Human gene expression and
genomic imprinting
Lecture 3
PROMOTERS – are combinations of short sequence elements (usually
located in the immediate upstream region of the gene- often within 200 bp
of the transcription start site) which serve to initiate transcription.
Position of cis-acting elements within promoter sequences
• TATA box, usually found at a position about 25 bp upstream (-
25) from the transcriptional start; it is typically found in genes
which are actively transcribed by RNA pol II
• GC box found in a variety of housekeeping genes, it appears to
function in either orientation
• CAAT box often located at position -80; it is usually the
strongest determinant of promoter efficiency
Basal transcription apparatus
• Various general transcription factors (TF) are
required to bind to a promoter sequences in the
immediate vicinity of a gene, in order to subsequently
position and guide the RNA polymerase that will
transcribe the gene
Basic terms
Sense strand – the DNA strand of a gene that is
complementary in sequence to the template
(antisense) strand, and identical to the transcribed
RNA sequence except (that DNA contains T where
RNA has U).
Template strand – in transcription , the DNA strand
that base pairs with nascent RNA transcript
Quoted gene sequences are always of the sense strand,
in the 5´ 3´direction
Cis-acting gene sequences -specific recognition elements
recognized by tissue-specific TFs
• ENHANCERS – positive transcriptional control elements which
are particularly prevalent in mammals; they serve to increase
the basal level of transcription which is initiated through the
core promoter elements
• They function is independent of both their orientation and the
distance (in some extent)
• SILENCERS – serve to reduce transcription levels;
• RESPONSE ELEMENTS – modulate transcription in response to
specific external stimuli; they are usually located upstream of
the promoter element (often within 1 kb of the transcription
start site)
• A variety of such elements respond to the specific hormones
(e.g. retinoic acid or steroid hormones such glucocorticoids)
Chromatin conformation: DNA
methylation and the histone code
•
Nucleosome – structural unit
of chromatin; it consists of a
central core of eight histone
proteins (2x H2A,H2B, H3 a
H4) around which a strech of
146 bp of dsDNA is coiled;
adjacent nucleotides are
connected by a short length of
spacer DNA
•The strings of beads,
approx. 10 nm in diameter,
are in turn coiled into a
chromatin fiber ; the
interphase chromosomes
seems to consists of these
chromatin fibers
Histone code
• A common shorthand is used for histone
modifications. Specific aminoacid residues
are identified by the type of histone, the
one-letter amino-acid code and the
position of the residue counting from N
terminus. Thus, H3K9 lysine is lysine 9 in
histone H3
Histone modification
• Histone acetylation
• Histone acetyltransferases (HATs) (K=lysine); reduced affinity
between histones and DNA ; transcriptional co- activators
• Histone methyltranseferases (HMTs)
• Histone deacetylases (HDACs); promoting transcriptional
repression ; are recruited as part of corepressor complex in
response to the DNA methylation
Enzymes responsible for DNA
methylation
• DNA methyltransferases DNMT
DNMT1
• Methzlation is sex-specific regulated - pohlavne špecifická regulácia Dnmt1
metyltransferázy
– 1so
somatic
– 1sp
sperm
– 1oo
egg
DNA methylation is accomplished by DNA
methyltrasnferases at CpG islands
• Genomic regions with high frequency of CpG dinucleotides; CpG
islands are typically 300 – 3 000 bp in length
The usual formal definition of a CpG island is a region with at least 500
bp and with a GC percentage that is greater than 55%
Methyl-CpG binding proteins with methylCpG-binding domain (MBD)
•MEPC2 on X chromosome – loss of function mutations in MEPC2 is
responsible for dominantly inherited Rett syndrome
Normal delivery, heterozygous girls develop
normally for their first year but the regress
Other main criteria include loss of purposeful
hand skills, loss of spoken language, gait
abnormalities, and stereotypic hand
movements.
80-90% - dominant de novo germline loss of function mutations (from fathers
germline) in MECP2 na Xq28
MECP2 is a transcription factor – methyl-CpG binding protein
• Epigenetic mechanisms of gene control
describes heritable states which do not
depend on DNA sequence
• (Genetic mechanisms explain heritable states
(characters) which result from changes in DNA
sequences (mutations))
• DNA methylation
Gene repression
• (Host defense against transposons or foreign DNA)
Epigenetic mechanism and long range control
of gene expression
• Epigenetic changes- are heritable, but that do not depend on
change in genome sequence
• DNA methylation – general method of maintaining repression
of transcription
•
- involved in mechanism operating on some
genes to ensure that only one of the two parentally expressed
alleles is normally expressed (monoallelic expression), even
although the nucleotide sequence of the nonexpressed allele
may be identical to that of the expressed
• X-chromosome inactivation
• Genomic impriting
Genomic imprinting
• „Imprint“ - nonequivalence in expression of alleles at
certain gene loci dependent on the parent of origin
• Only one parental allele is expressed
• Epigenetic process (cause – parentally specific DNA
methylation
•
Genomic imprinting- nonequivalence in expression
of alleles at certain gene loci dependent on the
parent of origin
• Uniparental disomy is pathogenic
Some conceptuses have normal 46,XX or 46, XY karyotype
but may have inherited two copies of the same
chromosome from just one of the parents . This may
result in abnormal phenotypes which are different
according to parental origin of the relevant chromosome.
Angelman syndrome
(OMIM 105830)
• mental retardation,
• lack of speech,
• growth retardation,
• hyperactivity,
• spasticity
• inappropriate laughter
Prader-Willi syndrome
•
•
•
•
Mental retardation
hypotonia,
gross obesity,
excessive and
indiscriminate eating
habits,
• male hypogenitalism
• small hands and feets
In approximately 70 percent of cases there is
cytogenetic deletion involving the proximal long arm of
chromosome 15q11-13 the same picture for AS and
PWS
•The same deletions – different syndromes
Deletion of certain chromosomal regions produces a different
phenotype when on the maternal or paternal chromosome.
Subchromosomal mutations causing differential
abnormal phenotypes according to parent of
origin
• Deletion of certain chromosomal regions produces a different
phenotype when on the maternal or paternal chromosome.
• The same deletions – different syndromes
Prader-Willi syndrome – deletion 15q12 on the paternal chromosome
Angelmano syndrome - deletion 15q12 on the maternal chromosome
Subchromosomal mutations causing differential
abnormal phenotypes according to parent of
origin
• Deletion of certain chromosomal regions produces a different
phenotype when on the maternal or paternal chromosome.
• The same deletions – different syndromes
Prader-Willi syndrome – deletion 15q12 on the paternal chromosome
Angelmano syndrome - deletion 15q12 on the maternal chromosome
5q12 IS AN IMPRINTED REGION
Chromosomal mutations:
De novo deletion 4-4,5Mb; deletion on paternal chr. 15 causes PWS, on
maternal AS
Uniparental disomy – both homologues of a particular pair (chr. 15)
are inherited from one parent.;
Maternal UPD - lacking the paternal 15, the person develop PWS
PAternal UPD – lacking the maternal 15, AS
Pathogenic gene expression
Genetic changes in the regulatory mechanism of the control elements of gene
expression – examples
• 1. mutations within the promoter region
• 2. mutation within enhancers, silencers and response elements
• 3.non-physiological gene expression – control of inappropriate enhancer, silencer
or responseelements e.g. gene translocation
• 4. mutations in conserved splicing sequences
DETECTION of PATHOLOGICAL GENE
EXPRESSION – diagnostic, prognosic and
predictive consequences
Aberrant expression of repressor is sometimes associated with
neoplasia.
The t(15;17) chromosomal translocation that occurs in
promyelocytic leukemia fuses the PML gene to a portion of the
retinoid acid receptor (RAR).
This event causes unregulated transcriptional repression in a
manner that precludes normal cellular differentiation. The
addition of the RAR ligand, retinoic acid, activates the receptor,
allows cells to differentiate and ultimately undergo apoptosis.
This mechanism has therapeutic importance as the addition of
retinoic acid to treatment regimens induces a higher remission
ate in patients with PML.
Proto-oncogenes activation
in lymphoma and leukemia
•An intact gene is translocated
near to a regulatory domain
of an other gene, which is for
this particularly cell type
specific (e.g. IgH) and which is
highly expressed within the
cell .
•Chimerical gene fusion which leads
to a production of fusion transcripts
and proteins, such fusion protein can
get a new pathological function.
• T(8;14) Burkitt lymphoma .
• MYC comes under controll of Ig
genes locus control region
(similar like enhancer) _high
expression of transcirption
factor MYC – activation of
genes
Quantification of c-myc
expression
Activation by translocation that creates a
novel chimeric gene
• This mechanism is rare in carcinomas, but common in hematologic
tumors and sarcoma.
• The best known example is the Philadelphia (Ph) chromosome, a
small acrocentric chromosome seen in 90% of patients with chronic
myeloid leukemia.
• This chromosome is one product of a balanced reciprocal í;22
translocation. The breakpoint on chromosome 9 is within an intron of
the ABL oncogene. The translocation joins the 3´part of the ABL
genomic sequence onto 5´part of the BCR (breakpoint cluster
region( gene on chromosome 22, creating a novel fusion gene.
• This chimeric gene is expressed to produce a tyrosine kinase
related to ABL product but with abnormal transforming properties.
Methods of mRNA analysis
•
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Nothern Blot
Reverse Transcription PCR
Real Time PCR
Microarrays