Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

Background: The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of gene

Modular Structure of the Human Lamin B2 Replicator

The cis-acting elements necessary for the activity of DNA replication origins in metazoan cells are still poorly understood. Here we report a thorough characterization of the DNA sequence requirements of the origin associated with the human lamin B2 gene. A 1.2-kb DNA segment, comprising the start site of DNA replication and located within a large protein-bound region, as well as a CpG island, displays origin activity when moved to different ectopic positions. Genomic footprinting analysis of both the endogenous and the ectopic origins indicates that the large protein complex is assembled in both cases around the replication start site. Replacement of this footprinted region with an unrelated sequence, maintaining the CpG island intact, abolishes origin activity and the interaction with hORC2, a subunit of the origin recognition complex. Conversely, the replacement of 17 bp within the protected region reduces the extension of the protection without affecting the interaction with hORC2. This substitution does not abolish the origin activity but makes it more sensitive to the integration site. Finally, the nearby CpG island positively affects the efficiency of initiation. This analysis reveals the modular structure of the lamin B2 origin and supports the idea that sequence elements close to the replication start site play an important role in origin activation

Ring1-mediated ubiquitination of H2A restrains poised RNA polymerase II at bivalent genes in ES cells

8 páginas, 5 figuras -- PAGS nros. 1428-1435Changes in phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAP) are associated with transcription initiation, elongation and termination1, 2, 3. Sites of active transcription are generally characterized by hyperphosphorylated RNAP, particularly at Ser 2 residues, whereas inactive or poised genes may lack RNAP or may bind Ser 5-phosphorylated RNAP at promoter proximal regions. Recent studies have demonstrated that silent developmental regulator genes have an unusual histone modification profile in ES cells, being simultaneously marked with Polycomb repressor-mediated histone H3K27 methylation, and marks normally associated with gene activity4, 5. Contrary to the prevailing view, we show here that this important subset of developmental regulator genes, termed bivalent genes, assemble RNAP complexes phosphorylated on Ser 5 and are transcribed at low levels. We provide evidence that this poised RNAP configuration is enforced by Polycomb Repressor Complex (PRC)-mediated ubiquitination of H2A, as conditional deletion of Ring1A and Ring1B leads to the sequential loss of ubiquitination of H2A, release of poised RNAP, and subsequent gene de-repression. These observations provide an insight into the molecular mechanisms that allow ES cells to self-renew and yet retain the ability to generate multiple lineage outcomesPeer reviewe