Reduced CRYL1 expression in hepatocellular carcinoma confers cell growth advantages and correlates with adverse patient prognosis.

Homozygous deletion screening has been widely utilized to define tumour suppressor genes (TSGs) in cancers. Although these biallelic deletions are infrequent, their identification has facilitated the discovery of many important TSGs. We have systematically examined the genome of hepatocellular carcinoma (HCC), a highly malignant tumour that is rapidly fatal, for the presence of homozygous deletions. Array-CGH analysis on early passage of HCC cultures and cell lines led us to identify six homozygous deleted (HD) regions. A high concordance between array-CGH and expression of HD genes was demonstrated, where crystallin Lambda1 (CRYL1; located on chromosome 13q12.11) displayed the most frequent down-regulation. We found that reduced mRNA expression of CRYL1 was common in HCC tumours when compared with their adjacent non-tumoural liver (p = 0.0097). Significant associations could also be drawn between repressed CRYL1 and advanced tumour staging, increased tumour size, and shorter disease-free survival of patients (p < 0.037). Moreover, homozygous deletions on CRYL1 could be detected in 36% of HCC cases, where recurrent HDs were identified on exons 1, 5, and 8. Examination of other causal events suggested histone deacetylation and promoter hypermethylation to be likely inactivating mechanisms as well. Re-expression of CRYL1 in the SK-Hep1 cell line, where biallelic loss of CRYL1 was found, induced profound inhibition of cellular proliferation and cell growth (p < 0.0015). By Annexin V staining, CRYL1 restoration readily increased pro-apoptotic cells with an induction of PARP cleavage. Flow cytometry further revealed that CRYL1 could prolong the G(2)-M phase, possibly through interruption of the Cdc2/cyclin B pathway. Given that regional chromosome 13q12-q14 loss is a causal genomic event in HCC tumourigenesis, our finding may have implications for identifying a novel TSG CRYL1 within this important locus.

The Rix1 (Ipi1p-2p-3p) complex is a critical determinant of DNA replication licensing independent of their roles in ribosome biogenesis.

Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G1 transition and for pre-RC maintenance in G1 phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.

Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen in Saccharomyces cerevisiae.

DNA replication in eukaryotic cells is tightly regulated to ensure faithful inheritance of the genetic material. While the replicators, replication origins and many replication-initiation proteins in Saccharomyces cerevisiae have been identified and extensively studied, the detailed mechanism that controls the initiation of DNA replication is still not well understood. It is likely that some factors involved in or regulating the initiation of DNA replication have not been discovered. To identify novel DNA replication-initiation proteins and their regulators, we developed a sensitive and comprehensive phenotypic screen by combining several established genetic strategies including plasmid loss assays with plasmids containing a single versus multiple replication origins and colony color sectoring assays. We isolated dozen of mutants in previously known initiation proteins and identified several novel factors, including Ctf1p Ctf3p, Ctf4p, Ctf18p, Adk1p and Cdc60p, whose mutants lose plasmid containing a single replication origin at high rates but lose plasmid carrying multiple replication origins at lower rates. We also show that overexpression of replication initiation proteins causes synthetic dosage lethality or growth defects in ctf1 and ctf18 mutants and that Ctf1p and Ctf18p physically interact with ORC, Cdt1p and MCM proteins. Furthermore, depletion of both Ctf1p and Ctf18p prevents S phase entry, retards S phase progression, and reduces pre-RC formation during the M-to-G1 transition. These data suggest that Ctf1p and Ctf18p together play important roles in regulating the initiation of DNA replication.