Eukaryotic Gene Regulation Eukaryotic control of gene expression is similar to bacterial control but more complicated Still involves activators and repressors and their associated binding sites, but there are many more and the interactions are more complex Also, regulation can take place at more levels due to the separation of the genome from the cytoplasm and the increased number of processing steps

Eukaryotic Gene Regulation

Eukaryotic Gene Regulation Three common regulatory DNA sequences Core promoter Right next to TSS Usually a TATA box + some other binding sites Binds RNA polymerase II and associated TFs Proximal elements Just upstream (within ~200 nt) Highly varied Enhancers and Silencers Can range from within the gene itself to ~200 – 100,000 nt away from promoter

Eukaryotic Gene Regulation Three common regulatory DNA sequences All are referred to as cis-acting regulatory sequences Cis-acting – impact genes on the same chromosome Proteins that interact with those regulatory sequence are trans-acting

Eukaryotic Gene Regulation Three common regulatory DNA sequences Enhancers and Silencers contain sequences that are bound by regulatory proteins They act from a distance via DNA loops and protein intermediaries

Eukaryotic Gene Regulation Enhancers and Silencers acting from a distance do so via DNA loops and protein intermediaries They contain sequences that are bound by regulatory proteins Sonic hedgehog (SHH) is a gene that directs limb formation in mammals It’s expression is regulated by an enhancer sequence that is ~1Mb away from the gene

Eukaryotic Gene Regulation Different regulatory sequences can direct the same genes to be expressed in different ways under different circumstances or in different tissues SHH is expressed in both brain and limb development but under different circumstances and at different times Tissue-specific enhancers will be bound by tissue-specific TFs to modulate these differences

Eukaryotic Gene Regulation Different regulatory sequences can direct the same genes to be expressed in different ways under different circumstances or in different tissues Locus control regions are specialized enhancers that regulate multiple genes in a coordinated fashion Multiple globin genes produce globins with slightly different oxygen affinities, which are expressed at different times during development

Eukaryotic Gene Regulation Are mutations good or bad? Lactose tolerance Most adults in non-European populations are lactose-intolerant Normal in mammals – Lactase gene is “switched off” after weaning Some human populations have high prevalence of lactase persistance in adults High prevalence is associated with cultures that began herding cattle 4-6 thousand years ago In these cultures, lactose tolerance confers an advantage Mechanism/mutation In European populations, the difference between persistence and non-persistence results from the difference in a single nucleotide located 13,910 bases upstream of the lactase gene. T = lactase persistence, C = lactase non-persistence Another SNP -14,000 bp upstream of the lactase gene is associated with lactase persistence in some African populations. Gerbault et al. 2011 Phil. Trans R. Soc. B 366 863-877

Eukaryotic Gene Regulation Insulators – Cis-acting sequences located between enhancers and the promoters of genes that need to be protected from their action Ensure that only the target gene is regulated by the enhancer Encourage loops or are bound by proteins that prevent interaction of the enhancer with the wrong promoter

Eukaryotic Gene Regulation Regulation via chromatin remodeling Recall that chromatin can be either loosely compacted (euchromatin ) or densely compacted (heterochromatin) Euchromatin – transcriptionally active Regions can switch back and forth depending on the needs of a cell

Eukaryotic Gene Regulation Epigenetic control Some proteins ‘tag’ histones and DNA by adding or removing methyl, acetyl and phosphoryl groups These tags alter (remodel) chromatin Epigenetic modifications Alter chromatin structure Are transmissible during cell division Are reversible Are directly associated with gene transcription DO NOT alter the DNA sequence

Eukaryotic Gene Regulation If DNA is packed into chromatin, how do activators, repressors, etc. access the binding sites? 1. Some sites are just accessible in the linker DNA that extends between nucleosomes 2. Chromatin remodeling enzymes can move histones around 3. Chromatin modifiers can add or remove acetyl or methyl groups to alter packing Generally, Adding acetyl groups  increased transcription Removing acetyl groups + adding methyl groups  silencing

Eukaryotic Gene Regulation Open chromatin vs. closed chromatin Open loose association b/t DNA and histone DNA accessible to TFs Transcriptionally active Closed DNA bound tightly to histones DNA inaccessible Transcriptionally inert How do we tell which regions are which?

Eukaryotic Gene Regulation How do we tell which regions are open or closed? DNase I is an enzyme that cuts naked DNA Only cuts in regions that are not bound by histones  open DNase I hypersensitive sites are common in regions of transcribed genes, promoters, etc.

Eukaryotic Gene Regulation How are the nucleosomes moved around to expose/hide binding sites? Chromatin remodelers Reposition or eject histones via multiple mechanisms and multiple enzyme complexes

Eukaryotic Gene Regulation Chromatin remodelers Imitation switch (ISWI) complex Can ‘measure’ spaces between nucleosomes Arranges nucleosomes into regular spaced pattern that serves to close chromatin

Eukaryotic Gene Regulation Chromatin remodelers SWR1 complex Replaces H2A with H2A.Z variant histone Interactions with other histone proteins are disrupted Makes the histone octamer easy to displace

Eukaryotic Gene Regulation Chromatin remodelers Switch/Sucrose non-fermenting (SWI/SNF) complex Described in yeast Slides or ejects histones to open chromatin Consists of multiple proteins that vary by species

Eukaryotic Gene Regulation Chromatin modifiers Don’t remove histones or move them Instead, they chemically alter them by adding or removing chemical groups Alter the strength of the DNA-histone interactions, leading to open or closed promoters Most common chemical modifications Acetyl and methyl groups Chromatin writers, erasers, readers

Eukaryotic Gene Regulation Histone acetyltransferases (HATs) Add acetyl groups (writers) Addition of acetyl groups neutralizes positive charge on histone tails, relaxes histone-DNA interaction Recruited by activators Histone deacetylases (HDACs) Remove acetyl groups (erasers) Recruited by repressors

Eukaryotic Gene Regulation Histone acetyltransferases (HATs) Add acetyl groups (writers) Addition of acetyl groups neutralizes positive charge on histone tails, relaxes histone-DNA interaction Recruited by activators Histone deacetylases (HDACs) Remove acetyl groups (erasers) Recruited by repressors

Eukaryotic Gene Regulation Histone methyltransferases (HMTs) Add methyl groups (writers) Addition of methyl groups can lead to either open or closed chromatin depending on which amino acids are methylated and how many methyl groups are transferred Histone demethylases Remove methyl groups (erasers)

Eukaryotic Gene Regulation Imprinting DNA methylation in mammalian cells Methylated DNA bound by MeCP2 MeCP2 recruits histone deacetylases and methylases Compacted chromatin, genes turned off

Eukaryotic Gene Regulation Gene silencing Imprinting – selective expression of one parental allele Neighboring genes, Igf2 and H19, are on and off depending on parental source What is involved in this regulation? Downstream enhancer CTCF – regulatory protein ICR – imprinting control region

Eukaryotic Gene Regulation Gene silencing Activators bound to enhancer could potentially activate both genes Maternal chromosome is unmethylated in this region Lack of methylation allows binding of CTCF to ICR CTCF blocks activation of Igf2 … allows activation of H19 Paternal chromosome is methylated in this region Methylation blocks binding of ICR … blocks activation of H19 via MeCP2

Eukaryotic Gene Regulation Gene silencing Beckwith-Wiedemann syndrome (BWS) ~1/15,000 births Increased risk of cancer (Wilms’ tumor) Hemihypertrophy

Eukaryotic Gene Regulation RNA-mediated control Small RNAs have been found to be key components of gene regulation Discovered when researchers wanted to induce a particular color petal in petunias by injecting transcripts that would encode the pigment Instead, they found that all pigment production stopped Referred to as RNAi (RNA interference) Can act transcriptionally or post-transcription The basic idea Short RNAs complementary to the target gene direct proteins to that gene or to the transcript to eliminate production of the gene product The small RNA/protein complex either A. enters the nucleus to shut down transcription of the gene or, B. targets the transcripts of the gene for destruction or, C. prevents translation of the transcript.

Eukaryotic Gene Regulation RNA-mediated control Source of the small RNAs? Various hairpin forming transcripts in the genome  microRNAs (miRNA) Externally supplied dsRNA  small interfering RNAs (siRNA) These ‘source’ RNAs are usually ~100-200 bp Two different but overlapping pathways Both pathways involve Processing of the original RNA Formation of a RISC (RNA-Induced Silencing Complex) Discard one strand of the RNA Targeting and silencing

Eukaryotic Gene Regulation RNA-mediated control siRNA pathway Processing of the original RNA with Dicer Formation of a RISC (RNA-induced silencing complex) Formation of single strand Targeting and silencing https://www.youtube.com/watch?v=EtFHIT2mcsM

Eukaryotic Gene Regulation RNA-mediated control miRNA pathway Processing of the original RNA with Dicer and Drosha Formation of a RISC (RNA-induced silencing complex) Formation of single strand Targeting and silencing https://www.youtube.com/watch?v=cK-OGB1_ELE Do NOT click - https://www.youtube.com/watch?v=VfzC-P3dhzs

Eukaryotic Gene Regulation RNA-mediated control

Eukaryotic Gene Regulation RNA-mediated control Thought to have evolved to protect against viruses and TEs VERY useful as an experimental tool RNAi vs knockouts