1. 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

2. Eukaryotic Gene Regulation

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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 Phil. Trans R. Soc. B

10. 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

11. 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

12. 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

13. 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

14. 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?

15. 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.

16. 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

17. 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

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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)

24. 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

25. 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

26. 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

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

28. 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.

29. 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 ~ 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

30. 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

31. 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
Do NOT click -

32. Eukaryotic Gene Regulation
RNA-mediated control

33. 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