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ORGANIZATION AND

EVOLUTION OF THE
NUCLEAR GENOME
PRINCESS LORRAINE GARCIA
BS-BIOLOGY IV

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
[Protein-rich 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

Chromosome Territories

Replication Sites

act
Ex
tr

trix

Transcript Tracks

Nuclear
Ma

ing

Splicing Factors

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

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 B-cdc2
(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.

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 S392 by cdc2 kinase is essential for nuclear envelope
breakdown.
Normal lamin A gene
Mutant lamin A gene

Anti-lamin A

DNA (DAPI)

Anti-lamin A

DNA (DAPI)

Table 1: Distribution of mitotic


phenotypes

Experimental basis for a role of


nuclear lamin dephosphorylation in
nuclear envelope assembly (Burke &
Gerace
paper)
Assembly of nuclear
envelope
in mitotic extractsIf 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
Incubate at 330C and measure
assembly.
Disrupt
mitotic
Mitotic CHO
cells

extract

nuclear envelope assembly


around the chromosomes and
dephosphorylation of lamins by
the 2-D gel shift assay

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)