Transcript Imprinting and Dosage Compensation-2015

Imprinting
Expression of only one allele of a locus
Only ~100 genes in mammals are imprinted
Parthenogenesis is not possible in mammals due to incorrect expression of imprinted genes
Most imprinted genes are involved in growth control or postnatal behavior
Imprinted genes involves allele specific methylation and are
resistant to genome-wide demethylation in germ cell development
Some imprinted gene clusters are regulated by methylation-regulated insulators
Some clusters of imprinted genes contain long ncRNAs that control allele-specific expression
Kinship Theory of Imprinting
Conflict exists between the interests of the paternal and maternal genes
For optimal fitness of the father, paternal genes maximize
acquisition of maternal resources to ensure larger sized offspring
Maternal genes are sparing in the demands of maternal resources,
so that the mother has a better chance to bear further offspring
Paternally-expressed genes generally stimulate growth
Maternally-expressed genes generally repress growth
Imprinted Expression of the H19 and Igf2 Genes
ICR is methylated in the male germ line
ICR is protected from methylation
in the female germ line by CTCF
CTCF binds to the unmethylated ICR in
females and forms an insulator that prevents
the activation of Igf2 by a downstream enhancer
from Bartolomei, Genes Dev. 23, 2124 (2009)
In males, the downstream enhancer
activates Igf2 and H19 expression
is repressed by DNA methylation
A long ncRNA Controls Imprinting at the Igf2r Locus
ICR in the Airn promoter
is methylated in females
Airn is expressed in males and silences
Igf2r, Slc22a2 and Slc22a3 in females
Airn is a long ncRNA that might associate
with proteins that modify histones
from Bartolomei, Genes Dev. 23, 2124 (2009)
Imprinting of the PWS-AS Locus
from Ferguson-Smith and Surani, Science 293, 1086 (2001)
The AS-ICR is required for methylation and inactivation
of the PWS-ICR in females to repress nearby genes
The AS-ICR is nonfunctional in males allowing
the PWS-ICR to activate nearby genes
The PWS-ICR promotes expression of an antisense Ube3a transcript in males
Dosage Compensation Mechanisms
Genomes compensate for different
numbers of sex chromosomes by
adjusting gene expression levels
from Straub and Becker, Nature Rev.Genet. 8, 47 (2007)
The Basics of X-inactivation in Mice and Humans
from Lee, Genes Dev. 23, 1831 (2009)
Stepwise Progression of X Inactivation in Differentiating ES Cells
One X chromosome is converted
to facultative heterochromatin
Xist transcription off the inactive X
initiates chromatin modification events
X inactivation is maintained epigenetically
from Brockdorff, Trends Genet. 18, 352 (2002)
The Basic Events of X Chromosome Inactivation
The Xic is the minimum
region to trigger X inactivation
Two Xic are necessary for XCI to occur
Xist RNA is transcribed from Xi
Xist coats Xi in cis
PRC2 is recruited to Xi
and methylates H3K27
from Lee, Science 338, 1435 (2012)
Tsix is expressed from the
opposite strand from Xist and
acts as an Xist repressor
Regulation of Xist Expression
RepA recruits PRC2 to Xist ?
Tsix is expressed from one allele
and recruits Dnmt3a to silence Xist
Tsix blocks recruitment
of PRC2 to Xist by RepA
Xist-PRC2 spreads in cis
from Lee, Science 338, 1435 (2012)
Identification of Proteins that Bind Xist in vivo
UV-crosslink SILAC-labelled ES cells after induction of Xist
Purify Xist using a biotinylated antisense probe in
denaturing conditions to disrupt non-covalent interactions
Separately purify proteins that bind to a control RNA
Identify proteins that specifically bind Xist by mass
spectrometry and calculation of a SILAC ratio for
each protein based on the intensity of all heavy and
light peptides originating from Xist of the control RNA
from McHugh et al., Nature 521, 232 (2015)
Initiation of X-Inactivation
from McHugh et al., Nature 521, 232 (2015)
SAF-A binds Xist and tethers Xist to Xi
SHARP binds to Xist and recruits SMRT to Xi
Xist-SHARP-SMRT recruits or activates HDAC3
Histone deacetylation compacts chromatin and silences transcription
PRC2 is recruited to Xi to maintain silencing
Xist Spreads to Coat the Inactive X Chromosome
The early binding sites for Xist are
spatially close to the Xist transcription site
Xist spreads by exploiting threedimensional chromosome topology
The few genes that escape silencing
loop out of the condensed core
from Dimond and Fraser, Science 341, 720 (2014)
X Chromosome Inactivation in Female Mouse Embryos
Xp is initially inactivated after
fertilization due to a maternal imprint
A maternal pool of Rnf12
initiates imprinted Xp inactivation
Xp is reprogrammed at the blastocyst stage
Random X chromosome inactivation
takes place in the ICM due
to reactivation of Rnf12 from Xp
Monoallelic expression of Xist is maintained
from Augui et al., Nature Rev.Genet. 12, 429 (2011)
Xi is reprogrammed in the female germ line
Xi reprogramming correlates with
expression of pleuripotency factors
Calico Cats
One of the genes controlling fur
color is on the X chromosome
B – orange
b - black
Female mammals are genetic mosaics
Random X inactivation early in embryonic development
leads to patchworks of skin cells expressing each allele
The Dosage Compensation Complex in Drosophila
SXL in females prevents MSL2 translation
MSL2 in males stabilizes
roX, MSL1, and MSL3
DMSL complex binds to high
affinity sites on X chromosome
from Gilfillan et al., FEBS Lett. 567, 8 (2004)
DCC spreads to nearby
sites on active chromatin
MOF mediates H4K16 acetylation
DCC is Localized to the X Chromosome
DCC localization is determined
by staining of polytene
chromosomes with anti-MSL1
DCC associates almost exclusively
with transcribed regions
from Straub and Becker, Nature Rev.Genet. 8, 47 (2007)
DNA Replicates by a Semiconservative Mechanism
Grow cells in 15N and transfer to 14N
Analyze DNA by equilibrium
density gradient centrifugation
Presence of H-L DNA is indicative
of semiconservative DNA replication
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-29
The 11th Commandment
The Replicon Model
Sequence elements determine where
initiation initiates by interacting with
trans-acting regulatory factors
from Aladjem, Nature Rev.Genet. 5, 588 (2007)
Mechanics of DNA Replication in E. coli
Leading strand is synthesized
continuously and lagging strand
is synthesized as Okazaki fragments
The 5’ to 3’ exonuclease activity
of Pol I removes the RNA primer
and fills in the gap
DNA ligase joins adjacent completed fragments
from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-9
Initiation of DNA Replication in E. coli
DnaA binds to high affinity sites in oriB
DnaA facilitates the melting
of DNA-unwinding element
DnaC loads DnaB helicase
to single stranded regions
from Mott and Berger, Nature Rev.Microbiol. 5, 343 (2007)
DnaB helicase unwinds the
DNA away from the origin
DnaB is an ATP-dependent Helicase
DnaB unwinds DNA in
the 5’-3’ direction
DnaB uses ATP hydrolysis
to separate the strands
SSB proteins prevent the separated
strands from reannealing
from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-8
RNA Primer Synthesis Does Not Require a 3’-OH
Primase is recruited to
ssDNA by a DnaB hexamer
from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-12
Coordination of Leading and Lagging Strand Synthesis
Two molecules of Pol III are
bound at each growing fork
and are held together by t
The size of the DNA loop increases
as lagging strand is synthesized
Lagging strand polymerase is
displaced when Okazaki fragment
is completed and rebinds to synthesize
the next Okazaki fragment
from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-11
Interruption of Leading Strand Synthesis by RNA Polymerase
Most transcription units in bacteria
are encoded by the leading strand
Natural selection for co-directional
collisions in the cell
from Pomerantz and O’Donnell, Nature 456, 762 (2008)
Replisome Bypass of a Co-directional RNA Polymerase
from Pomerantz and O’Donnell, Nature 456, 762 (2008)
Replisome Bypass of a Co-directional RNA Polymerase
Replication fork recruits the 3’terminus of the mRNA to continue
leading-strand synthesis
The leading strand is synthesized
in a discontinuous fashion
from Pomerantz and O’Donnell, Nature 456, 762 (2008)
Bidirectional Replication of SV40 DNA from a Single Origin
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-32
Mechanism of DNA Unwinding by Large T Antigen
A double hexamer of large
T antigen binds to SV40 origin
Two single hexamers unwind
dsDNA and translocates along
ssDNA in a 3’-to-5’ direction
The helicase can overcome a
covalent block to unwinding
from Trakselis and Graham, Nature 492, 195 (2012)
Replication of SV40 DNA
T antigen binds to origin and melts
duplex and RPA binds to ss DNA
Primase synthesizes RNA primer
and Pol a extends the primer
PCNA-Rfc-Pol d extend the primer
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-31