Transgenic Analysis of Gene regulation
Long-range enhancer identification
Pax6 – expressed in a variety of tissues – tissue specific enhancers for retina and
diencephalon
- Standard transgenic analysis of the promoter region and the more upstream
introns has uncovered three regions (EE, ENN and NRE) with enhancer activity
in the lens and pancreas (EE), the forebrain, hindbrain and spinal cord (ENN) and
peripheral retina(NRE)
- Advantage has been taken of the availability of the complete genome sequences
for humans, mice and Fugu fish to identify conserved non-coding regions
downstream of Pax6.
1) Comparative genomic analysis – syntenic regions of mouse, human and fish genomes
compared to identify potential regulatory elements
2) Mapping regulatory mutations (eg. GWAS identified obesity SNP maps 188kb
downstream of MC4R)
3) Functional or expression analysis of BAC or YAC transgenes able to carry 300-
1000kb of genomic sequence (Kleinjanet et al., 2006)
- The functionality of potentially conserved region can be assessed using BAC or
YAC transgenics.
- Some familial cases of ANIRIDIA are associated with chromosomal inversions
that have breakpoints up to 150 kb downstream of PAX6
- The Sey phenotype can be rescued with human genomic DNA cloned in a YAC
vectors that extends 200kb downstream of of PAX6 but not by DNA that only
extends 13) kb downstream.
These results suggest that conserved region 150- 200 kb downstream of the PAX6
promoter may play a role in regulating PAX6 expression even though they are
located closer to the promoter of another gene (ELP4)
HS234 is a 4.5kb region >150 kb downstream of the Pax6 P0 promoter which drives
transgene expression in early eye development
section of the trace produced by a computer programme that searches for regions
of high sequence similarity when two
genomes are aligned
C117 Box123 is a 2.9 kb region ~77kb
downstream of the Pax6 P0 promoter
which drives transgene expression in the olfactory system, central retina and
forebrain
- C117 Box123 is a 2.9kb region of high sequene identity 77kb downstream from
P0
, - Box123 drives transgene expression in central retina after E10 and expression
in the olfactory system and diencephalon
Interestingly, transgenes made using the Box123 region often exhibit ectopic
expression in the midbrain
- suggests that enhancers that drive midbrain expression are present in this
region, but that Pax6 expression in the midbrain is normally inhibited by
suppressor elements, which lie elsewhere in the genome.
Expression silencing mechanisms
a) Transcription factor binding cis-regulatory elements (eg. NRSE)
b) Trans-acting non-coding RNA (microRNA)
- Mature miRNAs are short, single stranded RNA molecules approximately 22
nucleotides in length – miRNAs sometimes encoded by multiple loci, some of
which are organised in tandemly co-transcribed clusters
- miRNA genes transcribed as RNA polymerase II as large primary transcripts
(pri-miRNA – 5’cap, a 3’ polyA tail with secondary hairpin like structures)
processed by a protein complex containing RNase III (Drosha), to form an
approximately 70 nucleotide precursor miRNA
- Precursor transported to the cytoplasm where it is processed by second RNase
II enzyme (Dicer) to form mature miRNA approximately 22 nucletodies
- Mature miRNA incorporated into a ribonuclear particle to form the RNA-inuced
silencing complex (RISC) which mediates gene silencing
- Usually induce gene silencing by binding to target sites found within 3’UTR of
the targeted mRNA – interaction prevents protein production by suppressing
protein synthesis and/or initiating mRNA degradation – can target as many as
100 different mRNAs
- Unlike siRNAs, many miRNAs do not exactly match their targets – may only need
an essential “seed region”
- C. elegans genome contains 100-300 miRNA encoding genes
- Human genome contains ~1000 miRNA genes many expressed only in specific cell
types or stages – computational analysis suggests miRNAs repress more than 1/3
of human genes
Testing the inhibitory activity is mediated miRNA in tissues – block miRNA
processing by using a conditional knockout allele of Dicer – inactivate Dicer in
specific tissues of mice using the cre-lox system
miRNA microarray analysis can be done to identify candidate miRNAs – miRNA
extracted from cells of test tissue – miRNA reverse-transcribed with biotin-
labelled nucleotides to obtain labelled target cDNA
Long-range enhancer identification
Pax6 – expressed in a variety of tissues – tissue specific enhancers for retina and
diencephalon
- Standard transgenic analysis of the promoter region and the more upstream
introns has uncovered three regions (EE, ENN and NRE) with enhancer activity
in the lens and pancreas (EE), the forebrain, hindbrain and spinal cord (ENN) and
peripheral retina(NRE)
- Advantage has been taken of the availability of the complete genome sequences
for humans, mice and Fugu fish to identify conserved non-coding regions
downstream of Pax6.
1) Comparative genomic analysis – syntenic regions of mouse, human and fish genomes
compared to identify potential regulatory elements
2) Mapping regulatory mutations (eg. GWAS identified obesity SNP maps 188kb
downstream of MC4R)
3) Functional or expression analysis of BAC or YAC transgenes able to carry 300-
1000kb of genomic sequence (Kleinjanet et al., 2006)
- The functionality of potentially conserved region can be assessed using BAC or
YAC transgenics.
- Some familial cases of ANIRIDIA are associated with chromosomal inversions
that have breakpoints up to 150 kb downstream of PAX6
- The Sey phenotype can be rescued with human genomic DNA cloned in a YAC
vectors that extends 200kb downstream of of PAX6 but not by DNA that only
extends 13) kb downstream.
These results suggest that conserved region 150- 200 kb downstream of the PAX6
promoter may play a role in regulating PAX6 expression even though they are
located closer to the promoter of another gene (ELP4)
HS234 is a 4.5kb region >150 kb downstream of the Pax6 P0 promoter which drives
transgene expression in early eye development
section of the trace produced by a computer programme that searches for regions
of high sequence similarity when two
genomes are aligned
C117 Box123 is a 2.9 kb region ~77kb
downstream of the Pax6 P0 promoter
which drives transgene expression in the olfactory system, central retina and
forebrain
- C117 Box123 is a 2.9kb region of high sequene identity 77kb downstream from
P0
, - Box123 drives transgene expression in central retina after E10 and expression
in the olfactory system and diencephalon
Interestingly, transgenes made using the Box123 region often exhibit ectopic
expression in the midbrain
- suggests that enhancers that drive midbrain expression are present in this
region, but that Pax6 expression in the midbrain is normally inhibited by
suppressor elements, which lie elsewhere in the genome.
Expression silencing mechanisms
a) Transcription factor binding cis-regulatory elements (eg. NRSE)
b) Trans-acting non-coding RNA (microRNA)
- Mature miRNAs are short, single stranded RNA molecules approximately 22
nucleotides in length – miRNAs sometimes encoded by multiple loci, some of
which are organised in tandemly co-transcribed clusters
- miRNA genes transcribed as RNA polymerase II as large primary transcripts
(pri-miRNA – 5’cap, a 3’ polyA tail with secondary hairpin like structures)
processed by a protein complex containing RNase III (Drosha), to form an
approximately 70 nucleotide precursor miRNA
- Precursor transported to the cytoplasm where it is processed by second RNase
II enzyme (Dicer) to form mature miRNA approximately 22 nucletodies
- Mature miRNA incorporated into a ribonuclear particle to form the RNA-inuced
silencing complex (RISC) which mediates gene silencing
- Usually induce gene silencing by binding to target sites found within 3’UTR of
the targeted mRNA – interaction prevents protein production by suppressing
protein synthesis and/or initiating mRNA degradation – can target as many as
100 different mRNAs
- Unlike siRNAs, many miRNAs do not exactly match their targets – may only need
an essential “seed region”
- C. elegans genome contains 100-300 miRNA encoding genes
- Human genome contains ~1000 miRNA genes many expressed only in specific cell
types or stages – computational analysis suggests miRNAs repress more than 1/3
of human genes
Testing the inhibitory activity is mediated miRNA in tissues – block miRNA
processing by using a conditional knockout allele of Dicer – inactivate Dicer in
specific tissues of mice using the cre-lox system
miRNA microarray analysis can be done to identify candidate miRNAs – miRNA
extracted from cells of test tissue – miRNA reverse-transcribed with biotin-
labelled nucleotides to obtain labelled target cDNA
