Eukaryotic gene regulation PART 1 for course BIMA71 2013 at Lund University, Sweden

1. EUKARYOTIC GENE
REGULATION I
Jill Howlin PhD
Canceromics Branch
Department of Oncology, Lund University
Medicon Village
http://www.med.lu.se/english/klinvetlund/canceromics/research

2. Lecture structure
Eukaryotic Gene Regulation I
A
Revision of the basics
• What is a gene, chromosome etc.
• Transcription
• mRNA processing & Splicing
• Translation…
• DNA mutations
Eukaryotic Gene Regulation II
B
Gene Structure and function
• Structure of typical gene in the genome and its
regulatory units
• How gene structure influences regulation..
A
Control of gene expression
• Producing specificity: Transcription factors
• Other forms of regulation of gene
expression, non-coding RNA, epigenetic
regulation: imprinting & methylation etc….
B
Analysis of Gene Expression & Regulation
• Analysis of gene expression in the genomics era:
transcriptomics
RNA-seq
• Studying gene regulation, DNA-protein interaction

3. Source material:
• Molecular Biology of the Cell. 5th edition. Alberts et al.
• The Cell: A Molecular Approach. 2nd edition. Cooper GM.
• Human Molecular Genetics. 4th edition. Strachan and Read
• Virtual Cell Animation Collectio http://vcell.ndsu.nodak.edu/animations/
(also available as free iphone/ipad app from itunes)
• Wikipedia http://en.wikipedia.org/
• ENCODE explorer http://www.nature.com/encode/#/threads

4. BIM12

5. PART IA
revision

6. the basics…..
• What is a gene?
• How many genes do we have?
• Cell Nucleus and the Chromosome
• What is DNA?
• The double helix
• DNA-RNA-PROTEIN -the central dogma
• What is a mutation?
• DNA replication and inheritance

7. What is a GENE?
• discrete segments of DNA (or RNA) that comprise a functional unit
of hereditary material
• make a complementary RNA molecule that serves as a template
for making a protein
• the total complement of genes in an organism or cell is known as
its Genome
• the human genome approx. 3Gbp DNA and around 20,000
protein coding genes

8. A brief History of Genetics
Gregor Mendel (father of modern genetics) published his ‘Experiments in Plant
Hybridization’ in 1866
Charles Darwin published ‘Origin of the Species’ in 1859
the theories of both men were understood together and merged in early 20th
century
Around 1910 Thomas hunt Morgan genes are on chromosomes and are the mechanical
basis of heredity
The Hershey–Chase experiments 1952 by Alfred Hershey and Martha Chase confirmed
that DNA was the genetic material

9. The Double Helix
• the double helix allowed scientists to understand how DNA was replicated
• 1962 Nobel Prize James Watson, Francis Crick & Maurice Wilkins.
• Rosalind Franklin generated the X-ray diffraction images used to formulate
Watson and Cricks’ hypothesis
Photo 51

10. DNAdeoxyribonucleicacid
Sugar-phosphate
polymer/backbone
Base
pairs A - T
C - G

11. DNA Replication
DNA polymerase can only extend an
existing DNA strand, it cannot begin
the synthesis
helicase unwinds DNA at
the replication fork, the
DNA ahead is forced to
rotate
enzymes that solve the
physical problems of
twisted & coiling DNA
100 to 200 nucleotides long
on the lagging strand the new DNA is made in installments

12. The Cell Nucleus
DNA (and RNA) is referred to as nucleic acid because its found in the nucleus of a
cell
Site of protein synthesis
Where ribosomes are produced

13. DNA Packaging
30nm
1nm per turn, 3.6nm pitch
Nucleosome
147bp DNA wrapped
around a complex of
8 core histones
(H2A, H2B, H3, H4)
Histone H1
The nucleosomes fold
to a 30nm fiber..
that form loops
300nm in length…
..the 300nm fiber is
compressed and folded
further to produce a
250nm wide fiber
700nm
Tight coiling of the 250nm fiber
produces the chromatid of a
chromosome
1400nm Chromsome

14. • Humans have 23 pair of chromosomes
and (22 autosomes and one pair of sex
chromosomes)
Chromosomes

15. Cell Division & Genetic Inheritance

16. Chromosome disorders
Abnormal number aneuploidy
• Down Syndrome 3 x chromosome 21
• Klinefelter syndrome 46/47, XXY, or XXY syndrome
Abnormal structure deletions, duplications, translocations
• Jacobsen Syndrome 11q deletion disorder
Most chromosome abnormalities occur as an accident in the egg or sperm

17. Somatic error
Gross chromosomal abnormalities
• thousands of clustered chromosomal
rearrangements
• single catastrophic event of
fragmentation and reassembly
• cancer
BIMA30 2011-09-14/20
Chromothripsis shattering

18. Expression of Genetic Information
Chromosomal DNA functions in two ways:
- to allow replication of the genetic material (cell division &
inheritance)
- allow expression of genetic information in an organism
the central dogma

19. Double stranded DNA
Single stranded RNA
Amino acids (protein)
The Central dogma

20. RNA ribonucleicacid
• Similar to DNA but contains the sugar ribose
instead of deoxyribose
• RNA also contains the base uracil (U) in
place of thymine (T)
• RNA molecules are less stable than DNA and
are typically single-stranded

21. The Genetic Code
DNA and RNA is organized in sets of three nucleotides, known as codons
DNA (base pairing A-T,G-C) while RNA (A-U,G-C)
Each codon correspond to a specific amino acid or to a signal (e.g.: START, STOP)
AUG, or the amino acid Met is the typical ‘START’ signal
Three ‘STOP codons’ tell the translation machinery that the end of the gene has
been reached.
64 (43) possible codons (4,4,4)
&
20 standard amino acids
genetic code is redundant

22. Deciphering the code..
5’ 3’
N C
coding strand /sense strand/Crick strand template strand/antisense strand / Watson strand
-NH2
positively charged amino group
-COOH
Negatively charged carboxylgroup

23. RNA codons

24. transcription
• DNA to RNA
basic mechanism-
DNA helix unwinds
Direction of
transcription
RNA polymerase
RNA strand
Template DNA strand

25. cis-acting factors onthesamemoleculee.g.:promoter
RNA Polymerase II is the enzyme that synthesizes RNA from genes
encoding proteins
It recognizes and requires certain regulatory sequences in a genes
promoter such as:
-TATA box
-GC box
-CAAT box

26. • RNA Polymerase II requires the assembly of the basal transcription
apparatus to initiate transcription
• This consists of general transcription factors:
-TFIIA
-TFIIB
-TFIID
-TFIIF
-TFIIH
trans-acting factors producedelsewhere

27. transcription
animation

28. mRNA
processing

29. Capping & Poly-A-tail
• RNA capping, 5’ methyl cap, is the first modification of the mRNA as soon as it
emerges from the polymerase
• In the nucleus, the mRNA cap binds a protein complex, the CBC (cap-binding
complex)
• Following splicing and cleavage of the mRNA, RNA binding proteins and processing
enzymes generate the 3’ poly-adenylation signal, Poly-A tail
• Poly-A-binding proteins

30. splicing
• following the 5’ capping, the processed mRNA must still undergo splicing before
translation into protein can occur
• removal of the intronic regions
• cleavage of the exons
• mature spliced mRNA also has 5’ and 3’ UTRs , untranslated regions

31. Splice junctions & the Spliceosome
splicing machinery or Spliceosome
large Protein–RNA complex (snRNA –snRNP)
made up of small nuclear RNAs and >50
proteins
3 consensus sequence sites:
• Most introns start with a GT (GU for RNA)
and end with AG (always the same)
• An A forms the branch point of the lariat
produced by splicing (can vary)
lariat

32. splicing
animation

33. translation
• mRNA to protein
• mature mRNA is DECODED by the ribosome to produce a specific
polypeptide (amino acid chain)
• proceeds through initiation, elongation and termination
• tRNAs, transfer RNAs: function as adaptors between the mRNA template
and the amino acids

34. tRNA
70 to 80 nucleotide long
Always CCA at the 3’ end
Binds to the codon in the mRNA sequence

35. translation
animation

36. Wobble hypothesis
There are 64 = 43 possible codons
49 different tRNAs
In 1966, Francis Crick proposed the
Wobble hypothesis
the 5' base on the anticodon, which
binds to the 3' base on the mRNA, is
not as spatially confined as the other
two bases, and can have non-standard
base pairing

37. Mutations
Permanent changes in the DNA sequence
Mutations can be :
spontaneously occurring (mistakes in replication, failure of DNA repair)
or induced (by radiation such as UV, or by chemical exposure to agents that bind or
react with DNA)
Types of mutations include:
• point mutations
• insertions
• deletions
• translocation

38. The effect on the resulting protein can vary and and includes the following
consequences:
• Frame shift mutation : a disruption in the reading frame
The sun was hot but the old man did not get his hat
T hes unw ash otb utt heo ldm and idn otg eth ish at
Or
Th esu nwa sho tbu tth eol dma ndi dno tge thi sha t
• Nonsence mutation
premature STOP or truncated protein
• Missence
Single point mutation resulting in different amino acid
• Neutral mutation
e.g.: AAA to AGA, lysine to argine
• Silent mutation
No effect on final protein

39. Mutations causing disease
BIMA30 2011-09-14/20
heritable genetic disorders (single mutation)
SCD sickle cell anaemia: a point mutation in the β-globin chain of
haemoglobin, causing the hydrophilic amino acid glutamic acid to be replaced
with the hydrophobic amino acid valine, GAG to GTG (Malaria resistance)
Cystic Fibrosis: most commonly (ΔF508) a deletion that results in a loss of
phenylalanine in the protein encoded by the CFTR gene.

40. SNPs…naturalvariation
=> 1% of population
accounts for 90% of all human genetic variation
every 100 to 300 bases
coding (gene) and noncoding regions
many SNPs have no effect on cell function
GWAS
BIMA30 2011-09-14/20

41. GWAS genome-wideassociationstudies
• SNP arrays, NG sequencing
• focus on the associations between SNP and a particular disease
• influences the risk of disease occurring in an individual
BIMA30 2011-09-14/20

42. BIMA30 2011-09-14/20
Types of SNPs…associatedwiththediseasephenotype
SNPs usually occur in non-coding regions more frequently than in coding regions
SNPs rather than cause disease may confer differential susceptibility e.g.: Alzheimer's
disease and ApoE SNPs

43. END OF PART IA

44. PART IB
gene structure and function

45. Common misconceptions:
• The only purpose of a gene is to encode a protein
• One gene encodes one mRNA and gives rise to one specific protein
• DNA that is not a gene is considered ‘junk DNA’
"Biologists should not deceive themselves with the thought that some new class of biological
molecules, of comparable importance to proteins, remains to be discovered. This seems highly unlikely."
(Francis Crick, 1958)

46. • 2003 (14th April) completion of Human Genome Project
(99% /99.99% accuracy)
• 2012 (5th September) The ENCODE project
regions of transcription, transcription factor association, chromatin structure and
histone modification in the entire genome
80% of the components of the human genome now have at least one
biochemical function associated with them!
BIMA30 2011-09-14/20
Human Genome annotation

47. Size approx. 3Gbp
GENCODE consortium aims to identify all protein-coding, long non-coding RNA
and short RNA genes: >51,096 genes: 20,026 protein-coding and 31,070 non-
coding (GENCODE version 8, March 2011)
A lot fewer genes than originally thought!
~20,000 protein–coding (<1.5%)
Non-protein-coding sequences make up the vast majority of DNA
The mitochondrial genome:
16.6kb
Not a chromosome, but one circular DNA molecule
37 genes (13 protein-coding genes)

48. © 2012, Published by Cold Spring Harbor Laboratory PressHarrow J et al. Genome Res. 2012;22:1760-1774
Human Genome publicdatasets

49. Gene Structure and Function protein-codinggenes
promoters, enhancers, r
epressors
alternative splicing,
transcript variants
mRNA processing &
editing
protein isoforms,
proteasome-mediated
destruction

50. • Pseudogenes
< 12,000
genes in the genome that never become proteins
• RNA genes DNA encoding non-protein coding RNA
Approx 9,000 lncRNA and 9000 sRNA
transfer RNA (tRNA)
ribosomal RNA (rRNA)
snRNA
snoRNAs, microRNAs
siRNAs
piRNAs
Xist
Gene Structure and Function non-protein-codinggenes

51. © 2012, Published by Cold Spring Harbor Laboratory PressHarrow J et al. Genome Res. 2012;22:1760-1774
GENCODE datasets

52. Pseudogenes approx12,000
They are dysfunctional relatives of known genes in the genome that never become
proteins

53. They result from:
1 .Reterotransposition
mRNA transcript of a gene is spontaneously reverse transcribed back into DNA and
inserted into chromosomal DNA (looks like cDNA structure, exons, no promoter)
2. Gene duplication
Arising from gene duplication events and subsequent acquisition of deleterious
mutations (has introns, exon structure and promoter)
3. Disabled Genes
A gene disabled by a mutation that does not have a deleterious effect on the
organism and gets fixed in the population
L-gulono-γ-lactone oxidase (GULO)- biosynthesis of vitamin C (GULOP gene in humans)

54. Pseudogenes in Gene Regulation
• Pseudogene-derived small interfering RNAs (siRNA)
Some pseudogenes actually encode siRNAs that regulate the expression of protein-coding
mRNAs- ‘parent’ genes
• An miRNA decoy function was proposed for pseudogenes when it was
demonstrated that their transcripts were biologically active units in
tumor biology e.g.: PTEN

55. Control of transcription promoters,enhancers,repressors..
• Control of transcription in eukaryotes is complex and is dependent on both -cis and -
trans factors including: regulatory & promoter sequences, RNA polymerase, the
general transcription factors and gene specific activators and repressors
• RNA pol II is responsible for transcription of mRNA or protein-coding genes [Pol I (rRNA)
and III (tRNA and some small RNAs)]

56. • Pol II requires assembly of the general TFs on the promoter in order to initiate
transcription
• specific gene transcription may also be regulated by activator and repressor proteins
binding at near OR distant sites
RNA Pol II promoters vary significantly in structure but often include one or more of:
TATA box
GC box
CAAT box
However none are essential and many promoters lack them all!!

57. Transcriptional repressors compete
with transcriptional activators

58. Transcript variation onegene=manymRNAs
Mainly due to the fact that: 1 Gene > 1 promoter & > 1 splicing pattern
At least half of all genes
have 2 or more promoters
Alternative splicing, as well as creating different protein isoforms, can also generate different 5’ and 3’ UTRs

59. Transcript variation …manyproteinisoforms
• Equally however the same final protein sequence can come from slightly different transcripts! As long as
CDS not affected

60. Roleof mRNA processingin generegulation?
• seems wasteful
• exon-intron arrangement facilitates new genetic recombination
• evidence in protein domains
• variation - several protein variants can be produced from one gene
• The 5’, 3’ modifications of mRNA also have a role in stability, transport and
recognition

61. Influenceof mRNA structurein generegulation
Export ready
(correctly spliced and
polyadenylated)
mRNA is assembled in
nucleus
It moves through the
nuclear pore complex
as a curved fiber, 5’ cap
first
Final mRNA checks
Translation Initiation factors
• 5’ cap distinguish mRNA from other types (pol I and III)
• 5’ cap binds CBC (cap-binding complex)
• the Poly-A-tail is bound by Poly-A binding proteins
• splicing junctions are marked by EJCs exon junction complexes
processing, exp
ort & initiation
of translation

62. Quality control
• In the cytosol, the 5’cap and Poly-A-tail are recognized by the translation initiation
machinery
• EJCs serve to label the mRNA
• Nonsense-mediated decay (NMD) rids cells of mRNAs with premature termination codons
• hUpf complex triggers NMD in the cytoplasm when recognized downstream
Exon junction complexes
Correct stop codon
Premature stop codon Upf proteins
Upf triggers mRNA degradation

63. mRNA stability & degradation
• The stability of mRNAs is very variable
• A deadenylase enzyme acts to shorten the Poly-A tail in the cytoplasm
• Decapping enzymes - uncapped mRNA is rapidly degraded by exonucleases
• 3’ binding of miRNA mediated degradation
• RNAi mediated degradation
AREs can stimulate Poly-A tail removal
AU-rich elements 50–150 nt (rich in
adenosine and uridine)
3-UTRs of many mRNAs with a short half-
life <10% genes, e.g: growth factor

64. Alteration of the amino acid sequence of the encoded protein so it differs from the genomic DNA
A-I deamination of adenine to produce inosine:
>100o genes
RNA editing
Adenosine deaminases
acting on RNA (ADARs)
C-U deamination of cytosine to
produce uracil
Apoplipoprotein B: each
isoform has a different role in
lipid metabolism

65. Control at the protein level ..lostaftertranslation
• In principle, every step required for the process of gene expression can be
regulated
• The steps following translation are no different
• Protein folding is initiated as synthesis proceeds, often with the help of molecular
chaperones (such as Hsp70)
• The ubiquitin-proteasome system efficiently destroys incorrectly or incompletely
folded proteins and acts to limit the life of normal or correctly folded proteins
• Abnormally folded proteins can form disease causing protein aggregates

66. Summary

67. END OF PART IB