Download Nuclear Organization and Genomic Function

Bio 405/505 Advanced Cell &
Developmental Biology II
The Cell Nucleus Lectures
Dr. Berezney
Lecture 1: Introduction to Nuclear
Organization and Genomic Function;
Nuclear Lamin Proteins; Figures from
Gerace et al., 1984 Heald & McKeon,
1990; Burke and Gerace, 1986
“We’re pretty good at thinking
about how individual genes are
turned on and off. We’re not as
good at thinking about how the
whole genome is coordinated.”
Quote of Jeanne Lawrence in “The Cell Nucleus
Shapes up“ Science 1993, Vol 259, pp 1257-1259
Correlating Functional Genomics With the
Cell Nucleus: The New Frontier
Major Breakthroughs in Cell
Nucleus Research
• Hierarchy of Genomic Organization from the
Nucleosome to the Chromosome Territory
• Functional Organization in the Cell Nucleus
• Role of Nuclear Matrix Architecture [Proteinrich Factories] in Genomic Organization
and Function
Genomic Organization And
Function in the Cell Nucleus
Interphase Nucleus
Chromosome territories
Mitotic Chromosomes
Bowl of Spaghetti Model for Organization of
Chromatin in the Interphase Cell Nucleus
Chromosome Territory Model for
Organization of Chromatin in the
Interphase Cell Nucleus
Chromosome 1 (red),
Chromosome 9 (green)
Visualizing Genomic Function in
the Cell Nucleus
Cells grown on cover-slips
Label functional sites with fluorescent
probes
Examine by fluorescence microscopy
Computer image analysis
How are multiple genomic processes organized and
coordinated in space and time in the cell nucleus?
Chromosome Territories
Splicing Factors
Replication Sites
Nuclear
Transcript Tracks
Transcription Sites
MAINTAINING IN SITU FUNCTIONAL DOMAINS
ON THE NUCLEAR MATRIX
Domains)
Domains)
3-D Model of a 1 mbp Multi-Loop Chromatin Domain
Many Nuclear Structures exhibit Constrained Motion
The Cell Nucleus as a
Hierarchical Epigenetic System
Epigenetics is the study of reversible
heritable changes in gene function that
occur without a change in the sequence
of nuclear DNA. It is also the study of
the processes involved in the unfolding
development of an organism.
Hierarchical Epigenetics of
the Cell Nucleus
Alterations of nuclear organization at all levels
affect gene regulation which in turn affects cell
function and phenotypic expression
• Molecular level (DNA methylation and histone
acetylation)
• Chromatin domains (unfolding of chromatin
loops)
• Chromosome Territories (changes in
shape/gene positions)
• Global Organization of CT (3-D interactions)
Transcription Factories: Gene Regulation By
Higher Order Arrangement of
Chromatin Loops and
Loop Domains
FUTURE DIRECTION
Defining protein factors that
mediate the dynamic
assembly, organization,
functional properties and
regulation of chromatin
loop domains
Towards A Systems Biology of the Cell
Nucleus: Image Informatics, the Missing Link
Knowledge Base
for Normal &
Disease States
Assembly and Disassembly of Nuclear Envelope
• Nuclear envelope (NE) is a cell cycle
dependent structure that disperses at the
onset of mitosis (late prophase) and
reassembles around the reforming nucleus
in the late telophase.
• Inhibition of protein synthesis by
cycloheximide in late G2 phase has no
apparent affect on nuclear assembly in
telophase indicating that no new protein
synthesis is required for reassembly of the
nuclear envelope.
• This reassembly involves ~ 10,000 nuclear
pores in a matter of minutes.
• The correlations of breakdown of the
nuclear envelope, chromosome formation
mitosis & NE reassembly after mitosis are
essential for cell division and the ability of
cells to divide in an orderly manner.
Assembly and Disassembly of Nuclear Envelope
contd…
• The proteins that compose the
nuclear lamina (lamins A, B,C)
are involved in the
disassembly/reassembly of the
nuclear envelope during cell
cycle via phosphorylation
(P)/dephosphorylation (deP).
• Yeast genetic studies have
identified cdc2 as an essential
gene for cell division in yeast.
This is a cyclin dependant
protein kinase called cyclin Bcdc2 (cdk1) kinase (cyclins are
regulatory proteins that mediate
the enzymatic activity of protein
kinases) that plays a major role
in the regulation of cell cycle.
• Lamin phosphorylation/
dephosphorylation during cell
cycle by cdc2 (cdk1) kinase.
Gerace et al., 1984, Figure 7
Phosphorylation (P)/De(P) of the nuclear lamins correlates with
nuclear envelope assembly/disassembly (Gerace et al. paper)
2-D Gel Shift – Phosphorylation of
the nuclear lamin proteins in late
prophase correlates with the
disassembly of the nuclear envelope
and dephosphorylation of the lamins
correlates with the nuclear envelope
reassembly. This is indicated by the
increased phosphorylation during
prophase and the dephosphorylation
during telophase of the nuclear lamins
in a 2-D gel shift experiment (AP =
alkaline phosphatase, acidic is left;
basic is right).
Gerace et al. , 1984
Figure 6
Experimental basis for a role of nuclear lamin phosphorylation in nuclear envelope disassembly (Heald & McKeon)
DNA transfection experiments – in which human lamin A gene mutated at
two sites ( S-22 and S-392 which are the phosphorylation sites for cdc2
kinase) to alanine or isoleucine (cannot be phosphorylated) are then
transfected into mammalian cells. Results show that mitosis proceeds up to a
point with no breakdown of nuclear envelope. Therefore phosphorylation
of S-22 and S-392 by cdc2 kinase is essential for nuclear envelope
breakdown.
Normal lamin A gene
Anti-lamin A
DNA (DAPI)
Mutant lamin A gene
Anti-lamin A
DNA (DAPI)
Wild-type phenotype of CHO transfected cells with human lamin A
(Heald & McKeon paper, Figure 2)
I
P
M
A
T
Phenotype of cells transfected with double Ser-22/Ser-392 point
mutations (Heald & McKeon paper, Figure 4)
I
P
M
A
T
Table 1: Distribution of mitotic phenotypes
Heald & McKeon Paper Conclusions
• Mutations in S-22 and S-392 that prevent
phosphorylation at these sites block the
disassembly of the nuclear lamina during
mitosis.
• A model is proposed for the regulation of lamin
assembly in which phosphorylation just outside
the ends of the α-helical domain of the lamin
proteins (i.e., S-22 & S-392) leads to the
disassembly of the nuclear lamin at the levels
of the lamin coiled-coil dimers and higher.
Structure of Intermediate Filaments
Experimental basis for a role of nuclear
lamin dephosphorylation in nuclear
envelope assembly (Burke & Gerace paper)
Assembly of nuclear envelope in mitotic extracts- If
mitotic cells are incubated in vitro, the assembly of nuclear
envelope around chromosomes can be tracked in association with
dephosphorylation of nuclear lamins as observed by shifts in the PI
of the lamin proteins on 2-D gels. If dephosphorylation of nuclear
lamins is inhibited there is a corresponding inhibition of nuclear
envelope assembly.
Mitotic CHO cells
Disrupt mitotic
extract
Incubate at 330C and measure nuclear
envelope assembly around the
chromosomes and dephosphorylation
of lamins by the 2-D gel shift assay
Experimental basis for the role of nuclear lamin dephosphorylation in nuclear
envelope assembly (Burke & Gerace paper) contd….
Figure 5
Dephosphorylation of lamins during
the course of in vitro assembly
Figure 1
Electron microscopy of nuclei during in vitro
reassembly
Effects of ATP and ATP analogues on lamin assembly
(Burke & Gerace,1986, Table 1)
Effects of ATP and γ-S-ATP on In vitro nuclear assembly
(Burke & Gerace,1986, Figure 6) contd….
20 mM PEP/PK at 30 min
5 mM γ-S-ATP
Inhibition of nuclear envelope assembly in homogenates
depleted of specific lamins (Burke & Gerace,1986, Table 2)
Inhibition of nuclear envelope assembly in homogenates
depleted of specific lamins (Burke & Gerace,1986, Figure 9)
Anti-lamin A/C
Anti-lamin B
Burke & Gerace Paper Conclusions
• Depletion of lamins in extracts inhibits in vitro
assembly of the NE. (Table 2 & Figure 9)
• Assembly of nuclear envelope in vitro in mitotic
extracts requires the concomitant
dephosphorylation of the nuclear lamins. This is
indicated by tracking the de-P (2-D gel shifts) as
assembly occurs (EM) in vitro and blocking de-P
which inhibits NE formation. (Figures 1, 5, 6 &
Table 1)