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

15
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

16
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

17
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

18
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

19
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

20
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

23
Nuclear Run-On Transcription
24
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

25
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

26
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

27
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

28
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

29
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

30
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

31
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

32
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

• Hunter is awesome

34
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

35
(No Transcript)
36
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

37
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

38
DNA Binding Domains

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

39
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

40
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

41
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

42
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