Download DNA Replication/Transcription/RNA Splicing

Bio 405/505 Advanced Cell &
Developmental Biology II
The Cell Nucleus Lectures
Dr. Berezney
Lecture 3 : Background & Figures; Huang &
Spector, 1996; Wei et al., 1999; Wei et al.,
1998; Osborne et al., 2004;
Conventional View of Eukaryotic Transcription
and RNA Splicing as two Separate Processes
1. DNA  Transcription  pre-mRNA
2. pre-mRNA  RNA Splicing mRNA
Current View of Eukaryotic Transcription and RNA
Splicing as Being Functionally and Spatially
Linked as a Highly Coordinated Process
DNA Transcription/RNA Splicing  mRNA
HUANG S & SPECTOR D
Intron-dependent recruitment of premRNA splicing factors to sites of
transcription
Journal of Cell Biology (1996) 133, 719-732
Huang & Spector , JCB 1996, Figure 1
Constructs used in transient transfections
Major Conclusions of Huang & Spector, 1996
Conclusion 1
Association of nascent RNA transcripts
with splicing factors is intron dependent
during transient or stable expression
(Figs 2-5).
RNA
SC-35
merged
Huang & Spector, Fig 2
β-galactosidase
β-globin (cDNA)
CMVTAT (partial intron)
AD VA RNA
Intronless RNA’s or RNA’s with a partial intron are not spatially associated with
splicing factors in the cell nucleus
RNA
SC-35
merge
Huang & Spector, Fig 3
β-globin (genomic DNA)
CGTAT
β- tropomyosin
β-tropo/U2 snRNP
Transiently expressed RNA transcripts containing
introns are spatially associated with splicing factors
Huang & Spector , JCB 1996, Figure 4
Association of transcripts and splicing factors
Huang & Spector, Fig 5
RNA
SC-35
merged
β-globin
minus
β-globin
plus
Intron dependent association of RNA transcripts and splicing factors in
stably transfected cell lines
Major Conclusions of Huang & Spector , 1996
Conclusion 2
The foci of expressed RNA are also the sites
of transcription (Fig 6).
Huang & Spector, JCB 1996, Figure 6
RNA
BrUTP
merged
β-globin
minus
β-tropo
plus
Transiently expressed RNAs co-localize with sites of
transcription
Major Conclusions of Huang & Spector , 1996 contd…
Conclusion 3
The foci of intron-containing RNA are
sites of pre-mRNA splicing and colocalize with mature mRNA
(Fig 7 & 8).
Huang & Spector , JCB 1996, Figure 7
Diagram illustrating the design of hybridization probes for
the specific detection of β- tropomysin pre-mRNA and
mature RNA. Introns 6 & 7 were used as pre-mRNA specific
probes Splice junction constructs 12 Nt from 3’ and 5’ ends,
respectively, between exons 5 & 6 or 5 & 8 were used as
mRNA specific probes
Huang & Spector, Fig 8
β-tropo mRNA (a)
& pre-mRNA (b)
β-tropo pre-mRNA
(d) & SC-35 (e)
β-tropo mRNA (g) &
SC-35 (h) after 5 hr in
α-amanitin
Sites of transiently expressed intron-containing pre-mRNA & mRNA
co-localize with each other and with sites of pre-mRNA splicing
Major Conclusions of Huang & Spector, 1996
• Association of nascent RNA transcripts with splicing
factors is intron dependent during transient or stable
expression (Figs 2-5)
• The foci of expressed RNA are also the sites of
transcription (Fig 6)
• The foci of intron-containing RNA are sites of pre-mRNA
splicing and co-localize with mature mRNA (Fig 7 & 8)
• Taken together these findings support a model for
coordinate transcription and RNA splicing at discrete
assemblies arranged within the nuclear architecture.
A Model for Transcription-Coupled Pre-mRNA Splicing
Ma H, SAMARABANDU J, DEVDHAR RS, ACHARYA R,
CHENG P-C, MENG C & BEREZNEY R
Spatial and temporal dynamics of DNA
replication sites in mammalian cells.
Journal of Cell Biology (1998) 143, 1415-1425.
Review paper and powerpoint lecture 4 (Section II) from
Bio 402/502 website
3-D Model of a 1 mbp Multi-Loop Chromatin Domain
Functional Model of ~1 Mbp
Chromatin Domains
G1
Non-Replicating
Chromatin Domain
S
Replicating
Chromatin Domain
S or G2
Non-Replicating
Chromatin Domains
Replication
factory
DO THE HIGHER ORDER CHROMATIN
DOMAINS (1 Mbp) FUNCTION AS GENE
TRANSCRIPTION FACTORIES AND IF SO,
WHAT IS THEIR RELATIONSHIP TO RNA
SPLICING SITES (NUCLEAR SPECKLES)?
Wei et al.
Three-dimensional visualization of
transcription sites and their association with
splicing factor-rich nuclear speckles
Journal of Cell Biology (1999) 146, 543-558
Major Conclusions of Wei et al. , 1999
Conclusion 1
Transcription Sites (TS) labeled with BrdUTP
in permeabilized 3T3 mouse fibroblast cells
are characterized by inhibitor and enzyme
digestions studies as pol I- and II- RNAP
mediated TS (Figure 1).
Figure 1 (Wei et al., 1999): Properties of Transcription
Sites in 3T3 Mouse Fibroblast Cells
Major Conclusions of Wei et al. , 1999
Conclusion 2
Approx. 2,000 sites (94% pol II-mediated TS)
are detected following segmentation and are
arranged in clusters and 3-D network arrays
(Figures 2 & 3).
Figure 2 (Wei et al., 1999) Segmentation of Transcription Sites
Figure 3 (Wei et al., 1999) 3-D Visualization of TS and
Individual Sites
Major Conclusions of Wei et al. , 1999
Conclusion 3
The number of TS, their 3-D organization and
arrangement into higher order functional
zones are maintained after extraction for
nuclear matrix (Figures 4, 5 & 6).
Figure 4 (Wei et al., 1999) TS After Nuclear Matrix Extraction
and Synthesis on Nuclear Matrix
Figure 5 (Wei et al., 1999) 3-D Visualization of TS and
Individual Sites After Nuclear Matrix Extraction
Figure 6 (Wei et al., 1999) Replication and Transcription
Sites Higher Order Zones After Nuclear Matrix Extraction
Major Conclusions of Wei et al. , 1999
Conclusion 4
Significant levels (43%) of pol II TS are
associated with splicing factor-rich nuclear
speckles (Figure 8, Table II) and are found
along the periphery as well as inside
individual speckles that are optically
sectioned (Figure 9).
Figure 8 (Wei et al., 1999) Association of TS With Nuclear
Speckles
Table II (Wei et al., 1999) Quantitation of TS Associated with
Nuclear Speckles
Figure 9 (Wei et al., 1999) Evaluation of TS Inside
Speckles By Optical Sectioning
Major Conclusions of Wei et al. , 1999
Conclusion 5
A similar distribution of TS along nuclear
speckles was detected following in vivo
labeling of TS with BrdU (Figure 10).
Figure 10 (Wei et al., 1999) In vivo Labeling of TS
and Association With Nuclear Speckles
Major Conclusions of Wei et al. , 1999
Overall Conclusion
Taken together these findings support a
model in which coordinate transcription/RNA
splicing of pre-mRNA occurs in close
association with the nuclear speckles as
functional transcription/splicing factories
that are associated with the nuclear matrix.
Major Conclusions of Wei et al., 1999
• Transcription Sites (TS) labeled with BrdUTP in permeabilized 3T3
mouse fibroblast cells are characterized by inhibitor and enzyme
digestions studies as pol I- and II- RNAP mediated TS (Figure 1).
• Approx. 2,000 sites (94% pol II-mediated TS) are detected following
segmentation and are arranged in clusters and 3-D network arrays
(Figures 2 & 3).
• The number of TS, their 3-D organization and arrangement into higher
order functional zones are maintained after extraction for nuclear
matrix (Figures 4,5 & 6).
• Significant levels (43%) of pol II TS are associated with splicing factorrich nuclear speckles (Figure 8, Table II) and are found along the
periphery as well as inside individual speckles that are optically
sectioned (Figure 9).
• A similar distribution of TS along nuclear speckles was detected
following in vivo labeling of TS with BrdU (Figure 10).
• Taken together these findings support a model in which coordinate
transcription/RNA splicing of pre-mRNA occurs in close association
with the nuclear speckles as functional transcription/splicing factories.
Replication Timing Experiments in
Mammalian Cells Have Demonstrate that
Actively Transcribed Genes are
Preferentially Replicated in Early S-Phase
This raises the key question: When are the
Genes in These Early S Replicated
Chromatin Domains Transcribed?
WEI X, SAMARABANDU J, DEVDHAR RS, SIEGEL AJ,
ACHARYA R & BEREZNEY R
Segregation of transcription and
replication sites into higher order
domains.
Science (1998) 281, 1502-1505.
Simultaneous Replication and Transcription at
Chromatin Domains during S-Phase
RS
TS
RS/TS
Major Conclusions of Wei et al. , 1998
Conclusion 1
The ~ 1,000 RS & 2,000 TS at any moment in
early S are spatially separate chromatin
domains (Fig 1).
Wei et al. , Science 1998, Figure 1
Spatial seperation of replication and transcription sites throughout S phase
These Findings Are Consistent With Model 2
Major Conclusions of Wei et al. , 1998 contd..
Conclusion 2
Individual RS & TS are arranged into
separate nuclear zones of replication or
transcription (Fig 3).
Wei et al. , Science 1998, Figure 3
Cluster distribution of replication and transcription sites extending into
several optical sections
These Findings Support A Novel Model 3
Major Conclusions of Wei et al. , 1998 contd..
Conclusion 3
These segregated zones are arranged into
spatially separate 3-D networks that extend
throughout the nuclear interior (Fig 4).
Wei et al. , Science 1998, Figure 4
Replication and transcription sites in early S phase
separate into distinct higher order domains in 3D
Wei et al. , Science 1998, Figure 4 contd….
Replication and transcription sites in early S phase
separate into distinct higher order domains in 3D
Major Conclusions of Wei et al. , 1998 contd..
Overall Conclusions
•Replication timing and transcription of newly
replicated genes likely involves a corresponding
dynamic networking of replication & transcription
processes in which nuclear zones of replication
and transcription undergo coordinate rezoning.
•These networks of functional replication &
transcription zones are the organizational basis for
the regulation of replicational and transcriptional
activity.
Major Conclusions of Wei et al, 1998
• The ~ 1,000 RS & 2,000 TS at any moment in early S are
spatially separate chromatin domains (Fig 1).
• Individual RS & TS are arranged into separate nuclear
zones of replication or transcription (Fig 3).
• These segregated zones are arranged into spatially
separate 3-D networks that extend throughout the nuclear
interior (Fig 4).
• Replication timing and transcription of newly replicated
genes likely involves a corresponding dynamic networking
of replication & transcription processes in which nuclear
zones of replication and transcription undergo coordinate
rezoning.
• These networks of functional replication & transcription
zones are the organizational basis for the regulation of
replicational and transcriptional activity in the cell.
Transcription Factories
Gene Regulation By
Higher Order
Arrangement of
Chromatin Loops and
Multi-Loop Domains
Based On Transcription
Osborne et al., Nature Genetics 36, 1065 - 1071 (2004)
Active Genes Dynamically Colocalize to Shared
Sites of Ongoing Transcription
The intranuclear position of many genes has been correlated with their
activity state, suggesting that migration to functional subcompartments
may influence gene expression. Indeed, nascent RNA production and
RNA polymerase II seem to be localized into discrete foci or 'transcription
factories'. Current estimates from cultured cells indicate that multiple
genes could occupy the same factory, although this has not yet been
observed. Here we show that, during transcription in vivo, distal genes
colocalize [close proximity?] to the same transcription factory at high
frequencies. Active genes are dynamically organized into shared nuclear
subcompartments, and movement into or out of these factories results in
activation or abatement of transcription. Thus, rather than recruiting and
assembling transcription complexes, active genes [may] migrate to
preassembled transcription sites. More generally, these findings indicate
a dynamic interplay between multiple actively transcribed genes and the
transcriptional machinery to form highly regulated transcription factories.
Gene Probes From a ~40 Kbp Region
on Mouse Chromosome 7
•
•
•
•
•
•
Hbb-b1 (β-like globin, highly transcribed in erythroid cells)
Eraf (essential globin-stabilizing protein)
Uros (heme biosynthetic enzyme essential for globin)
Igf2 (insulin-like growth factor, unrelated to globin)
Kcnq1ot1 (long QT intronic transcript, unrelated to globin)
Hba (α-globin gene on chromosome 11)
Major Conclusions of Osborne et al. , 2004
Conclusion 1
Actively transcribed genes show significant
levels of co-localization (proximity)
(Fig 1 and 2)
Fig 1. Transcription frequencies of genes on mouse chr 7
(b) RNA FISH on adult anemic
spleen erythroid cells with
intron probes for Uros. Note cell
nuclei with zero, one or two
signals (green). DAPI staining is
blue. Scale bar, 5 µm. (c) The
percentage of alleles with a gene
transcription signal by RNA
FISH for Hbb-b1, Hba, Eraf,
Uros, Igf2 and Kcnq1ot1 in
erythroid cells. (d) Relative
primary transcript levels for
Hbb-b1, Eraf, Uros and Igf2,
measured by quantitative RTPCR of intron sequences.
Average values are shown with
Eraf set to 1.
Fig 2. Colocalization of transcribed genes with
Hbb-b1 (red) on mouse chr 7 using RNA FISH
Eraf
Uros
IgF2
Kcnq1ot1
Hba
% of gene signals that overlap with Hbb-b1 gene signals
Major Conclusions of Osborne et al. , 2004
Conclusion 2
Co-localization (proximity) of genes
correlates with an active transcriptional state
(Fig 3)
Fig 3. Colocalization of genes is transcriptiondependent- 3-D measurement of distances
between Hbb-b1(red) and Eraf (green)
Major Conclusions of Osborne et al. , 2004
Conclusion 3
Actively transcribed genes show significant
levels of co-localization (proximity) with RNA
pol II (Fig 4)
Fig 4. Actively transcribed genes associate
with RNAP II foci
BLACK (RNA FISH % alleles)-GRAY (% foci overlap with pol II)
Major Conclusions of Osborne et al. , 2004
Conclusion 4
Co-localization (proximity) of actively
transcribed genes and RNA pol II (Fig 5)
Fig 5. Actively transcribed genes colocalize to
shared transcription factories
DNA FISH: (Hbb (green), Eraf (red), pol II (blue)
Distances Hbb Eraf
RNA FISH: (Hbb (red), Eraf (green), pol II (blue)
Major Conclusions of Osborne et al. , 2004
Conclusion 5 (Overall)
Multiple actively transcribed genes are
dynamically associating with individual
transcription factories. It is conceivable that
that there is a dynamic and coordinate
assembly of chromatin loops (actively
transcribed genes) and transcription
factories.
(Fig 8)
Fig 8. Model of dynamic associations of genes with
transcription factories
Schematic representation of chromatin loops (black) extruding from a
chromosome territory (gray). Transcribed genes (white) in RNAP II factories
(black circles). Potentiated genes (free loops) that are not associated with RNAP
II factories are temporarily not transcribed. Potentiated genes can migrate to a
limited number of preassembled RNAP II factories to be transcribed (dotted
arrows). We propose that both cis and trans associations are possible.
Transcription
Factories
Gene Regulation
By Higher Order
Arrangement of
Chromatin Loops
and Loop Domains