Biomedical subjects

Jeanette Gowen Cook

Publications and source records attributed to Jeanette Gowen Cook.

4 recordsLinked to original sources

PubMed2022

Chromosomal localization of cohesin is differentially regulated by WIZ, WAPL, and G9a.

BACKGROUND: The cohesin complex is essential for proper chromosome structure and gene expression. Defects in cohesin subunits and regulators cause changes in cohesin complex dynamics and thereby alter three-dimensional genome organization. However, the molecular mechanisms that drive cohesin localization and function remain poorly understood. RESULTS: In this study, we observe that loss of WIZ causes changes to cohesin localization that are distinct from loss of the known WIZ binding partner G9a. Whereas loss of WIZ uniformly increases cohesin levels on chromatin at known binding sites and leads to new, ectopic cohesin binding sites, loss of G9a does not. Ectopic cohesin binding on chromatin after the loss of WIZ occurs at regions that are enriched for activating histone modifications and transcription factors motifs. Furthermore, loss of WIZ causes changes in cohesin localization that are distinct from those observed by loss of WAPL, the canonical cohesin unloading factor. CONCLUSIONS: The evidence presented here suggests that WIZ can function independently from its previously identified role with G9a and GLP in heterochromatin formation. Furthermore, while WIZ limits the levels and localization pattern of cohesin across the genome, it appears to function independently of WAPL-mediated cohesin unloading.

Cell Cycle Proteins↗

PubMed2017

Rapid DNA replication origin licensing protects stem cell pluripotency.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

Cell Cycle↗

PubMed2003

The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells.

Chromosomal DNA replication requires the recruitment of the six-subunit minichromosome maintenance (Mcm) complex to chromatin through the action of Cdc6 and Cdt1. Although considerable work has described the functions of Cdc6 and Cdt1 in yeast and biochemical systems, evidence that their mammalian counterparts are subject to distinct regulation suggests the need to further explore the molecular relationships involving Cdc6 and Cdt1. Here we demonstrate that Cdc6 and Cdt1 are mutually dependent on one another for loading Mcm complexes onto chromatin in mammalian cells. The association of Cdt1 with Mcm2 is regulated by cell growth. Mcm2 prepared from quiescent cells associates very weakly with Cdt1, whereas Mcm2 from serum-stimulated cells associates with Cdt1 much more efficiently. Cdc6, which normally accumulates as cells progress from quiescence into G(1), is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells. We further show that Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region we had previously shown to be essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and we provide evidence that the mechanism of this inhibition involves blocking the binding of Cdt1 to both Mcm2 and Cdc6. These results identify novel molecular functions for both Cdc6 and geminin in controlling the association of Cdt1 with other components of the replication apparatus and indicate that the association of Cdt1 with the Mcm complex is controlled as cells exit and reenter the cell cycle.

Cell Cycle Proteins↗

PubMed2002

Analysis of Cdc6 function in the assembly of mammalian prereplication complexes.

Eukaryotic DNA replication requires the previous formation of a prereplication complex containing the ATPase Cdc6 and the minichromosome maintenance (Mcm) complex. Although considerable insight has been gained from in vitro studies and yeast genetics, the functional analysis of replication proteins in intact mammalian cells has been lacking. We have made use of adenoviral vectors to express normal and mutant forms of Cdc6 in quiescent mammalian cells to assess function. We demonstrate that Cdc6 expression alone is sufficient to induce a stable association of endogenous Mcm proteins with chromatin in serum-deprived cells where cyclin-dependent kinase (cdk) activity is low. Moreover, endogenous Cdc6 is sufficient to load Mcm proteins onto chromatin in the absence of cdk activity in p21-arrested cells. Cdc6 synergizes with physiological levels of cyclin E/Cdk2 to induce semiconservative DNA replication in quiescent cells whereas cyclin A/Cdk2 is unable to collaborate with Cdc6. Cdc6 that cannot be phosphorylated by cdks is fully capable of inducing Mcm chromatin association and replication. Mutation of the Cdc6 ATP-binding site severely impairs the ability of Cdc6 to induce Mcm chromatin loading and reduces its ability to induce replication. Nevertheless, the ATPase domain of Cdc6 in the absence of the noncatalytic amino terminus is not sufficient for either Mcm chromatin loading or DNA replication, indicating a requirement for this domain of Cdc6.

Adenoviridae↗