CUL4B Regulates Cell Proliferation

DNA replication is the core event of normal cell division and proliferation. During evolutionary process, cell set up and develop a very multiplicity system: through G1/S, G2/M phase transition to regulate and control DNA replication. DNA replication contains initiation, extension and termination, and is tighly regulated by several cell cycle proteins and cell proliferation growth factors. These proteins interact with each other, and constitute a specific and cell cycle dependent regulator to guarantee the complete and precise replication of DNA. The initiation of DNA replication takes place at many sites on chromosomes which are called the origins of replication. During this process,pre-replication complex(Pre-RC) assemble and bind to replication origins. It starts by the binding of the six-subunit origin-recognition complex (ORC) to DNA in late mitosis. As a cell enters G1 phase, the initiation factors 6 (CDC6) will bind to the origin-bound ORC, followed by the recruitment of DNA replication factor 1 (CDT1) and the loading of the DNA replicative helicase minichromosome maintenance protein (MCM) complexes. Proper DNA replication depends mainly on the regulation of the pre-RC formation.CUL4B encodes a scaffold protein that organize cullin-RING ubiquitin ligase (E3) complexes(CRLs). CRLs constitute a major subclass of RING finger E3s that regulate diverse cellular processes, including cell cycle progression, transcription, signaltransduction, development and DNA repair. Recent genetic studies showed that germ-line mutation in CUL4B can cause X-linked mental retardation syndrome. The findings that X-chromosome inactivation was extremely skewed in carriers and only the wild-type allele was detected in leukocyte cDNA from those carriers suggest that cells lacking CUL4B are impaired in proliferation and strongly selected against in vivo. We found that RNA interference (RNAi) silencing of CUL4B led to an inhibition of cell proliferation and a prolonged S phase, due to the over accumulation of cyclin E. These data suggest that CUL4B may plays an important role in cell proliferation and DNA replication. To further understand the function of CUL4B in regulating cell proliferation, we, first determined the expression pattern of human CUL4B during cell cycle by Real-time PCR and Western blot. CUL4B transcripts were detected in all phases of cell cycle, and there were no systematic variations through the cell cycle. Similar results were obteined at protein levels. Cytoplasmic, nuclear soluble and chromatin-bound protein extracts were separated and analyzed by Western blot. In addition to cytoplasmic and nucleosolic fractions, CULB was also detected in chromatin-bound fraction, suggesting that CUL4B is a chromatin-bound protein. Furthermore, CUL4B was colocalizes with sites of DNA synthesis. These results suggested that CUL4B is important for DNA replication and S-phase progression. To test this hypothesis, we examined the DNA replication dynamics in CUL4B-depleted cells using a DNA fiber-labelling assay, and found that CUL4B knockdown reduces the new origin firing. Further analyses showed that the downregulation of CUL4B leads to a reduction in the level of chromatin-bound CDC6 and an accelerated degradation of nuclear CDC6 by APCCDHI. CUL4B regulates the nuclear CDC6 independent of CDC6 phosphorylation by CDK2. The E3 ligase activity of CUL4B is not required for its regulation of nuclear CDC6. Although CUL4A and CUL4B protein are highly homologous, they regulate nuclear CDC6 via different mechanisms.Taken together, this study provided the first evidence that CUL4B has a novel function in regulating DNA replication by maintaining the structural integrity of pre-replication complexes.