#Chromatin Loops as Allosteric Modulators of Enhancer-Promote

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eukaryotic gene transcription regulation

eukaryotic gene regulation – more complex variations on and additions to prokaryotic mechanisms

increased cellular/multicellular complexity calls for solutions to the following:

DNA helices are stored in conditionally-inaccessible chromatin structures

transcription occurs in the nucleus → mRNA must pass through nuclear membrane → translation occurs in the cytoplasm

cell specialization necessitates cell-type-specific kinds and levels of gene expression 

eukaryotic solutions to the above circumstances include:

regulation of transcription initiation, like in prokaryotes

post-transcriptional modification of primary mRNA transcript via splicing – remove introns, add 3′ polyA tail and 5′ cap

post-transcriptional regulation of mRNA’s nuclear membrane bypass

translatability of mature mRNA transcript

organelle-specific localization of translated protein in specific cell types

post-translational modifications to protein amino acid chain / secondary+ structure

basic mechanism of eukaryotic transcription

three different types of RNAPs transcribe mRNAs in eukaryotes: 

pol I synthesizes rRNAs for ribosomes

pol II synthesizes protein mRNAs

pol III synthesizes helper tRNAs and other small nucleic acid aids (5S rRNA, snRNA)

each recognizes different types of promoter sequences that correspond to ribosomes, proteins, and helper RNAs, respectively

promoter region – characteristic (G-box + CAAT box + TATA • 7 box) DNA sequence + transcription initiation point, right before gene-encoding region

pol II binding to TATA box → transcription initiation → low basal level of gene expression in cell

i.e., basic RNAP binding DNA sequence, analogous to prokaryotic promoter regions

all promoters have some TATA-esque region; promoters may or may not have a G-box, CAAT box, and TATA • 7 box in that order and presence

enhancer region – some protein-binding DNA sequence that modulates basal gene expression levels when bound by regulatory proteins

enhancer can be at any proximity to gene-encoding region up to 10kb’s

±enhancement of basal level of expression depends on type and specific combination of / interactions between proteins binding at the region

enhancers can be 5′ or 3′, i.e., orientation-independent, and have multiple protein binding sites

regulating basal expression levels via transcription factors

transcription factor (TF) – any protein that binds to DNA and affects transcription

basal transcription factor – assists in RNAP’s promoter-binding efficacy

key basal factor: TATA box-binding protein (TBP) and TFIID, which bind TATA • 7 sequence in promoter

TBP+TFIID recruit other basal factors – TBF-associated factors (TAFs) – to form basal factor complex (BFC)

TAF recruitment order is key to construction of active transcription complex for pol II initiation, and is thus highly regulated

pol II does not bind at promoter’s initiation site until BFC has formed | BFC orients pol II towards +1 transcription initiation site when it binds TFIID

necessity of basal factor complex for pol II mRNA synthesis = basal factor gene conservation throughout eukaryotic line

» interactions between RNAP+BasalTF complex at promoter, and TFs bound to enhancer regions (eTFs)

DNA between promoter and enhancer regions is looped and shifted to create direct interaction between two regions despite enhancer location distal to promoter

direct RNAP+B–eTF interaction → eTF binds or phosphorylates the BFC bound to pol II

indirect RNAP+B–eTF interaction via multi-subunit Mediator protein complex which binds RNAP+B and eTF like a bridge for interactions between them

activator transcription factor – increases basal level gene expression

can recruit RNAP+B complexes to key promoter regions in DNA sequence

and/or increase RNAP+B’s promoter binding efficacy by interacting with the BFC

and/or recruit co-activators that open chromatin histone-nucleosomes to allow RNAP+B access to gene-encoding regions

said co-activators are either histone acetyltransferases (HATs) that modify histone tails or chromatin remodeler enzymes that displace entire nucleosomes relative to promoter region

» domains within activator structure for DNA and BFC or co-activator binding

DNA-binding domain – helix-turn-helix and zinc finger amino acid motifs, generally structured in all DNA-binding TFs but with sequence-specific areas in specific amino acids

activation domain – some amino acid structure that folds in specific ways to bind the BFC or a particular co-activator

dimerization domain – some amino acid structure – often a “leucine zipper” – that allows for binding with another TF to form multimeric activator complex

homodimers are those that contain all same activator protein; heterodimers contain 2+ types of activator proteins

repressor transcription factor – decreases basal level gene expression

[rare in eukaryotes: repressor directly blocks RNAP+B complex binding to promoter regions]

general mechanism: repressor binds enhancer DNA region and recruits non-DNA-binding co-repressor to prevent RNAP+B binding to promoter or modify chromatin storage of relevant gene-encoding region

co-repressors that reduce chromatin accessibility are histone deacetyltransferases (HDACs) that modify tails

» indirect repressor mechanisms to lower basal level expression

competition – repressor binds to enhancer in competition with activator that binds the same or overlapping DNA sequence

quenching – repressor binds or phosphorylates activator, inhibiting its expression-increasing activity

direct interaction – repressor binds RNAP+B and inhibits its initiation-bind / efficacy

heterodimerization – repressor either binds activator and forms activator heterodimer, or homodimerizes with itself to form repressive or neutral complex without activator function

time-, development-, and cell-specific gene regulation

overall gene expression is determined by levels and tissue-specific expression of activators and repressors, as well as enhancer region’s chromatin accessibility

cell- or time-specific chromatin-openness of enhancer determines TF binding and resulting expression modulation

cell- or time-specific levels of TFs determine up- or down-regulation of gene-encoding region

gene-encoding region may have multiple enhancers, or one TF can bind enhancers for multiple gene-encoding regions

DNA rearrangement mutations that affect regulatory regions alter phenotype

large translocation mutation can place regulatory region of one gene in front of the transcriptional region of another, altering second gene’s expression levels

large deletion mutation can remove regulatory region all together and produce inability to express gene beyond basal level, altering phenotype

inversion mutations can shift enhancer region so far away from promoter that even distal modulation of transcription is not possible

transcription factors can themselves be modified to indirectly regulate gene expression

allosteric interactions with small molecules (i.e., steroids like cholesterol) affect TF activity and efficacy as a further regulatory mechanism

kinase phosphorylation of TFs changes protein conformation and either stimulates or inhibits TF activity

TF cascade – once encoded, one set of TF proteins upregulate another set of TF proteins, which upregulate another set of – and so on

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Chromatin Loops as Allosteric Modulators of Enhancer-Promoter Interactions [NEW RESULTS]

— bioRxiv Subject Collection: Biophysics