ch 21: transcriptional regulation of eukaryotic gene expression

prokaryotes and eukaryotes

DNA binding proteins are used for transcriptional regulation in _____

prokaryotes and eukaryotes

transcriptional regulation controlled by activators in _____

prokaryotes and eukaryotes

transcriptional regulation via recruitment of RNA pol, open complex formation, promoter escape ____

chromatin structure

more transcriptional regulation in eukaryotes at levels of ______

RNA pol II

_____ in eukaryotes doesn’t interact with activator proteins directly (unlike cAMP/CRP in bacteria)

TFs, mediator, chromatin modifying enzymes

activators in eukaryotes don’t interact with RNA pol II directly but rather with _____, _____, and _____

monocistronic

eukaryotic mRNA is _____ (only one protein encoded by the mRNA)

off (activation required)

default state of eukaryotic mRNA expression is ____

heterochromatin

• transcriptionally “silent” DNA that is tightly condensed

• 10% of chromatin: telomers, centromeres, inactivated X chrom

• often methylated at CpG dinucleotide repeats

euchromatin

• less tightly condensed DNA (not necessarily actively transcribed)

• histones loosen chromatin structure (more likely to be acetylated)

regulatory sequences (like promoters)

nucleosome remodelers expose _____ in histone by modified chromatin structure

bromodomains

proteins that recognized acetylated histones to increase gene expressionenzy

histone tails

enzymes change histone and chromatin structure by modifying ____

histone code

each histone modification has a specific function

methylation, phosphorylation, ubiquination, acetylation

specific sites of histone tails can be modified by ____, ____, ____, and ____

HATs (histone acetyltransferases)

acetylates histone to increase expression (heterochromatin → euchromatin)

HDACs (histone deacetylases)

deacetylates histone to decrease expression (euchromatin → heterochromatin)

H2AZ and H3.3

histone variants of H2 and H3 in euchromatin

alternative histone chaperones

assist in histone variant assembly

open (locks chromatin in more open state)

histones replaced with variants that will maintain more ____ chromatin state

larger

regulatory distance (distance between regulatory sequence and coding sequence) is ____ in eukaryotes

enhancers

• activate transcription in particular cell types/times/response to specific signal

• can be >10,000 bp upstream or downstream of core promoter

general transcription factors

regulatory molecule that binds at all promoters

activators

• bind to enhancers

• doesn’t interact with RNA pol II directly

• recruits proteins to modify chromatin structure/interacts with mediator/TFs

AD (activation domains), BD (binding domains)

activators are modular (made up of different units) and contain ____ and _____

common DNA binding motifs (in activators)

homeodomain (a type of helix-turn-helix), zinc finger, leucine zipper, helix-loop-helix

5+

bacteria only need 1 activator/repressor but higher eukaryotes typically have ___ regulatory sequences outside of the core promoter

1600

• a limited number of transcriptions factors can regulate many genes

• 20,000 human genes but only ____ transcription factors

different expression outcomes

• different combinations of activators can bind different enhancers → _________

• combinatorial control

EMSA (electrophoretic mobility shift assay)

• technique used to detect interactions between protein and RNA/DNA in vitro (outside normal living conditions)

• can be used to detect combinatorial control (different combinations of activators binding to different enhancers)

nucleosome remodelers

activators recruit ____ that expose regulatory sequences

histone modifying enzymes (like HATs)

activators recruit the mediator, general TFs, and _____ to loosen chromatin and

ChIP (chromatin immunoprecipitation)

• detects binding of specific proteins to DNA in vivo (in cells/whole organism)

• detects binding sites, distribution of TFs, modification to histones

ChIP-exo (ChIP + exonuclease)

• identifies protein-DNA interactions with near base pair precision

• removes DNA that is not bound by protein

ChEC-seq (chromatin endogenous cleavage-seq)

• DNA near protein of interest is cleaved by MNase → isolate/sequence small fragments of DNA

• uses MNase (micrococcal nuclease) attached to protein of interest via a flexible linker

• MNase completely inactive until Ca2+ ions added

Ca2+ ions

MNase attached to protein of interest in ChEC-seq is completely inactive unless ____ are added

RNA pol II

DNA-binding transcriptions factors don’t directly interact with ____

mediator

giant co-activator that stimulates TFIIH (involved in open complex formation and promoter escape)

TFIIH

transcription factor involved with open complex formation and promoter escape

liquid/liquid phase separation (nuclear condensates)

caused by intrinsically disordered regions of proteins (increases the local concentration of TF)

repressor binding site, activator

repression mechanisms compete for ______ blocking the activator from binding to the activator binding site → prevents ____ from interacting with mediator or general TFs

activators, RNA pol, corepressors, DNA, condensation

repressors displace ______ → corepressors inhibit _____ → HDAC associates with ____ → RNA pol dissociates from ____ → histone deacetylation leads to chromatin _____

methylation

____ of DNA results in gene silencing

CpG (→ C^-CH3pG)

____ is often methylated to silence genes (more methylation = more repression)

methylated (more methylation = more repression)

the DNA in inactivated X chromosome is _____

fragile X mental retardation (FMR)

methylation of expanded CGG repeat causes ____

DNA methylase

methylates CpG to silence genes

methyl-CpG binding protein

____ binds to methylated DNA (C^-CH3pG) AND recruits histone deacetylases (HDACs)

bi-sulfite sequencing

• DNA methylation can be detected by _____

• this treatment converts C → U but methylated C won’t convert

Gal80p, Gal4p

• galactose utilization gene in yeast in repressed by ____

• when this repressor is absent, ____ can activate transcription

Gal4p, TFIID

Gal80p represses Gal gene transcription by binding to the activation domain of ____ which prevents it from interacting with ____

Gal3p, Gal80p, Gal4p

• when galactose is present, ____ is ligand sensor that binds to galactose (effect) and ATP → causes a conformational change in _____ allowing ____ to interact with TFIID

Mig1, Tup1

____ and ____ repress transcription of genes for alternative carbon sources when glucose is present

Mig1

• repressor with DNA binding domain (zinc finger) that binds upstream of start site

• represses genes for other carbon source when glucose present

Tup1

• corepressor than binds to Mig1

• recruits HDAC to deacetylate histones

• represses genes for other carbon source when glucose present

phosphorylated, cytoplasm

• when no glucose present, Mig1 repressor is ____ and held in ____

• Mig1 represses genes for other carbon sources when glucose present

dephosphorylated, nucleus

• when glucose is present, Mig1 repressor is ____ and moves into _____

• Mig1 represses genes for other carbon sources when glucose present

insulators

prevent unwanted cross talk between regulators of different genes

CTCF (CCCTC binding factor)

• binds to insulator DNA and prevents activators from communicating

• activation message to insulated DNA

• creates loops in DNA

CTCF (CCCTC binding factor)

_______ binding to insulator can block spread of histone/DNA modification (HATs, HDACs, methylases)

inherited

DNA methylation state is ____ with each round of DNA replication

maintenance methylases

recognize hemi-methylated DNA during replication and methylate daughter strand to match which is why methylation state is inherited

silenced

• DNA methylation during egg/sperm formation (in the parent) causes either the copy from mom or copy from dad to epigenetically ______ in the offspring

• regardless of mutation, certain genes are always silenced in egg and others in sperm

imprinting

copy from mother or copy from father is silenced while other is actively transcribed

egg/sperm formation (of the offspring)

epigenetic tags on imprinting genes remain for life of organism but are reset during _____ (it won’t be passed to their offspring)

insulator

methylation of ____ prevents binding of CTCF (CCCTC binding factor)

IGF2 (insulin-like growth factor 2)

• gene for this is always silence in mother and only inherited from father

• promoter for this gene can be accessed by activators because the insulator is methylated (prevents CTCF from binding to block cross talk)

• key role in fetal growth/development

Prader-Willi syndrome

• only paternal copy of genes involved in ____ are expressed

• chronic feeling of hunger, cognitive disabilities

Angelman syndrome

• only maternal copy of genes involved in ____ are expressed

• due to missing/defective ubiquitin ligase

• intellectual/development disability, unusually happy disposition

mother

• genes from ____ tend to limit nutrient use and growth in order to protect her own health and reserve energy for future offspring

• mediated by imprinting where it silences certain genes from mother to promote growth where necessary

father

• genes from ____ tend to promote growth to favor maximizing the fetus’s growth even if it drains more from the mother

• mediated by imprinting where it silences certain genes from father where excess growth isn’t necessary

JAK-STAT pathway (Janus kinase)

• Janus kinase (JAK) phosphorylates cytoplasmic side of receptor

• STAT binds receptor and also gets phosphorylated by JAK

• STAT-P dimerizes and enters nucleus

• STAT-P binds to enhancer activating genes involved in immune system function

cytokines

• small protein chemical messengers that play crucial role in immune system

• involved in JAK-STAT pathway

JAK (Janus kinase)

• involved in JAK-STAT pathway for immune system

• phosphorylates cytoplasmic side of receptor after cytokine binds

STAT

• involved in JAK-STAT pathway for immune system

• binds to cytoplasmic side of receptor after JAK phosphorylates

• also gets phosphorylated by JAK

STAT-p

• involved in JAK-STAT pathway for immune system

• dimerizes and enters nucleus to bind enhancers → activate immune system genes

Ras-MAPK (mitogen activated protein kinase)

pathway that promotes mitosis/cell cycle