Eukaryotic Gene Regulation

1
Chapter 23

• Eukaryotic Gene Regulation

2
Levels of Regulation

• 5 main levels
• genome level
• transcription level
• RNA processing and export
• translation level
• post-translational events
• Last 3 are also categorized as post-transcriptiona
  l controls

3
Genome Control

• Small fraction of genes are used in each cell but
  they all have the same genes
• except sperm and egg cells haploid
• Nucleus is totipotent
• put adult nuclei in an egg cell and can get
  tadpole development
• clone
• Can do in plants by isolating a single cell and
  grow in a dish without transplanting nucleus into
  a new cell

4
Gene Amplification

• Selective amplification of certain genes
• Genomic control as regulates the change in the
  make of the structural organization of genome
• rRNA genes in Xenopus usually number 500 copies
  for each gene but during oogenesis they increase
  4000x
• needed for the amount of ribosomes that are
  needed to make the proteins

5
rRNA Genes

• Present as circles in the nucleoli
• Use RNA rather than protein as to get abundant
  protein we can use the mRNA over and over to get
  enough protein

6
Additional Genome Modification

• Some organisms will delete genes they no longer
  need
• RBCs remove all DNA once they have enough mRNA
  for hemoglobin for their lifespan
• Copopods get rid of heterochromatin in all cells
  with the exception of cells destined to become
  gamates

7
DNA Rearrangement

• Alter the genome
• See in yeast that control mating and vertebrates
  that make antibodies

8
Yeast Mating Type

• 2 haploid cells meet and fuse
• both a and ? genes are present in the HML? and
  HMRa locus but mating type is dependent on which
  gene is in the MAT locus
• can get DNA rearrangement to change the mating
  type cassette movement
• SIR gene products keep the extra copies of the
  genes from being expressed to keep from altering
  the phenotype
• ? and a encode secretory proteins and surface
  receptors

9
Ab Formation

• Ab made of 2 heavy chains and 2 light chains
• Proteins are made from selecting gene segments
  from a small number of segments that will yield
  numerous Ab
• millions of combinations
• Heavy chain use a V, J, D and C segment
• Light chain use only the V, J and C segments

10
Recombination Aides Transcription

• Enhancer region is near the C segments but the
  promoter is upstream of the V region
• Need enhancer to activate transcription of Ab
  genes into mRNA and prior to rearrangement, the
  enhancer and promoter are too far apart to work
  together

11
Genome Decondensation

• Need some degree of chromatin infolding to get
  the necessary transcription machinery access to
  the DNA
• Saw first in fruit fly salivary glands
• cell becomes larger and daughter strands are not
  separated into new cells but rather, remain
  attached to form polytene chromosomes

12
Polytene Chromosomes

• See areas of highly condensed inactive DNA and
  areas that are open and not so condensed
• Green areas are areas of transcription
  fluorescent Ab to RNA polymerase

13
Chromosomal Puffs

• Puffs are large areas of decondensed DNA that is
  being actively transcribed
• Under the influence of ecdysone insect steroid
  hormone that triggers transcription

14
DNase 1 Sensitivity

• DNase I is an endonuclease that degrades dsDNA
  that is not protected by proteins (histones)
• Active genes will be degraded and others left
  untouched
• tissue specific in terms of DNA being degraded

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Chromatin Changes

• Chromatin uncoiling is prerequisite for DNA
  transcription
• DNase 1 hypersensitivity is common upstream of
  transcription start sites and are 10x more
  susceptible to DNase 1 that other DNA
• usually no chromatin present to protect the DNA
• area of hypersensitivity

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DNA Methylation

• Methylation of various Cytosine residues in DNA
• most vertebrates contain small amount -CH3 in the
  non-coding regions _at_ 5 end of genes
• -CH3 of the parent strand will dictate the -CH3
  of new daughter strand
• Epigenetic changes change the gene expression
  without altering the DNA sequence

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X Chromosome

• Most recognized -CH3 DNA sequence
• 2nd X-chromosome is heavily -CH3 and this causes
  the chromosome to become a tight mass of
  heterochromatin called a Barr Body
• same X remains inactive for the all descendents
  of that cell following Barr Body formation in
  embryogenesis

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Methylation

• Can use restriction enzymes to determine -CH3
  patterns as on the same gene in different tissues
  they are specific
• Usually inactive genes are -CH3 and active are
  not
• -CH3 prevents transcription
• not always true, some active genes are -CH3 while
  some un-CH3 genes are inactive
• Methylation is one of many factors regulating
  gene expression

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Changes in Histones

• Acetylation is the addition of an acetyl group to
  the R groups on histone amino acids
• See increase in acetylation in active gene areas
• Treat cells with Na Butyrate increases the
  acetylation and causes changes in nucleosome
  formation and DNA becomes more susceptible to
  DNase 1 activity
• Phosphorylation and -CH3 also can influence gene
  expression
• no acetyl or methyl group leads to
  heterochromatin formation
• H1 histone absence also leads to transcription
• the looser the packing of DNA the more accessible
  to RNA polymerase and other machinery

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High-Mobility Group (HMG) Proteins

• Non-histone proteins that move rapidly in
  electrophoresis
• Remove the HMG proteins and loose gene activity
• add HMG protein from a different tissue, get gene
  expression similar to that cell type
• tissue specific proteins

21
Association With Nuclear Matrix

• Active genes are found in association with the
  nuclear matrix
• DNA sequences called matrix attachment regions
  (MARs) hold the active genes near the nuclear
  matrix

22
Transcriptional Control

• 2nd main level of control
• Different gene sets in different cell types
• See differential gene expression in different
  cells
• see a different set of proteins in different
  cells so you would assume a different set of
  nuclear RNA in those cells if it was
  transcriptional control rather than translational
  control

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Nuclear Run-On Transcription
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RNA Determination

• Northern analysis can tell how much mRNA is being
  made in a cell
• Use microarrays now for more information and no
  radioactivity
• Normal green
• Abnormal red
• Yellow equal
• Black none

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Proximal Control Elements

• Close to the promoter, may be upstream or
  downstream depending on gene
• Seen in protein-coding genes that require RNA
  polymerase II
• Transcription factors are responsible for
  determining the start of transcription, not RNA
  pol II
• transcription factor is not part of pol like
  sigma factor in prokayotes
• Transcription factors and pol assemble at only
  the core promoter, get basal level of
  transcription

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3 Common Types

• CAAT box, GC box and the octamer
• Transcription factors bind outside core promoter
  are called regulatory TF
• usually increase transcription or may decrease
• Combination of reg. TF and proximal control
  elements is specific to each gene

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Enhancers/Silencers

• Lie variable distances from the core promoter and
  proximal control elements
• near or great distance
• upstream or downstream
• can even be in a reverse orientation
• Enhancer stimulates expression of gene
• Silencer inhibits expression of gene

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Enhancers

• Sequence varies but have common properties
• Octamer and GC box can also act as enhancer
  region
• TF that bind enhancers are called activators
• Study by moving enhancer around and see what
  happens to gene expression levels

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Silencers

• Bind reg. TF and repress gene expression
• TF is called a repressor
• SIR gene products is a repressor and HML? and
  HMRa are the silencers

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Coactivators

• Reg. TF and RNA pol complex
• Enhancer loops around to be near promoter and
  coactivator proteins mediate the interaction
• Coactivators make the promoter more accessible to
  the pol complex

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Coactivator Molecules

• Histone acetyl transferase (HAT) acetylates
  histones and nucleosome decondenses
• Chromatin remodeling proteins
• SWI/SNF different families
• Mediator bridge both activator proteins and
  enhancer along with RNA pol II

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Mediator

• Activator bind enhancer and form an enhanceosome
  that causes DNA to bend
• Enhanceosome interacts with SWI/SNF and HAT to
  alter chromatin structure
• Activator binds mediator and positions the RNA
  pol and TF for transcription

33
Combinatorial Model

• Multiple DNA control elements and TF in
  combinations establish a specific and precise
  control of gene expression
• General TF and reg. TF used for constitutive
  genes and frequently used genes
• Tissue specific genes have TF or combinations of
  them are unique for that cell type

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(No Transcript)
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Structural Motifs

• Portions of TF that bind DNA and activate or
  repress transcription
• Regulatory factors have 2 activities in separate
  protein domains
• bind specific DNA sequence DNA binding domain
• ability to regulate transcription transcription
  regulation or activation domain

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Activation Domain

• Figured out using swapping experiments
• take DNA binding domain and place in various
  parts of TF
• see gene expression only if the activation domain
  was present
• Activation domain has a high proportion of acidic
  amino acids on one side of the ? helix
• mutations that increase acidity increases
  expression
• mutations that decrease acidity decrease
  expression

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DNA Binding Domains

• 2 structure or motif
• 4 common patterns
• helix-turn-helix
• zinc finger
• leucine zipper
• helix-loop-helix

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Helix-Turn-HelixMotif

• 2 ? helices and a turn
• 1 ? helix is recognition helix and binds by
  H-bonds
• 1 ? helix stabilizes structure with hydrophobic
  interactions

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Zinc Finger Motif

• ? helix and 2 segments of ? sheet with a Zn
  between them on a Csy or His residue
• May have 2 to several dozen per TF
• Protrude from protein and interact with specific
  DNA sequence

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Leucine Zipper Motif

• 2 polypeptides (homomeric or heteromeric) with ?
  helix that has regularly spaced Leu residues
• Form coiled-coil structure by zipping up the
  Leu
• 2 ? helices at end of each peptide interacts with
  specific DNA sequence

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Helix-Loop-Helix Motif

• Short ? helix, long loop of amino acids and long
  ? helix
• hydrophobic regions that connect 2 polypeptides
  (same or different)
• 4 helix bundle and 2 DNA recognition sequences
  2-part DNA binding domain

42
DNA Response Elements

• Activate a group of genes at the same time
• development and functioning of specialized cells
• genes usually scattered in the genome
• Response elements turn on and off genes in
  response to environmental or developmental signal
• place the RE next to genes that need to be
  regulated can controlled together
• important in embryonic development and tissue
  responding to environmental and physiological
  conditions

43
Hormone Response Elements

• Respond to steroid hormones
• Nuclear receptor proteins mediate the actions
  of steroid hormones
• progesterone, estrogen, testosterone and
  glucocorticoids
• Chemical messenger binds to receptor protein and
  enters the cell, transmits signals to usually
  activate transcription with an occasional
  inactivation
• Steroid hormone receptor acts as a TF binding to
  DNA control sequence Hormone Response Element

44

• Steroid response elements have similar sequences
  and are upstream of promoter
• steroid hormone receptor is a zinc finger type TF
  with a hormone binding site, DNA binding site and
  a transcription activator site

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Cortisol Activationof Gene

• Glucocorticoid receptor (GR) is in cytosol and is
  bound to hsp so cannot enter the nucleus
• When cortisol is present, binds to GR and hsp is
  removed and now can enter nucleus and bind to
  response element
• Forms a GR-S dimer and activates transcription

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Common Theme

• Inverted repeat 2 copies in opposite directions
• Most other steroid receptors are already in the
  nucleus without the hormone but require it to
  release inhibition
• Occasionally can inhibit by binding to gene
• GC doesnt form dimers but depresses initiation
  oby recruiting enzyme to remove acetyl groups
  chromosomes condense and no room for
  transcription machinery

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Protein Phosphorylation

• Involves cAMP to stimulate protein kinase A
• CREB binds to cAMP response element and is
  phosphorylated by PKA, recruits transcriptional
  activator CREB binding proteins (CBP)
• CBP catalyzes histone acetylation to recruit RNA
  pol and transcription

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Signal Transducers and Activators of
Transcription (STATs)

• Interferons are the signaling molecules for STATs
  
• IFN make neighboring cells to resistant to virus
  infection
• IFN binds the receptor activates Janus Activated
  Kinase (JAK) which adds PO4 to STATs which
  dimerize and move to nucleus and bind DNA to
  increase transcription
• JAK-STAT pathway has considerable specificity
  activates a unique set of genes
• STATs are also phosphorylated by other signaling
  molecules

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Other Important Phosphorylations

• SMADs after TGF? binding
• MAP Kinase controlling cell growth and division

50
Heat Shock Response Elements

• Increase in temperature activates genes
• Heat shock or stress-response genes regulates
  genes at different chomosomal sites
• Gene products help to minimize damage due to
  thermal denaturation
• Hsp70 is a molecular chaperone that helps refold
  proteins
• Heat-shock response element is a binding site
  upstream of other common promoter sequences
• heat shock TF binds to the HSRE it is inactive at
  permissive temperature but as the temperature
  increases causes conformation change that allow
  binding and further activated by phosphorylation

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Basic Principle

• Genes in different chromosomal locations are
  activated by the same signal if the same response
  element is near each of them

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Homeotic Gene

• Genes responsible for body plan
• Mutations in the homeotic gene causes changes in
  body plan
• bithorax gene complex 2 sets of wings
• antennapedia gene complex legs instead of
  antenna
• Homeotic genes encode a family of TF that
  activate/inhibit transcription of developmentally
  important genes

53
Homeobox

• Near the 3 end of homeotic genes
• Homeobax encodes the homeodomain that binds to
  DNA
• highly conserved during evolution
• helix-turn-helix motif

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Post-Transcriptional Control

• All are regulatory points after transcription
• Fine tune gene expression without drastic
  transcriptional changes to transient changes in
  environment

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Control of RNA Processing and Transport

• 1º transcript to mRNA
• 5 cap, 3 polyA tail, chemical modifications
  (CH3), exon selection, intron removal and RNA
  editing
• RNA splicing is important can make different
  mRNA from 1º transcript that allows for more than
  one type of protein product
• Splicing is controlled by proteins that bind to
  the splice site and protect it from splicing

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Alternative Splicing

• IgM can either be secreted or membrane bound
  depending on the splicing that occurs at the 3
  end
• Use alternative splice site if it is to
  membrane bound, then need the exons that contain
  the hydrophobic membrane spanning region

57
RNA Export

• Splicing and processing of the 1º transcript is
  important for export
• 5 capping and 3 polyA defects have a decrease
  in export of those messages to the cytoplasm
• If introns are removed from a gene, then the
  message is not transported to the cytoplasm but
  if you put at least one intron back into the gene
  there is transport to the cytoplasm of the mRNA
• mRNA genes require a nuclear export signal

58
Translation Rates

• Several mechanisms
• alter ribosomes or protein synthesis factors
• regulate the activity and/or stability of mRNA

59
Heme-Controled Inhibitor (HCI)

• Globin synthesis is regulated by the presence of
  heme
• HCI is inhibited by heme that allows for
  translation of the globin mRNA
• When no heme, HCI phosphorylates eIF2 which
  prevents translation of globin and other messages
• no need for globin if there is no heme

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Initiation Factor Regulation

• Other kinases can also phosphorylate eIF2
• PO4 of eIF4 binds the 5 mRNA cap but rather than
  inhibiting translation it activates it
• seen in adenovirus infection
• Phosphorylation also regulates initiation and
  elongation factors as well as aminoacyl-tRNA
  synthetases
• All these are non-specific controls of translation

61
Ferritin

• Iron storage protein synthesis stimulated in
  the presence of iron
• Iron-response element (IRE) in the 5
  un-translated leader sequence
• forms a hairpin loop that is required for
  expression in presence of iron

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• IRE-binding protein binds to IRE without iron
  get low level translation
• Fe present binds to the IRE binding protein and
  removes it from the hairpin loop and ribosome can
  bind and do high level translation
• Translational repressor control specific mRNA
• response to specific changes in environment

63
mRNA ½ Life Regulation

• ½-life is the time to remove ½ mRNA time
  differs from mRNA to mRNA
• alters the stability of the message
• PolyA tail dictates the stabilty of mRNA
• long tail greater stability
• short tail less stability
• Other features in the 3 un-translated region
  also involved
• AU rich sequence triggers removal of polyA tail
  by degradative enzymes causes short ½ life

64
Fe Control of mRNA Degradation

• Iron comes in by transferrin receptor protein
• when Fe levels drop there is an increase in
  transferrin receptor synthesis
• mRNA protected from degradation
• use IRE (similar to ferritin IRE) in the 3 end
• increase in iron in the cell causes mRNA
  degradation

65
RNA Interference

• Use RNA to silence genes
• Can inhibit activity
• trigger mRNA degradation
• inhibit translation
• inhibit transcription of genes
• Response to introduction of dsRNA identified in
  plants with infection with dsRNA virus

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Specific Gene Inhibition

• Ribonuclease Dicer cleaves dsRNA into short
  fragments 21-22 bp called small interfering RNA
  (siRNA)
• siRNA combine with a group of proteins to make
  RISC (RNA-induced silencing complex)
• degrades one strand of RNA and the other strand
  will complementarily bind to target mRNA
• if match is close slicer comes in and degrades
  mRNA
• if not close match, inhibit translation but not
  degraded
• RISC can move to nucleus and siRNA can bind to
  DNA and cause methylation to histones DNA
  becomes transcriptionally inactive
• Also a useful lab tool to determine the affects
  of genes in cells and cell development

67
MicroRNAs (miRNA)

• miRNA transcribed into longer RNA called primary
  miRNA
• Fold into a hairpin loop and converted to mature
  miRNA by steps
• Drosha cleave into smaller hairpins
• move to cytoplasm and Dicer will cut into small
  pieces
• bind to ribonucleoprotein complex silence
  expression
• close similarity degraded
• partial match inhibited
• Most are targeted to genes for proteins involved
  in development of the organism

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Post-Translational Control

• Modification of protein structure
• phosphorylation reversible and irreversible
• permanent change such as proteolytic cleavage
• Folding by chaperone protein
• Targeting to intra- and extra-cellular locations
• Interactions with cAMP or Ca2
• Amount of protein is a balance between synthesis
  and degradation

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Ubiquitin Targeting of Proteins

• Ubiquitin is a small protein bound to target
  proteins for proteosome
• 3 components involved
• ubiquitin-activating enzyme E1
• ubiquitin-conjugating enzyme E2
• substrate recognition protein E3
• E1 is ATP dependent transfer ubiquitin to E2,
  move to target protein lysine residue with E3

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Proteosomes

• Ubiquitin is recognized by proteosomes protease
  in the cytoplasm
• 6 proteases and ATPase and binding site for
  ubiquitin
• removes ubiquitin and then proteases chop up the
  protein into small fragments in a ATP-dependent
  fashion
• Different E3 enzymes recognize different amino
  acid sequences in amino terminus can either
  increase or decrease degradation
• Degrons internal amino acid sequences that
  allow degradation
• anaphase-promoting complex acts as an E3 for
  mitotic cyclins

71
Other Methods

• Ubiquitin targeting is not the only method to
  remove proteins
• Microautophagy lysosomal membrane forms small
  vesicles that bring in proteins that are then
  degraded in the lysosome
• non-selective
• More selective process requires the recognition
  of a glutamine residue with 4 very basic, very
  acid or hydrophobic amino acids on each side