Genetics PCB 3063

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Genetics - PCB 3063

• Todays focus
• Gene Regulation
• We will focus on two major questions today
• How does the structure of the genetic material
  alter the expression of genes?
• What are additional mechanisms of gene regulation?

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Gene Expression

• We discussed multiple points at which the
  activity of gene products are controlled. These
  include
• Transcription
• Translation
• Processing of Transcripts (and/or Proteins)
• Degradation (of mRNA and/or protein)
• Control of protein activity (e.g.,
  phosphorylation)
• Although post-translational modification of
  proteins by processes like phosporylation is very
  important, it is the first four processes that
  are usually studied in the context of gene
  expression.
• Are there additional ways to regulate gene
  expression?

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Examining Transcriptional Regulation

• This basic method was extended for the Gal4p
  study that we have been discussing discussed.
• For this study, the researchers tagged the Gal4p
  protein so the could purify from the cell.
• Then, they chemically cross-linked it to DNA and
  purified it.
• This allowed them to purify the DNA that Gal4p
  was bound to in the cell.
• The DNA that Gal4p was bound to in the cell was
  labeled and used to probe the microarray.
• Does this examine transcriptional regulation?

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Examining Transcriptional Regulation

• This study established several interesting facts
• The Gal4p binding sites in the DNA are sometimes
  bound by Gal4p in the absence of galactose,
  others are bound only in the presence of
  galactose.
• So the trigger is more complex than simply
  whether or not the Gal4p protein can bind.
• This more complex regulation involves Gal80p, an
  inhibitor.

Two possible models for regulation of
the Gal4p-Gal80p complex by galactose.
The models differ only in the exact binding sites
for Gal80p.
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How do Eukaryotic Transcriptional Regulators Work?

• There are a few specific types of proteins that
  act to increase transcriptional activity
• Many proteins have an acidic domain.
• Surprisingly, these acid-blob proteins often
  require a hydrophobic residue embedded in an
  acidic region.
• Both Gal4p and the herpes simplex virus VP16
  protein (an transcriptional regulator for this
  virus) have acid blobs.
• Glutamine-rich and Proline-rich transcriptional
  activation domains have been characterized.
• These protein regions activate transcription when
  fused to other DNA-binding domains.
• Alternatively, they can be recruited by
  protein-protein interactions - e.g., a
  DNA-binding protein binds the enhancer, and it
  contains a region that recruits and acid-blob
  protein.

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Using Eukaryotic Transcriptional Regulators

• The yeast 2-hybrid system exploits these features
  of eukaryotic transcription factors to examine
  protein-protein interactions.
• The DNA-binding and transcription activating
  regions of Gal4p can be separated.
• Interestingly, if you fuse one protein to the
  Gal4p DNA-binding domain (BD) and a second
  protein that it interacts (physically) with to
  the Gal4p transcriptional activating domain (AD),
  one can see transcriptional activation

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How do Eukaryotic Transcriptional Regulators Work?

• Another interesting phenomenon that is sometimes
  seen with transcription factor is SQUELCHING.
• Overexpression of transcription activators like
  Gal4p can result in a general inhibition of
  transcriptional activity.
• How does this happen?
• Presumably, specific transcription factors like
  Gal4p act by recruiting basal transcription
  factors.
• In fact, some basal factors that physically
  interact with these transcription activating
  domains have been found.
• Basal factors are factors involved in recruiting
  RNA polymerase II to a large number of promoters.
• So overexpressing proteins with these
  transcription activating domains can actually
  turn gene expression off, by competing for these
  factors.

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How do Eukaryotic Transcriptional Regulators Work?

• At least one way is by altering the packing of
  DNA into chromatin.
• The role of chromatin structure in the regulation
  of transcription is an area of very active
  investigation.
• However, two important factors that play clear
  roles in transcriptional regulation are known
• DNA METHYLATION - A subset of cytosine (C)
  residues are modified by methylation.
• HISTONE ACETYLATION - Histones can be modified by
  acetylation.

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Chromatin

• Remember, DNA in eukaryotes packs into CHROMATIN.
• HISTONES form the NUCLEOSOME, which DNA loops
  around.
• EUCHROMATIN - less compact actively transcribed
• HETEROCHROMATIN - more compact transcriptionally
  inactive.
• Heterochromatin can be either constitutive or
  facultative.

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DNA Methylation

• Genes that are transcriptionally inactive are
  often METHYLATED.
• In eukaryotes, cytosine residues are modified by
  methylation.
• Typically, the sites of methylation are CG
  dinucleotides (vertebrates).
• This allows maintenance through replication.

CYTOSINE
METHYL-C
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Histone Acetylation

• HISTONES in transcriptionally active genes are
  often ACETYLATED.
• Acetylation is the modification of lysine
  residues in histones.
• Reduces positive charge, weakens the interaction
  with DNA.
• Makes DNA more accessible to RNA polymerase II
• Enzymes that ACETYLATE HISTONES are recruited to
  actively transcribed genes.
• Enzymes that remove acetyl groups from histones
  are recruited to methylated DNA.
• There are additional types of histone
  modification as well, such as methylation of the
  histones.

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Genetic Imprinting

• Remember that DNA methylation can be maintained
  through replication.
• This allows the packing of chromatin to be passed
  on - just like a gene sequence.
• However, differences in chromatin packing are not
  as stable as gene sequences.
• Heritable but potentially reversible changes in
  gene expression are called EPIGENETIC phenomena
• Vertebrates use these differences in chromatin
  packing to IMPRINT certain patterns of gene
  regulation.
• Some genes show MATERNAL IMPRINTING while other
  show PATERNAL IMPRINTING.
• The alleles of some genes that are inherited from
  the relevant parent are methylated, and therefore
  are not expressed.

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Prader-Willi Angelman Syndromes

• Both of these genetic disorders are caused by
  deletion of a region of chromosome 15.
• However, the syndromes differ
• Prader-Willi Syndrome - obesity, mental
  retardation, short stature. (abbreviated PWS)
• Angelman Syndrome - uncontrollable laughter,
  jerky movements, and other motor and mental
  symptoms. (abbreviated AS)
• Syndrome that develops depends upon the parent
  that provided the mutant chromosome.
• This is a common phenomenon for - imprinted genes
  tend to be found in clusters.

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PWS Mouse model
PWS
AS Mouse model
AS
From Annu Rev Genomics Hum Genet
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Prader-Willi Angelman Syndromes

• Prader-Willi Syndrome - develops when the
  abnormal copy of chromosome 15 is inherited from
  the father.
• Angelman Syndrome - develops when the abnormal
  copy of chromosome 15 is inherited from the
  mother.
• The differences reflect the fact that some loci
  are IMPRINTED.
• For imprinted loci, only the allele inherited
  from one parent is expressed.
• The PWS/AS region contains both maternally and
  paternally imprinted genes.

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Imprinted Genes in the Mouse

• Data from http//www.mgu.har.mrc.ac.uk/research/im
  printed/imprin.html

Red Maternal allele.
Blue Paternal allele.
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Methylation and Gene Regulation

• For imprinted genes, the pattern of gene
  regulation is dependent upon the parent that
  donated the chromosome.
• The methylation pattern is reprogrammed in the
  germ line.
• There are other cases where changes in the
  pattern of methylation alter gene expression.
• In mammals, one of the two X chromosomes in
  females is inactivated and the inactivated X
  chromosome is methylated.
• Methylation is common in filamentous fungi. E.g.,
  Ascobolus has a process called MIP that leads to
  the methylation of all C residues, whether they
  belong to symmetrical sequences or not, in
  repeated sequences (both repeats are methylated).

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X Chromosome Inactivation

• Female mammals inactivate one X chromosome.
• Thus, females arent truly homozygous for genes
  on the X, since one allele is expressed in some
  areas of the body and the other allele is
  expressed in other areas of the body.
• This makes female mammals MOSAICS.
• The most famous phenotype reflecting this is coat
  color for Calico and Tortoiseshell Cats...

Tortoiseshell
Calico
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X Chromosome Inactivation

• The Tortoiseshell pattern reflects a color gene
  (O for orange) on the feline X chromosome...

• Early in embryonic development, the X chromosomes
  are inactivated randomly.
• The tortoiseshell cat is heterozygous for the O
  locus, so patches of fur are expressing either
  the O (orange) or o (black) allele.
• Calicos have a specific allele of an autosomal
  gene that causes white spotting.

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X Chromosome Inactivation

• However, this phenomenon also impacts humans.
• The disease EDA (Ectodermal dysplasia 1,
  anhidrotic) reflects an allele of an X-linked
  gene that cause a number of glandual defects and
  problems with the development of hair and teeth.
• This is most famous for causing a defect in sweat
  glands, so females that are heterozygous for the
  EDA gene have patches of skin with no functional
  sweat glands.
• How would you expect EDA to affect males?

Note the dental phenotype
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How Does X Inactivation Work?

• X chromosome inactivation (XCI) was first
  reported in 1961 by Mary Lyon.
• The X chromosome inactivation is correlated with
  formation of a condensed BARR BODY named for
  Murray Barr, who had observed sex chromatin in
  females.
• An RNA gene called XIST acts in cis to recruit
  factors that silence the inactive X chromosome
  (Xi).
• Expression of XIST on the active X chromosome
  (Xa) is blocked by another RNA called TSIX.
• XCI is global - it affects almost all genes on
  the X chromosome.
• Once established, the pattern of XCI is
  irreversible in the soma.
• How Xist RNA paints the Xi and how Tsix blocks
  this epigenetic painting are two of the most
  compelling questions in the field of X
  chromosome inactivation J. T. Lee (2003)

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Gene Regulation DNA Rearrangement

• We discussed multiple points at which the
  activity of gene products are controlled. These
  include
• Transcription
• Translation
• Processing of Transcripts (and/or Proteins)
• Degradation (of mRNA and/or protein)
• Control of protein activity (e.g.,
  phosphorylation)
• We saw modifications to the DNA (and associated
  proteins) as a means of gene regulation.
• One might imagine that DNA sequence changes might
  be able to change gene expression.
• Has this been documented?

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Salmonella Phase Variation

• For Salmonella, the flagellum is the major
  antigen.
• This organism escapes the immune system of hosts
  is by switching the type of flagellin produced.
• Flagellin is the major protein of the flagellum.
• This switching occurs through a mixture of DNA
  rearrangements and transcriptional regulation

Arrow indicates the direction of transcription.

• H1 repressor (second gene in the H2 operon) turns
  transcription of the H1 flagellin gene off.
• Thus, in one arrangement H2 is expressed (but not
  H1) while in the other H1 is expressed (but not
  H2).

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Salmonella Phase Variation Mechanism

• Phase variation is mediated by the product of the
  hin recombinase gene.
• This gene product allows DNA between inverted
  repeats to recombine, flipping it

• The Hin recombinase is related to the Flp
  recombinase of S. cerevisiae.
• Flp resolves concatamers of the yeast 2 µm
  plasmid, which are replication intermediates.

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Anabaena N2 Fixation

• The cyanobacterium Anabaena is capable of fixing
  atmospheric nitrogen.
• Cyanobacteria produce oxygen during
  photosynthesis.
• This is a problem, because nitrogenase, the
  enzyme responsible for fixing nitrogen, is
  inactivated by oxygen.

• Anabaena solves this problem by fixing nitrogen
  in specialized cells.
• These cells are called heterocysts.
• Heterocysts do not produce oxygen, but they do
  express nitrogenase.

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Anabaena N2 Fixation DNA Rearrangement

• In heterocysts, two segments of DNA that
  interrupt genes involved in fixing nitrogen are
  deleted

• Deletion elements encodes recombinases (XisA and
  XisF) that excise the material between direct
  repeats.
• Do you think this can be reversed?

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Gene Cassettes in Eukaryotes

• S. cerevisiae switches mating types by moving a
  gene cassette into an expressed position

a-specific genes
a-specific genes

• There are two silent mating type cassetes (HML
  and HMR) that can recombine to the MAT locus.
• The MAT locus is expressed.
• The products of the MAT locus is a master
  regulator, MAT a turns a-specific genes on and
  a-specific genes off.

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TrypanosomesVariable Surface Glycoproteins

• Trypanosoma brucei is the protozoan that causes
  African sleeping sickness.
• These organisms are transmitted by the bite of
  the tsetse fly

• The antigen presented by trypanosomes are the
  variable surface glycoprotein (VSG)
• Trypanosomes have more than 1000 VSG genes, but
  only the VSG gene at a specific telomeric locus
  is expressed.
• New (unexpressed) VSG genes are moved into the
  expressed locus.

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Heritable and Non-Heritable Changes

• MUTATIONS are heritable changes in DNA.
• In contrast, some chemicals or conditions cause
  phenotypic changes that cannot be inherited.
• Non-heritable changes caused by chemical agents
  or other environmental conditions that resemble
  mutant phenotypes are often said to PHENOCOPY the
  mutation.
• For example, thalidomide causes severe birth
  defects that resemble the inherited condition
  phocomelia.
• SC phocomelia is sometimes called
  pseudothalidomide syndrome.
• Likewise, vitamin D deficiency can cause rickets
  (lack of proper bone calcification). This is a
  phenocopy of vitamin D resistant rickets, an
  X-linked mutation in humans.
• Ultimately, these conditions cause a change in
  gene expression that causes the phenotype