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

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 Mitotic Chromosomes Chromosome territories

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?

MAINTAINING IN SITU FUNCTIONAL DOMAINS ON THE NUCLEAR MATRIX Chromosome Territories Splicing FactorsReplication Sites Transcript TracksTranscription Sites Extracting Nuclear Matrix

Domains)

3-D Model of a 1 mbp Multi-Loop Chromatin Domain

Many Nuclear Structures exhibit Constrained Motion

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. The Cell Nucleus as a Hierarchical Epigenetic System

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 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. 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

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. Experimental basis for a role of nuclear lamin phosphory- lation in nuclear envelope disassembly (Heald & McKeon) Normal lamin A gene Mutant lamin A gene Anti-lamin A DNA (DAPI) Anti-lamin A DNA (DAPI)

I A P T M Wild-type phenotype of CHO transfected cells with human lamin A (Heald & McKeon paper, Figure 2)

I A P T M Phenotype of cells transfected with double Ser-22/Ser-392 point mutations (Heald & McKeon paper, Figure 4)

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 33 0 C and measure nuclear envelope assembly around the chromosomes and dephosphorylation of lamins by the 2-D gel shift assay

Electron microscopy of nuclei during in vitro reassembly Dephosphorylation of lamins during the course of in vitro assembly Experimental basis for the role of nuclear lamin dephosphorylation in nuclear envelope assembly (Burke & Gerace paper) contd…. Figure 5 Figure 1

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 min5 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)