Next lectures: Differential Gene expression

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Next lectures Differential Gene expression

• Chapter 5 and websites on syllabus
• Epigenetic control mechanisms
• Histone modification
• DNA methylation
• Nucleosome disruption machines
• Promoters and enhancers
• Old and new models of enhancer function
• Novel transcriptional control sequences

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DNA methylation

• Eukaryotic DNA methylation occurs on the 5
  position of cytosine in the CpG dinucleotide
• The major methylation enzyme is DNMT
• DNA methylation is seen in plants and animals
  but NOT in yeast or Drosophila
• DNMT knockout mice die in utero

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DNMT knockout miceLi, Bestor and Jaenisch (1992)
Cell 69915-926
DNMT is required for post-gastrulation
development. These mice do not progress past the
9th day of their 19 day gestation period
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DNMT is a maintenance methylaseFrom Jaenisch
(1997) Trends Genet. 13323-9
There are, likely, many DNA methyltranferase
activities that have yet to be identified. Some
are for maintenance and others de novo
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Role of DNA methylation in the genome

• Genomic stability
• Higher mutation rates in dnmt-/- ES cells
• Global demethylation in tumor cells
• Specific demethylation of oncogene promoters
• Transcriptional regulation
• Correlation between methylation and closed
  chromatin structures
• Blockade of factor recognition of DNA

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Chromatin structure and DNA methylation

• Pharmacological evidence
• Trichostatin A
• Blocks histone deacetylase activity
• Prevents DNA methylation dependent repression
• Sodium butyrate
• Mimicks histone acetylation
• Used to loosen up chromatin

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Mediators of methylation induced gene silencing

• Methyl CpG binding proteins are repressive
• MeCP2 (Knockout mouse also embryonic lethal)
• Has a methyl CpG binding domain and a
  transcriptional repression domain
• Interacts with the mSin3 co-repressor complex
  which associates with HDAC to repress
  transcription

From Bestor (1998) Nature 393311
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Proposed mechanism stable repression of gene
expression through development
From A. Razin (1998) EMBO J. 174905-4908
Transcription factors are transient while DNA
methylation is not
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How stable is DNA methylation?

• Specific DNA de-methylation events have been
  implicated in gene activation.
• Igk gene enhancer (Y. Bergman--Jerusalem)
• Growth control genes in tumors (Baylin and
  Herman--Johns-Hopkins)
• DNA de-methylation can be global (as above) or
  targeted to particular sequences
• Santoso, et. al.(2000) J. Biol. Chem. 2751952
• Schubeler, et. al. (2000) Mol. Cell. Biol. 209103

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How is DNA methylation regulated?
From Ng and Bird (1999) Curr. Opin. Genet. Dev.
9158-163
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A DNA de-methylase

• Bhattacharya, et. al. (1999) Nature 397579
• Has a methyl-binding domain (MBD) homologous to
  that of MeCP2
• Removes ONLY the methyl group from the cytosine
  without damage to the nucleotide or the DNA
  backbone
• Possibility of dynamic regulation of DNA
  methylation as is with histone acetylation

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Study of DNA methylation

• Restriction enzymes isoschizomers with
  differential ability to cut methylated DNA
• Msp I and Hpa II (CCGG)
• Hha I (CGCG)
• Bisulfite conversion followed by PCR
• McrBC restriction enzyme (cuts methyl-CpG)
• SssI methylaseTo artificially methylate DNA
• 5-azacytidineTo artificially de-methylate DNA

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DNA methylation has been implicated in the
following developmental processes

• X-chromosome inactivation (pp. 126-129)
• A.D. Riggs
• Genomic imprinting (p.126, website 5.9)
• S.M. Tighlman
• Tissue-specific activation of transcription
• Allele-specific gene expression
• Y. Bergman

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Other sequence elements that regulate
transcription

• Increase gene expression by indirect mechanisms
  (i.e. not via RNA polymerase)
• Suppress or eliminate position-effects
• Matrix/Scaffold attachment regions (MAR)
• Allow local factor access to sequences
• Boundary/Insulator elements
• Prevents cooperation of elements on either side
  of it
• Locus control regions (LCR)
• Eliminate position-effects via an ill-defined
  mechanism involving overcoming heterochromatin

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MAR/SAR

• A-T rich DNA sequences which associate with the
  nuclear matrix
• Hypothesized to define domains of regulatory
  influence in chromatin
• MARs that affect gene expression are often next
  to defined enhancers (Igm heavy chain)
• Improve transgene expression
• Limiting influence of integration site
• Trafficking gene to regions of nuclear activity

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Boundary/Insulator elements

• Position-dependent silencers--need to be placed
  in between a promoter and enhancer
• Imposes a cis-obligation on enhancers
• Found in genomic locations that suggest a role in
  separating regulatory influences in the genome
• End of the b-globin LCR
• In between differentially expressed genes

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Boundary/Insulators (continued)

• Vertebrate insulators bind the zinc finger
  protein CTCF which appears to be responsible for
  their function (Bell, et. al.)
• Prototype insulators identified in Drosophila
• Scs and Scs in the heat shock locus
• Gypsy (binds Su(HW) zinc finger protein)
• Fab-7

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LOCUS CONTROL REGION Confers high-level,
position- independent, copy number-dependent,
tissue-specific expression on a linked transgene
in chromatin. Action Contains recognition
sequences for many ubiquitous and
tissue-restricted transcription factors.
Overcomes heterochromatin induced
position-effect-variegation by providing an open
chromatin domain for a linked transgene
1987 Human ?-globin locus Grosveld, et.
al. 1988 Human CD2 locus Lang, et.
al. 1992 Adenosine deaminase Aronow, et.
al. 1994 Macrophage lysozyme Bonifer, et.
al. 1994 T cell receptor ??? Diaz, et.
al. 1994 Immunoglobulin HC Madisen, et.
al. 1995 Human growth hormone Jones, et.
al. 1999 l5/VpreB gene locus Sabbatini, et. al.
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(No Transcript)
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LCRComparison and contrastFrom Li, et. al.
(1999) Trends Genet. 15403
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In other words.

• Shared characterisitics of different cis-acting
  transcriptional control elements
• Lineage-specificity LCR, enhancer
• Timing and activation LCR, enhancer
• Facilitating factor access MAR, LCR
• Insulation boundary, LCR
• RNA pol II activity promoter, enhancer
• Needs chromatin LCR, MAR, boundary

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The first LCR--the globin locusFrom Li, et. al.
(1999) Trends Genet. 15403
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(No Transcript)
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HS can be mapped using restriction enzymesFrom
Ortiz, et. al. (1997) EMBO J. 165037-45
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Little is known about the mechanism of LCR
activity

• Collection of HS, each of which has distinct
  functions. All are required for LCR activity
• Very few proteins (outside of enhancer binding
  proteins) have been identified which functionally
  interact with LCRs
• HBP-1 (an HMG-Box protein) in the CD2 LCR
• EKLF (a zinc finger protein) in the globin LCR

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LCRsMany unanswered questions

• Knockout studies have caused controversy over its
  non-redundant roles in the genome
• Molecular basis for its effect on chromatin
• Little sequence homology between LCRs
• Fraser and Grosveld (1998) Curr. Opin. Cell Biol.
  10361-5
• Kioussis and Festenstein (1997) Curr. Opin.
  Genet. Dev. 7614-619
• Festenstein and Kioussis (2000) Curr. Opin.
  Genet. Dev. 10199-203

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Maintaining gene expression states

• Activating and repressive multiprotein complexes
  first found in Drosophila
• Trithorax group proteins are an activating
  complex Similarities to SWI/SNF
• Polycomb group proteins are the repressive
  complex Mammalian homologs found
• Do not establish repression, only maintain it.
• Reversed by the action of the trithorax proteins

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Transcriptional regulationputting it
togetherProposed order of regulatory events

• Methylation status/de-methylation
• Histone acetylation/de-acetylation
• Boundary and matrix attachment regions
• Locus control regions
• Transcriptional enhancers
• Promoters-------gt mRNA production
• SWI/SNF or Polycomb proteins

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Other forms of gene regulationPages 130-137 of
Gilbert

• Differential mRNA processing
• Differential mRNA stability
• Selective mRNA translation
• Selective mRNA localization/nuclear export
• Post-translational modifications
• Proteolytic cleavage
• Phosphorylation and other small additions