NURO 346: Biological Clocks

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NURO 346 Biological Clocks

• Lecture 2
• Molecular Genetic Basis of Circadian Rhythms
  Milestones to a Molecular Clock Mechanism, Novel
  Circadian Photoreception, CircadianTranscriptional
  Control of the Genome

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The Period Gene

• The story of specific clock genes begins with
  the work of Konopka and Benzer (1971) describing
  the Period locus..
• Their basic design was to mutagenize flies and
  then screen for inherited changes in eclosion
  timing.

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The Period Gene

• Locomotor activity rhythms were affected as well.

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The Period Gene
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The Period Gene

• Summary of Early Findings
• Per is the first single gene shown to control
  behavior.
• PER protein is located in the nuclei of brain and
  visual neurons.
• Per transcription, translation and nuclear
  localization is rhythmic.
• Accumulation of PER suppresses its own
  transcription.
• Later
• The PER sequence contains a PAS protein-protein
  interaction domain.
• Homologs of Per (Per1, Per2, Per3) exist in
  mammals.

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The Clock Gene

• Clock was identified in a screen of mutagenized
  mice done in the lab of J. Takahashi (Vitaterna
  et al.,1994).

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The Clock Gene

• Mice heterozygous for the mutation showed
  lengthened free-running period, while homozygotes
  showed either very long periods or complete loss
  of behavioral rhythmicity (Vitaterna et al.,1994).

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The Clock Gene

• Transgenic introduction of the wild-type Clock
  gene by bacterial artificial chromosome,
  rescues normal circadian function (Antoch et
  al., 1997).

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The Clock Gene

• Sequencing of Clock revealed that it has PAS
  domains, like Per.
• bHLH basic Helix-Loop-Helix structural domain
  for binding of DNA.
• Homology with ARNT and SIM transcription factors.
• King et al., 1997

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The Clock Gene

• The Clock mutation is a single nucleotide change.
• It leads to a shortened version of CLOCK missing
  exon 19.
• King et al., 1997

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The Clock Gene

• Yeast two-hybrid screen reveals that Clock
  dimerizes with other proteins (Gekakis et al.,
  1998).

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The Clock Gene

• Of the potential binding partners only Bmal1
  co-expresses with Clock in the SCN and retina
  (Gekakis et al., 1998).

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The Clock Gene

• Clock/Bmal1 heterodimers drive transcription from
  the mPer1 promoter.
• This is specific to E-box response element
  sequences
• Mutant Clock (Clock ?19) fails to drive Per1
  (Gekakis et al., 1998).

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The Clock Gene

• PER protein can inhibit E-box mediated
  transcription by Clock (Drosophila, Darlington et
  al., 1998).

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The Clock Gene

• The first mammalian single gene shown to control
  a complex behavior.
• Has PAS domains like Per.
• Has a partner Bmal1.
• A transcription factor that acts to increase Per
  transcription.
• Binds to E-box sequences in the Per gene
  promoter.

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The Tau Gene

• Tau was described as a spontaneous mutation in an
  individual hamster that had an unusually phase
  advanced phase angle of entrainment and short
  freerunning period (Ralph and Menaker, 1998).

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The Tau Gene

• Tau was cloned and characterized by the Takahashi
  lab (Lowrey et al., 2000). that phosphorylates
  PER proteins.

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The Tau Gene

• Tau was shown to be casein kinase 1 epsilon
  (Lowrey et al., 2000).

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The Tau Gene

• Tau phosphorylates PER proteins (Lowrey et al.,
  2000).
• Phophorylation of PER decreased degradation rates
  (Price et al., 1998, Drosophila).

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Elements of a Clock Mechanism

• Negative feedback? PER
• Positive drive? Clock/Bmal1
• Delay? Tau

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Mouse Cryptochrome Genes

• Cryptochromes (Cry) are blue light sensitive
  proteins related to DNA repair enzymes in plants.
• In the course of non-circadian studies two groups
  (Sancar, Van der Horst, 1999) produced Cry1, Cry2
  and Cry1/Cry2 knockout mice.
• There were profound effects on circadian rhythms.

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Mouse Cryptochrome Genes

• Crys interact with PER proteins and concentrate
  them in the nucleus.
• PERs and CRYs are co-negative elements in the
  molecular oscillator mechanism (Kume et al 1999).

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Testing Roles with Gene Knockouts

• Bmal1 knockout induces arrhythmicity
• (Bunger et al., 2000).

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Testing Roles with Gene Knockouts

• Like the Cry genes, Per1 and Per2
• Show partial redundancy.
• Per3 KO leads only to a change in
• Period, not arrhythmicity.

Bae et al, 2002
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Testing Roles with Gene Knockouts

• KO of Rev-Erb does not lead to arrhythmicity.
• Locomotor period is shortened and the responses
  to light are modified(?).

Preitner et al, 2002
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Necessary Mammalian Clock Proteins
Positive Elements CLOCK, BMAL1
Negative Elements PER1, PER2, CRY1, CRY2
Mutations in Per3, RevErb, RORA, CKIe/d modify
period, but do not abolish clock.
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Mammalian Autoregulatory Clock Gene Feedback Loops

• Reppert and Weaver, 2002

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• Clock Genes in Other Nervous Systems

Zebrafish, Sassone-Corsi 2004
Drosophila, Hardin 2004
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Human Clock Genes

• The human genome contains homologs of all the
  mammalian circadian clock genes.

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Clock Input

• How are light zeitgeber signals transduced into
  physiological signals for the clock? Are there
  specialized photopigments for circadian
  phototransduction?
• Once transduced how do light signals impinge on
  the molecular clock mechanism?

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Circadian Photoreception

• In mammals circadian phototransduction occurs in
  the eye and neural signals are conducted along
  the RHT to the SCN.
• It was long assumed, but not proven, that the rod
  and cone photoreceptors which contain opsin
  photopigments were the locus of circadian
  phototransduction.

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Melanopsin A Photopigment Specialized for
Circadian Photoreception

• Melanopsin is expressed in a restricted set of
  retinal ganglion cells (red) that project to the
  biological clock (SCN) as well as the nuclei
  regulating the pupillary light reflex (OPN) and
  is a critical regulator of these non-visual
  photic responses.
• Its stimulation produces depolarizing responses
  in GCs.
• Its absorbance maximum is in the blue.
• The transduction pathway is undefined.

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Molecular Basis of Entrainment

• Reppert and Weaver, 2002

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Clock Output

• Hands of the clock (overt rhythms in physiology
  and behavior) vs. gears of the clock (molecular
  components).
• Do clock genes influence the expression of other
  genes?

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Transcriptional Control

• Genome sequencing and DNA microchip arrays
  ushered in the era of Circadian Genomics.

Hybridize to DNA chip With probes for many genes
Extract and Label Total RNA at Different CTs
Quantify Temporal Patterns of Gene Expression
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Transcriptional Control

• In mice two tissues were sampled SCN and liver.
• 672/7000 genes were found to be rhythmic in both.
• Genes were phase specific. (Panda et al 2002).

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Transcriptional Control

• Rhythmic genes were also tissue specific.
• Out of more that 300 cycling genes in each tissue
  only 28 (including core clock genes) were
  rhythmic in both.

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Transcriptional Control

• The clock coordinates control of gene clusters
  that are functionally related to modulate tissue
  physiology.
• Oxidative metabolism components in the SCN.

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Transcriptional Control

• Phase specificity is conferred by specific
  sequences in gene promoters.
• E-boxes, Clk/Bmal1 for day-phase genes.

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Transcriptional Control

• Phase specificity is conferred by specific
  sequences in gene promoters.
• RevErba response elements for night-phase genes
  (Ueda et al 2002).