think of the core promoter as a control panel?
cause it not necessarily the primary thing controlling transcription but helping to regulate it by integrating and modifying signals
how was promoter proximal pausing identified?
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
outline establishment of PPP?
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)
discuss the theory of establishment of PPP due to DNA sequence?
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
discuss the theory of establishment of PPP due to nucleosome position?
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
discuss the theory of establishment of PPP due to it just happens?
it just happens and isnt regulated or anyhting
discuss maintenance/stabilisation of PPP?
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
what causes release of PPP into productive elongation?
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
if PPP is regulated, which stages would be regulated?
establishment - maybe
maintenance - prob not cause just paused if pausing factors present
release - likely by control of P-TEFb recruitment and activity
what evidence is there for regulation of the release stage of PPP?
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)
what three complexes can P-TEFb exist in in the cell?
inactive - in 7SK complex
active 1 - in association w BRD4
active 2 - in association w super elongation complex (SEC)
outline P-TEFb in the 7SK complex?
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
outline P-TEFb in association w BRD4?
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
outline P-TEFb in association with SEC?
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
what causes P-TEFb to alter which complex it’s associated with?
we don’t know but would be cool to
outline the hypothesis that PPP is a checkpoint?
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
how might PPP be both regulation and machinery?
probably both regulation and machinery
machinery as it acts like checkpoint but also provides opportunity for regulation
