Long-range gene regulation
Short Course in Medical Genetics
Melbourne, June 2011
Question
Why should long-range regulation of gene expression
be of interest to Clinical Scientists and Pathologists
interested in providing genetic diagnostic testing
services?
• Intrinsically interesting and important area…
• New technologies – Array/ NGS are going to reveal
“novel” genomic variants, some of which will be
disease-causing as a result of disruption to distant
regulatory elements rather than to transcriptional units.
• We need to be able to deliver on the expectation that
we can recognise a disease-causing genomic variant
when we see one
Regulation of gene expression
Labile regulation
• Moment-to-moment control by transcriptional
activators and repressors
• Control exerted by nuclear concentrations, covalent
modifications, subunit associations, etc.
Epigenetic regulation
• Has sufficient stability to be transmitted from
parental cell to daughter cells
• Involves alterations to the structure of chromatin,
including covalent modification of DNA and
histones
Labile gene expression
• Is controlled by regulatory elements
that can be located far away along the
chromosome
• In some instances, regulatory elements
may be on other chromosomes
Promoters for eukaryotic genes
encoding polypeptides
CG
box
CAAT
box
TATA
box
B-globin gene promoter region
Glucocorticoid receptor gene
Assembling the basal transcription
apparatus onto the promoter
Basal transcription
apparatus
comprises 27
polypeptides
Transcription factors may be either upstream or
downstream of the transcription start site
Distal regulatory elements
Enhancers
Silencers
Insulators
Locus control regions
Fractal globule
No knots!
Chromosome
A hierarchically coiled
single DNA molecule
Chromosomal
loops
Silencers
Enhancers
Chromatin loops
Characterising long-range gene
regulation
In a small but significant number of patients the
molecular lesions do not directly disrupt the
transcribed region of the gene
Instead, demonstrated interference with
transcriptional regulation of the gene
Clinical cases provided the first indications that
long-range effects regulated expression of
genes
Aniridia
Absence of the iris
Aniridia
Arises from heterozygous loss-of-function mutations within
the PAX6 gene on chromosome 11
Some cases – demonstrated absence of a PAX6 mutation
Cases with chromosomal translocations with breakpoints
up to 125 kb downstream of the last PAX6 exon
- Demonstrating the presence of distant control elements
PAX6 enhancers
Positioned within the intron of a neighbouring
unrelated gene (ELP4)
Some cases –demonstrated absence of a PAX6 mutation
Holoprosencephaly
Malformation of the forebrain arising from
failed or incomplete forebrain division
Pre-axial polydactyly
Acheiropodia
SHH Gene
Specifies the signaling protein Sonic
Hedgehog, which acts to establish the
body plan in embryogenesis
• SHH loss of function – holoprosencephaly
– preaxial
polydactyly
• SHH regulatory lesions
– acheiropodia
SHH Gene
SHH Gene
∂
∂
∂
∂
Genetic diseases triggered by aberrant gene
transcription can occur in several ways • Separation of cis-regulatory elements and promoters by
chromosomal translocations or inversions
• Deletion of long range cis-elements
• Deleterious mutations within cis-elements
• Disturbing the normal interactions of promoters and cisacting enhancers through appearance of a new
promoter
• Interfering with local chromatin structure by disturbing
antisense transcripts regulating more global chromatin
structures
ENCODE project
National Human Genome Research Institute (NHGRI)
Goal: To define all the functional DNA sequence
elements in the human genome
ENCODE pilot study
Studied 44 genomic regions encompassing 30Mb
(1% of the human genome)
• Most genomic regions were transcribed, at least
in some cells and at some developmental stage
• Multiple novel transcripts
– non-coding
– some previously unknown exons of known genes
• extra exons were often 50-100 kb upstream of TSS
(some 200 kb upstream).
• evidence of transcripts containing exons from more
than one gene
An example of ENCODE data
Array Genomic Scanning
(Molecular Karyotyping)
Use of arrays to detect constitutional aneuploidy in
lieu of cytogenetic analysis of chromosomes
- diagnostic assessment of infants and children
• developmental delay
• pervasive developmental disorders
• birth defects
- miscarried products of conception and stillborn
fetuses
- likely utility for classification and staging of cancer
- emerging use in prenatal diagnosis
Interpretive assessment of array data
Clinical interpretation is heavily weighted by • CNV population frequencies (databases;
literature)
• Gene content
“gene deserts”
Empirically determined reporting thresholds
based on genomic length around 100 - 200 kb for deletions
200 - 400 kb for duplications.
“Gene desert”
Sometimes contain highly conserved
sequences
– evolutionary protection from chromosomal
rearrangements
Should we be considering neighbouring upstream
and downstream regions?
There are instances where the process of
checking beyond array coordinates has yielded
clinically relevant information
Summary
Long-range regulation of gene
expression
- is interesting!
- has growing relevance for
clinical scientists and
pathologists offering genetic
diagnostic testing services
"a rough or fragmented
geometric shape that
can be split into parts,
each of which is a
reduced-size copy of the
whole”
Fractal globule
No knots!
ENCODE study tools
Multiple techniques used to identify all
functional sequence elements • Intensive transcriptome analysis
• Concerted efforts to identify both cis-acting
regulatory DNA elements and their respective
trans-acting regulatory proteins
• Efforts to define chromatin structure and some
of its epigenetic regulation
Clinical relevance of long-range
gene regulation
In a small but significant number of patients the
molecular lesions do not directly disrupt the
transcribed region of the gene
Instead, demonstrated interference with
transcriptional regulation of the gene
Clinical cases provided the first indications that
long-range effects regulated expression of
genes
Treasure the exceptional case!