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
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
DNMT knockout mice
Li, Bestor and Jaenisch (1992) Cell 69:915-926
DNMT is required for post-gastrulation development. These mice
do not progress past the 9th day of their 19 day gestation period
DNMT is a “maintenance” methylase
From Jaenisch (1997) Trends Genet. 13:323-9
There are, likely, many DNA methyltranferase activities that have
yet to be identified. Some are for maintenance and others de novo
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
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
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 393:311
Proposed mechanism: stable repression
of gene expression through development
From A. Razin (1998) EMBO J. 17:4905-4908
Transcription factors are “transient” while DNA methylation is “not”
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. 275:1952
– Schubeler, et. al. (2000) Mol. Cell. Biol. 20:9103
How is DNA methylation regulated?
From Ng and Bird (1999) Curr. Opin. Genet. Dev. 9:158-163
“A DNA de-methylase”
• Bhattacharya, et. al. (1999) Nature 397:579
• 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
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 methylase:To artificially methylate DNA
5-azacytidine:To artificially de-methylate DNA
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
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
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
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
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
LOCUS CONTROL REGION: Confers high-level, positionindependent, 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:
1988:
1992:
1994:
1994:
1994:
1995:
1999:
Human b-globin locus
Human CD2 locus
Adenosine deaminase
Macrophage lysozyme
T cell receptor 
Immunoglobulin HC
Human growth hormone
l5/VpreB gene locus
Grosveld, et. al.
Lang, et. al.
Aronow, et. al.
Bonifer, et. al.
Diaz, et. al.
Madisen, et. al.
Jones, et. al.
Sabbatini, et. al.
DEMONSTRATING LCR ACTIVITY
USING TRANSGENIC MICE
L CR
Gene expression
A. Copy num be r de pe nde nce
B. Tis s ue dis tr ibution
Chromatin Structure
I. DNas e Hype r s e ns itivity
LCR:Comparison and contrast
From Li, et. al. (1999) Trends Genet. 15:403
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**
The first LCR--the globin locus
From Li, et. al. (1999) Trends Genet. 15:403
The DNase I Hypersensitivity assay reveals sequences
available for interaction with soluble nuclear factors
Site 3
Site 2
Site 1
p ro b e
Tis s ue 1
Site 1
Site 2
Site 3
p ro b e
Tis s ue 2
Tis s ue 1
DNase I
-
+
Tis s ue 2
-
+
p ar ent
Site 3
Site 2
Site 1
HS can be mapped using restriction enzymes
From Ortiz, et. al. (1997) EMBO J. 16:5037-45
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
LCRs:Many 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. 10:361-5
• Kioussis and Festenstein (1997) Curr. Opin. Genet. Dev.
7:614-619
• Festenstein and Kioussis (2000) Curr. Opin. Genet. Dev.
10:199-203
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
Transcriptional regulation:putting it together
Proposed order of regulatory events
•
•
•
•
•
•
•
Methylation status/de-methylation
Histone acetylation/de-acetylation
Boundary and matrix attachment regions
Locus control regions
Transcriptional enhancers
Promoters-------> mRNA production
SWI/SNF or Polycomb proteins
Other forms of gene regulation
Pages 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
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