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Eukaryotic gene expression

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					      Eukaryotic gene expression
• Bacterial genes have a ground state that permits
  transcription
   – Without CAP site or operator, the sigma subunit will
     locate a gene
• Eukaryotic genes require complex systems to turn
  them on
   – Chromatin structure must be relaxed in order for RNA
     polymerase to gain access to DNA sequence information
   – Eukaryotic genes are positively regulated. They are not
     transcribed in the absence of active mechanisms.
   – The regulatory components and systems are more
     complex than bacteria
   – Transcription is removed from translation
      • There are no systems equivalent to attenuation in eukaryotes
   The evidence for
     alteration of    • DNase I cleaves chromatin at
                        the linker junctions between
chromosomal structure nucleosomes
 during transcription    – When run on an agarose gel, the
                               DNA resulting from cleavage
                               forms ladders reflecting discrete
                               units increasing in size by 200
                               nucleotides
                                • This means that nucleosomes cover
                                  the bulk of DNA and protect from
                                  Dnase digestion
                             – But this procedure reveals the
                               structure of DNA, and not any
                               specific genes
DNAse digestion of heat shock genes
                 • When genes are identified
                   within these ladders, they are
                   found in two forms
                    – Genes that are not transcribed are
                      also found to form ladders in
                      response to DNAse I
                    – Genes that are transcribed are
                      fragmented into smaller pieces
                        • The nucleosomes are gone in the
                          upstream regions of genes
                          undergoing transcription
                    – A heat shock gene was digested
                      over time following heat shock
                      and the upstream region identified
                      with a specific probe
                        • Following heat shock, the control
                          regions of the gene become
                          hypersensitive to digestion
Where there are no nucleosomes
               • The nucleosome free sites
                 are not throughout the
                 entire gene, but in certain
                 places called
                 hypersensitive sites
                  – Hypersensitive sites
                    correspond to regions of
                    DNA that bind transcription
                    factors
                  – Thus hypersensitive sites
                    are found upstream of the
                    coding region of genes
                  – They also may be found
                    wherever transcription
                    factors bind
                      • For eukaryotic genes, that
                        is not always just 5’ to the
                        transcriptional start
 Other alterations     • Loss of histone H1
                          – This is the histone that
to transcriptionally        exists between the
                            DNA/histone octomer coils
    active DNA         • Loss of methyl group from
                         5 methyl cytosine in CpG
                         islands
                          – Transcriptionally silent
                            DNA tends to have more 5
                            methyl cytosine than active
                            DNA
                     • The shutdown of g globin is
A clinical example     due to methylation of the
                       upstream region of the genes
  - thalassemia        before and after birth
                     • In the human disease
                       thalassemia, b and d globin
                       chains are lost due to
                       mutation of the globin genes.
                     • One therapy involves
                       administration of 5
                       azacytidine
                        – Incorporation of this nucleotide
                          results in a loss of methylation
                        – This results in the activation of
                          fetal globin genes which
                          assume some of the oxygen
                          carrying capacity of the mutant
                          globin
                          • Histones have two functional
Histone acetylation         domains
                             – One for binding other histones
                               and wrapping DNA around the
                               nucleosome core
                             – The other is a modification site
                               for control of histone assembly
      Acetylation sites
      Of Histone H4              • Multiple lysine residues are
                                   presented to the exterior of the
                                   histone
                          • Histones are acetylated prior to
                            import into the nucleus
                            following their synthesis on
                            ribosomes
                             – They are actively assembled on
                               DNA by an enzymatic
                               mechanism
   Acetylation near        • A nuclear histone
                             acetylase further acts on
transcriptionally active     histones H3 and H4
                              – Increasing acetylation
         genes                  decreases the affinity of
                                the histone octomer for
                                DNA
                              – This makes the DNA
                                more available for
                                binding interactions with
                                other proteins
                              – Histones are moved out
                                of the way by an ATP
                                driven process involving
                                a multiprotein complex
                                  • Repressors may
                                    stimulate deacetylation
                                  • Activators may
                                    stimulate acetylation
                     Eukaryotic promoters

• Why are they subject to positive regulation?
   – The genes within are sequestered because of chromatin structure
   – The size of the genome favors non-specific binding of regulatory proteins at
     random
       • In a diploid genome of 6 billion nucleotide pairs, a short sequence capable of binding
         regulatory proteins would occur many times by chance
       • So regulatory systems demand that multi-protein complexes form before a gene is
         transcribed
   – It is more efficient to negatively regulate the entire genome with a single
     mechanism (chromatin structure) and then specifically turn on the set of genes
     needed by the cell than to specifically negatively regulate every gene of a
     eukaryote
       • That would mean tens of thousands of repressors for each cell type
             Promoters and Enhancers
• Promoters include, for example, TATAA boxes, GC boxes and
  CAAT boxes that are responsible for positioning RNA polymerase II
  at the beginning of a gene
   – Polymerase II has no affinity for the TATAA box on its own.
   – Assembly of a transcriptional complex depends on the sequence around the 5’
     end of the gene
• Enhancers are sequences that are distant from the promoter but
  positively affect its function
   – They may be pointed in either orientation
  Three classes of •    Basal (general)
                        transcription factors
transcription factors   – These interact directly with
                          RNA polymerase II or with
                          each other in building a
                          complex around the
                          promoter
                        – They also recognize the
                          promoter sequences
                            • The TATA box is highly
                              conserved
                            • The TATA binding protein +
                              transcription factors for
                              polymerase II (TF II) assemble
                              and provide the minimal
                              assembly for transcription
                            • But transcription still requires
                              a positive signal
                                – This complex marks the spot
                                  where RNA polymerase is to
                                  bind and begin transcription
                   • Also known as DNA binding
   Enhancer          transactivators
                      – These bind enhancers that are far
binding proteins        away from the promoter
                          • They recognize the specific
                            enhancer sequence
                          • Some enhancer binding proteins
                            work on a large number of genes,
                            permitting coordinate control of
                            transcription
                          • Others are specific to a single
                            gene
                      – They then loop inward toward the
                        promoter so that the enhancer
                        binding protein can interact with
                        the basal transcription factors at
                        the promoter site
                          • Protein-protein interactions are
                            mediated through motifs such as
                            the leucine zipper and the helix
                            loop helix
Coactivator proteins

                • These bind RNA
                  polymerase II complexes
                  and enhancer binding
                  proteins and mediate the
                  signaling between them
                • RNA polymerase II may
                  carry the coactivator
                  proteins with it as it
                  transcribes
                • Coactivators are necessary
                  for transcription
The process of    • Remodeling chromatin
                     – May involve
transcriptional          • Demethylation of 5 methyl C
                         • Acetylation of histones
   activation     • Binding of basal transcription
                    factors
                  • Transactivator binding enhances
                    the remodeling of chromatin and
                    facilitates opening up chromatin
                    structure
                     – This helps other enhancer binding
                       proteins to interact with exposed
                       DNA sequence
                  • Transactivators interact with
                    coactivators and help RNA
                    polymerase position itself on the
                    transcription complex at the
                    TATA box
                • Inducibility and especially
Induction and     repression is not as common a
                  phenomenon in eukaryotic cells
  repression       – Especially higher eukaryotic cells
                • The larger the organism, the more
                  stable the environment a cell
                  experiences
                   – So it needn’t respond to radical
                     changes in the environment
                • However some transcriptional
                  regulation is still necessary
                   – Transactivators can serve the function
                     of inducers or repressors
                   – A repressor generally inhibits the
                     function of an inducer by some
                     mechanism
                       • Competitive binding
                           – To DNA
                           – To basal transcription factors
                       • Directly binding the activator
                             • Binding to a small
     Induction and             molecule can result in an
                               increase or decrease in the
       repression              ability of a transactivator
                               to work
                                – Steroid hormone receptor
                                  becomes a functional DNA
                                  binding transactivator in
                                  response to binding its
                                  ligand
                                   • Binding to a ligand displaces
                                     HSP90 (a heat shock protein)
                                     and permits translocation to
                                     the nucleus and subsequent
                                     DNA binding
AD: activator domain            – In the absence of ligand, it
DBD: DNA binding domain           interferes with transcription
LBD: ligand binding domain        and thus becomes a
                                  repressor
                                             A specific example
• The GAL genes of yeast
   – These are a set of individual genes under coordinate control
       • Eukaryotes don’t have operons
   – Each gene has a promoter and set of enhancers (called UAS)
       • Turning on one of the GAL genes means activating a set of enhancer
         binding proteins and coactivators that turn on all of the other GAL genes
   – The products of the genes are needed for importation of galactose and
     its metabolism
The GAL genes can be repressed

                 • The logic is the same as
                   with bacteria
                 • When glucose is
                   present, there is no
                   necessity to make
                   galactose importation
                   and metabolizing
                   enzymes, so the genes
                   are shut down
                    – This repression
                      overrides induction
            • Gal4p is a transactivator
Induction     that induces transcription at
              a GAL locus by interacting
              with the coactivator
              assembly at promoter
               – In the absence of galactose,
                 Gal4p is sequestered by
                 another regulatory protein
                 Gal80
               – Gal4p is displaced from
                 Gal80 by Gal3p when Gal3p
                 binds galactose
               – Thus in contrast to bacterial
                 inducers, the ligand binding
                 and the DNA binding
                 proteins are not the same
Minimum structure
of enhancer binding •   Each must
      proteins          – Bind its target DNA
                        – Bind the promoter complex and/or
                          activating proteins
                    • The ability of a protein to
                      perform each function is due to
                      functional domains in the
                      protein
                        – The domains permit interaction
                          with other proteins and specific
                          recognition of DNA sequence
                        – In addition to DNA and protein
                          interaction domains, there are 3
                          common types of activator
                          domains
                            • Acidic – Gal 4p
                            • Glutamine rich – SP1
                            • Proline rich - CFT1
         • This has a domain
           resembling a zinc finger
GAL 4p      – Instead of two cys and two his
              coordinating a Zn , it has 6
              cys residues
         • It is a homodimer, bound by
           interactions of two coiled
           coils
         • The two zinc fingers interact
           with a palindromic sequence
         • The protein is controlled by
           another domain that is rich
           in aspartic and glutamic acid
           residues
            – This was identified by
              constructing mutants of the
              Gal4p gene that substituted
              other amino acids in this
              domain
            – The mutants lost function
• SP1 binds the GC box
   – GC boxes are located close to the TATA sequence
   – SP1 is a very common enhancer binding protein
       • Many genes lack a GC box
   – There are 3 Zn fingers for DNA binding
   – Two glutamine rich activator domains
• CTF1 binds the CAAT box
   – The DNA binding domain is unique and is neither helix turn helix or a zinc
     finger
   – The activation domain is proline rich




                                              SP1 and CTF1
 Domain    • Since the domains for DNA
             binding and activation are
swapping     distinct, their domains may be
             separated on the level of DNA
              – By taking a domain for DNA
                binding and adding it to a domain
                for activation, a new protein may
                be engineered
              – This binds the DNA sequence
                specified by one gene, and
                responds to the signals of another
              – Such experiments permit the
                manufacture of proteins with
                unique control abilities
                  • Although not therapeutically
                    useful right now, they are
                    important experimental tools in
                    defining the way that genes
                    respond to external signals.
• Gene expression in
  multicellular organisms is           Regulated gene
  often controlled by
  intercellular signaling                expression
   – Some genes are directly
     responsive to environmental
     stimulus however
      • UV induction of DNA repair
        enzymes
      • Stress response (heat shock)
        genes
• Signaling takes two forms
   – Hormones may be bound by
      • Membrane bound receptors
      • Diffusible receptors
             • Diffusible receptors act by
Diffusible     directly binding a hormone
               and then moving into the
receptors      nucleus
                – Hormone binding induces a
                  conformational change that
                  permits the receptor to act as a
                  transcriptional activator
                   • Diffusible molecules can be
                     transactivators
                       – “trans” means something that
                         acts on a gene that originates
                         from another site. In this
                         fashion they resemble the
                         activators of bacteria
                   • However hormones are made by
                     one cell in order to command a
                     transcriptional response in
                     another cell
                   • Bacterial effectors are nutrients
                     or their metabolites or analogs
           • These are
 Steroid      – endocrine hormones
              – hydrophobic molecules that are
hormones        synthesized using cholesterol as a
                precursor
              – made by certain cell types and
                secreted in response to
                biochemical, developmental or
                neurological signals
              – carried by the blood from their cell
                of origin to target cells either
                dissolved or by a protein carrier
                  • Many are too hydrophobic to
                    dissolve directly in blood
           • They enter a cell by dissolving
             in the plasma membrane and
             diffusing to their receptor
                   • This is a DNA binding protein
   The steroid       with a hormone binding domain
                     at the carboxyterminal end of
hormone receptor     the protein
                   • There are several related types
                      – Each receptor has a specific
                        complement of transcription
                        factors it must interact with which
                        vary from one receptor to another
                        and one cell type to another
                   • They are all related in structure
                      – The DNA binding domain
                        contains two Zn fingers and is in
                        the middle of the protein
                      – The domain that interacts with
                        transcription factors is amino
                        terminal and varies in structure
                   • The hormone binding region is
                     highly variable in structure
                      – Each must recognize and bind its
                        cognate ligand
• Loss of responsiveness to a
  hormone can be caused by
  changes in any of the three
  domains
    – Hormone-ligand complexes may
      serve either positive or negative
M     regulatory functions
        • Mutations prevent transcriptional
U         activation or repression in
          response to hormone binding
T           – Mutation in the androgen
              binding domain of the androgen
              receptor creates androgen
A             unresponsiveness
            – Mutation in the DNA binding or
T             transcription activation domains
              would mean the protein could
I             bind androgen, but nothing
              would happen
                 » This results in
O                   developmental
                    abnormalities such as XY
N                   females
                 » To the left are four XY
                    siblings suffering from
S                   androgen insensitivity
           • Cis refers to sequence involved
 The cis     in gene expression
              – Trans elements interact with cis
elements        elements but arise from other genes
              – The glucocorticoid responsive
                element (GRE) and estrogen
                responsive element (ERE) share
                sequence homology
           • The cis elements that are
             important in hormone
             responsiveness are the binding
             sites for the hormone-receptor
             complexes
              – Hormone responsive elements: HRE
              – These are direct repeats that interact
                with the Zn finger domains
                  • The consensus sequences for these
                    receptors are very similar
                  • This reflects the similarity in the Zn
                    finger domains among the various
                    receptors
              • Following binding of a
                hormone, the receptor
 Receptor –     diffuses to the nucleus and
                binds the HRE
DNA binding   • The receptor is a dimer
                 – Each subunit of the dimer
                   binds to one of the two repeat
                   elements
                 – The strength of binding is
                   determined by the variation of
                   the HRE away from the
                   consensus sequence
                 – The stronger the binding
                   between the receptor and
                   HRE, the longer the receptor
                   will remain bound and the
                   longer transcription will be
                   activated
                     • Binding is an all or none
                       event
                     • If bound, activation due to
                       the receptor is full
                        • Transcription factors are subject to
 Phosphorylation of       phosphorylation on serine and
                          threonine residues
transcription factors   • This is the result of second
                          messenger activation of serine-
                          threonine kinases or ras activation
                        • In abnormal, though common,
                          conditions, such as mutations or
                          viral infections, gene expression is
                          deregulated and genes are
                          inappropriately expressed because
                          of deregulated phosphorylation
                          mechanisms
                           – This is because second messengers
                             activate a complex cascade of
                             enzymatic steps that can be perturbed
                             at many different points
                           – Here the transcription factor Elk-1 is
                             activated through phosphorylation
                      Repression
• This occurs at the transcriptional and translational
  level
   – Genes are usually turned off as a default at the
     transcriptional level
      • But this does not mean the mRNA is gone
          – It could have been stabilized through sequestration
   – Translational regulation permits rapid responsiveness
      • The primary transcript of a gene may take several minutes to
        synthesize because of its size
      • It also must be spliced and transported to the ribosomes
      • A sequestered transcript that is released in response to a signal
        is faster
                     • The distribution of mRNA
  Translational        within some cells creates a
                       distribution of protein
repression affects     inside a cell
  more than just        – This results in intracellular
                          protein gradients that are
    ribosomal             important in development

     proteins
  Regulatory     1. Inhibition of initiation factors
                    through phosphorylation
mechanisms of       – eIF phosphorylation inhibits its
                      function and can be reversed
 translational        through dephosphorylation
                 2. Inhibition of initiation factors
   initiation       by binding to specific factors
                    – Interference with eIF4E and eIF-
                      4G activity by 4E-BP’s.
                 3. Inhibition of specific mRNA
                    by binding of inhibitory
                    proteins to sequences in the
                    3’untranslated region
Phosphorylation of    • Maturation of red blood
                        cells involves a stage in
 eIF-2 inhibits its     which reticulocytes translate
                        mRNA left behind after the
     activity           loss of the nucleus
                      • Reticulocytes regulate the
                        amount of globin
                        synthesized by
                        phosphorylating eIF-2
                         – When there is heme
                           deficiency globin synthesis is
                           wasteful since hemoglobin
                           cannot be synthesized
                             • Low heme activates HCI
                               which phosphorylates eIF-2
                         – Phosphorylated eIF2 binds
                           eIF2 binding protein and is
                           unavailable for translational
                           initiation
     eIF4E inhibition
• eIF4E is necessary to bind
  the 5’ CAP in order to from
  an initiation complex for
  translation
   – Normally it binds eIF4G
• Maskin binds eIF4E
  (preventing it from binding
  eIF4G) when it is bound to
  an mRNA through
  interaction with CEPB
Developmental control •   The study of fruit fly
                          development resulted in
 of gene expression       the discovery of a number
                          of genes involved in
                          human disease
                          – Although fruit fly
                            development is greatly
                            different in the processes
                            leading to the final form,
                            the activation of genes and
                            the structure of gene
                            products and their
                            participation in the
                            formation of patterns and
                            structures have parallels in
                            human gene regulation and
                            the structure of human
                            regulatory gene products
Fly development is controlled by
        gene expression
• The conceptually difficult part of this is to
  understand how a single cell can create multiple,
  morphologically different structures starting from
  a seemingly symmetrical, undifferentiated state
  merely by dividing.
• It is easier to think of the process in parts and then
  add up the whole than to see a cell turn into a fly
  and attempt to understand the entire process at
  once
                      • Maternal genes
                         – Made by the female and
Three gene families        exist within the egg at
                           the time of fertilization
are responsible for         • Responsible for
                              establishing the polarity of
early development             the early embryo
                      • Zygotically acting genes
                         – Segmentation genes
                            • These establish a
                              repeating pattern of body
                              segments
                         – Homeotic genes
                            • These establish the
                              identity of the segments
           • This is a distinction in
             structure established
Polarity     between two poles.
              – The distinction needn’t be
                great, only a morphological
                difference is enough to
                create polarity
              – Without polarity, further
                structures would have no
                way of organizing
                themselves
                  • Segments would be
                    repeating structures that
                    are all the same
              – Establishing polarity is thus
                the earliest developmental
                event
                  • Polarity is actually
                    established by the
                    assymetry of the egg
                  • This yields pole cells on
                    one end of the zygote
               • This is obvious in the formation
                 of the fly abdomen
                  – Repeating abdominal segments are
Segmentation        very similar in appearance
                  – However this patterning extends
                    from end to end of the fly
                      • The patterns are given different
                        identities by homeotic genes
                      • Thus the head and abdomen begin
                        as segments similar to abdominal
                        segments
                          – But polarity makes them
                            different, and therefore the genes
                            that are expressed within each
                            segment differs
                  – Segments are created and further
                    divided into smaller segments
                      • Gap genes create the largest
                        segments
                      • Pair rule and segment polarity
                        genes subdivide the largest
                        segments
• These give rise to the dramatic
  mutants of Drosophila
• Once segments are established with
  the proper polarity, homeotic genes
  create unique structures
• Mutation of a particular homeotic
  gene results in the formation of a
  structure that is due to the action of
  another homeotic gene
   – The homeotic genes are controlled by
     their position within a gradient of polarity
   – So if one segment was destined to give
     rise to antennae, but lacked the homeotic
     gene due to mutation, it would create the
     next most available structure



        Homeotic genes
Antennaepedia   • Antennaepedia represents the
                  mutation of a gene that
                  would create an antennae
                   – Antennapedia is a transcription
                     factor that coordinately
                     controls expression of genes,
                     that when expressed result in
                     an antennae
                • In its absence, the next most
                  similar structure is a leg
                   – The normal leg is also formed
                     in the next segment
                   – The gene encoding the protein
                     controlling leg development is
                     expressed at lower
                     concentrations in the head
                     segment than antennapedia
                     gene, but in the absence of
                     antennapedia, it is the most
                     highly expressed protein
                     capable of activating genes
                     that result in a structure
                  • Maternal genes establish
 Key genes are      polarity due to formation of
                    gradients
expressed early      – Front to back and top to bottom
                       gradients establish anterior
                       posterior and dorsal ventral
                       gradients
                  • When cells are formed in the
                    blastoderm, they form within an
                    environment in which the
                    concentration of transcription
                    factors will vary along one axis
                     – This varies the type and numbers
                       of genes that are expressed within
                       any cell
                  • To the left is bicoid RNA
                    (upper) and bicoid protein
                    (lower) in the early embryo
                     – The RNA gradient is present in the
                       egg and establishes the protein
                       gradient
                • Bicoid is a maternal gene that
A few examples of controls expression of
                  segmentation genes
 developmental      – It is a transcription factor that
                      activates segmentation genes
      genes         – And a translational repressor
                • It appears in the anterior of an egg,
                  and its concentration falls of
                  towards the posterior
                • The gradient is maintained during
                  formation of the larvae
                • Experiments with bcd mutants
                    – (a) A failure of bicoid to be expressed
                      means a fly develops with two
                      posteriors rather than an anterior and a
                      posterior
                    – (b)injecting cytoplasm from a normal
                      embryo rescues the embryo (makes an
                      anterior)
                    – ( c) injecting bicoid mRNA also
                      rescues
                   • It represses translation of caudal
                     in the anterior of the fly larvae
                      – Caudal is a transcription factor
What bicoid does        found uniformly throughout the
                        larvae, and it creates the posterior
                        end
                   • And activates expression of
                     hunchback
                      – Hunchback is a transcription factor
                        that creates the anterior end
                   • The bicoid gradient means it has
                     these effects only in the anterior
                     end
                   • Without bicoid, caudal is not
                     repressed in the anterior end and
                     hunchback is not transcribed
     Nanos
• This is a translational
  repressor that is found at
  highest concentrations in the
  posteror of a fly larvae
• It acts in concert with the
  uniformly distributed
  pumilio gene product to
  translationally repress
  hunchback
• This results in establishment
  of high caudal gene product
  and inactivated hunchback
  mRNA, meaning the
  posteriorizing effect of
  caudal dominates
Some Gap genes
       • Gap genes create a gross
         form of segmentation in
         the early embryo
       • They are overlayed onto
         the pair-rule gene
         expression to create
         complex transcriptional
         signals
       • These are the expression
         patterns of the hb-z, Kr
         and kni genes
               • These are two segmentation
fushi tarazu     genes known as pair rule genes
                 that split a segment in two
  and eve         – Ftz establishes the “pair rule”
                      • Two segments form out of one
                      • Without it the fly forms 7 rather
                        than 14 segments
               • Ftz (blue) is expressed in each
                 segment, in the anterior half of
                 the segment
                  – This expression pattern is again the
                    result of the action of an anterior
                    posterior gradient, but now within
                    each segment
               • Eve (brown) for even-skipped
                 are expressed in the posterior
                 half of each segment
               • Both ftz and eve are
                 homeodomain transcription
                 factors that control expression of
                 genes expressed in the segments

				
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