Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2001-06-08
2003-04-29
Mosher, Mary E. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S007100, C435S007720
Reexamination Certificate
active
06555329
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a method for identifying compounds influencing chromosome dynamics in eukaryotic cells. In particular, the invention relates to the treatment and prevention of human conditions by modulating higher order chromatin dependent chromosome stability during mitosis and meiosis.
2. Related Art
Higher-order chromatin is essential for epigenetic gene control and for the functional organisation of chromosomes. Differences in higher-order chromatin structure have been linked with distinct covalent modifications of histone tails which regulate transcriptional ‘on’ or ‘off’ states (Grunstein, 1998; Turner, 1998; Strahl and Allis, 2000) and influence chromosome condensation and segregation (Karpen and Allshire, 1997; Wei et al., 1999).
Histones constitute a highly conserved family of proteins (H3, H4, H2A, H2B, H1) which are the major components of eukaryotic chromatin structure. Histones compact genomic DNA into basic repeating structural units, the nucleosomes. In addition to their DNA packaging function, histones have been proven to be integral components of the molecular machinery that regulates gene expression.
Post-translational modifications of histone N-termini, particularly of H4 and H3, are well documented and have functionally been characterised as changes in acetylation (Grunstein, 1998; Turner, 1998; Strahl and Allis, 2000), phosphorylation (Wei et al., 1999) and, most recently, methylation (Chen et al., 1999; Strahl et al., 1999). In contrast to the large number of described histone acetyltransferases (HATs) and histone deacetylases (HDACs), genes encoding enzymatic activities that regulate phosphorylation (Sassone-Corsi et al., 1999; Hsu et al., 2000) or methylation (Chen et al., 1999) of histone N-termini are only beginning to be identified. Moreover, the interdependence of the different histone tail modifications for the integration of transcriptional output or higher-order chromatin organisation is currently not understood.
Overall, there is increasing evidence that the regulation of normal and aberrant cellular proliferation is not only affected on the transcriptional level, but that also a higher level of regulation is involved, i.e. the organisation of chromatin structure through the modification of histone molecules. The determination of the proteins and the molecular mechanisms involved in histone modification will contribute to the understanding of the cellular proliferation program and will thus shed led light on the mechanisms involved in aberrant proliferation occurring in tumour formation and progression (Jacobson and Pillus, 1999).
Genetic screens for suppressors of position effect variegation (PEV) in Drosophila (Reuter and Spierer, 1992) and
S. pombe
(Allshire et al., 1995) have identified a subfamily of approximately 30-40 loci which are referred to as Su(var)-group (Wallrath, 1998) genes. Interestingly, several histone deacetylases (De Rubertis et al., 1996), protein phosphatase type 1 (Baksa et al., 1993) and S-adenosyl methionine synthetase (Larsson et al., 1996) have been classified as Su(var)s. In contrast, Su(var)2-5 (which is allelic to HP1) (Eissenberg et al., 1992), Su(var)3-7 (Cléard et al., 1997) and Su(var)3-9 (Tschiersch et al., 1994; Schotta and Reuter, 2000) encode heterochromatin-associated proteins. Su(var) gene function thus suggests a model, in which modifications at the nucleosomal level may initiate the formation of defined chromosomal subdomains that are then stabilised and propagated by heterochromatic SU(VAR) proteins (Henikoff, 1997).
Su(var)3-9 is dominant over most PEV modifier mutations (Tschiersch et al., 1994), and mutants in the corresponding
S. pombe
clr4 gene (Ivanova et al., 1998) disrupt heterochromatin association of other modifying factors and result in chromosome segregation defects (Ekwall et al., 1996). Recently, human (SUV39H1) and murine (Suv39h1 and Suv39h2) Su(var)3-9 homologues have been isolated (Aagaard et al., 1999). It has been shown that they encode heterochromatic proteins which associate with mammalian HP1 (Aagaard et al., 1999). The SU(VAR)3-9 protein family combines two of the most evolutionarily conserved domains of ‘chromatin regulators’: the chromo (Aasland and Stewart, 1995; Koonin et al., 1995) and the SET (Tschiersch et al., 1994; Jenuwein et al., 1998) domain. Whereas the 60 amino acids chromo domain represents an ancient histone-like fold (Ball et al., 1997) that directs eu- or heterochromatic localisations (Platero et al., 1995), the molecular role of the 130 amino acids SET domain has remained enigmatic. Overexpression studies with human SUV39H1 mutants indicated a dominant interference with higher-order chromatin organisation that, surprisingly, suggested a functional relationship between the SET domain and the distribution of phosphorylated (at serine 10) histone H3 (Melcher et al., 2000).
SUMMARY OF THE INVENTION
It was an object of the invention to gain further insight into the molecular pathways leading to histone modifications and higher-order chromatin organisation in order to harness these findings for interfering with aberrant gene expression and genomic instability through chromosome mis-segregation and thus provide new cancer therapies.
In particular, it was an object of the invention to investigate the function of members of the SU(VAR)3-9 protein family with the view to develop novel strategies to affect higher-order chromatin dependent chromosome stability. Such strategies can be employed in therapies for the treatment of conditions in which aberrant gene expression and genomic instability through chromosome mis-segregation are causally involved. (The term “chromosome stability” implies successful segregation of chromosomes resulting in the maintenance of a stable karyotype).
Examples 1 to 7 of the present invention show that mammalian SU(VAR)3-9 related proteins (human SUV39H1, murine Suv39h1 and murine Suv39h2) are SET domain-dependent H3-specific histone methyltransferases which selectively methylate lysine 9 (“K9”) of the H3 N-terminus. Methylation of K9 negatively regulates phosphorylation of adjacent serine 10 and reveals a ‘histone code’ that appears intrinsically linked to the organisation of higher-order chromatin. (In the following, histone methyltransferases are termed “HMTases” or, more generally, “MTases”).
After having identified Suv39h1 and Suv39h2 as mammalian histone H3 lysine 9 specific histone methyltransferases (Suv39h HMTases), it was shown that these HMTases are heterochromatin-enriched enzymes which transiently accumulate at centromeres during mitosis (Aagaard et al., 1999; Aagaard et al., 2000). Moreover, it was shown that methylation of histone H3 at lysine 9 (H3-K9) creates a high-affinity binding site for HP1 proteins (Lachner et al., 2001; Bannister et al., 2001), thereby defining the SUV39H1-HP1 methylation system as a crucial regulatory mechanism for the assembly and propagation of heterochromatin (Jenuwein, 2001). Overexpression of human SUV39H1 induces ectopic heterochromatin and results in chromosome mis-segregation in mammalian cell lines (Melcher et al., 2000). In addition to the essential mitotic functions described above, heterochromatin is also crucial for the dynamic reorganization of meiotic chromosomes. Meiosis is initiated by chromosomal movements from the nuclear lumen to the nuclear envelope, where chromosomes cluster via their pericentric satellite sequences (Hawley et al., 1992; Scherthan et al., 1996). At meiotic prophase, chromosomes condense, followed by homolog pairing and recombination (at pachytene) between maternal and paternal chromosomes. The onset of the meiotic divisions is preceded by desynapsis, further chromosome condensation and histone H3 phosphorylation at pericentric heterochromatin (Cobb et al., 1999). In particular for male germ cells, the haploid genome content is finally organized into one heterochromatic block in elongating spermatids. In Drosophila, heterochromatin and its associated satellite sequences have been proposed to assist
Eisenhaber Frank
Jenuwein Thomas
O'Carroll Dónal
Rea Stephen
Boehringer Ingelheim International GmbH
Sterne Kessler Goldstein & Fox P.L.L.C.
Yu Misook
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