Regulation of Breast Stem-Progenitor Cell Chromatin by Pygo2

Institution: University of California, Irvine
Investigator(s): Bingnan Gu, Ph.D. -
Award Cycle: 2008 (Cycle 14) Grant #: 14FB-0129 Award: $135,000
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Biology of the Normal Breast: the starting point



Initial Award Abstract (2008)

Our understanding of breast cancer has been greatly advanced since the recent identification of breast cancer stem cells (BCSCs), a minor pool of transformed stem cells that can initiate new breast tumors. The presence of BCSCs may explain the cause of metastatic breast cancers and why cancers recur after being treated with available therapeutic agents that can shrink the tumor mass but cannot eliminate BCSCs. A better understanding of the molecular control of BCSCs and their physiological counterparts may provide new targets for more effective therapies. Wnt signaling plays a critical role in the self-renewal of both BCSCs and normal mammary gland stem/progenitor cells. Aberrant activation of Wnt signaling results in breast cancers with an expanded stem cell pool. Pygopus 2 (Pygo2) is a recently identified activator of the Wnt pathway. Deleting Pygo2 from the mouse genome leads to reduced Wnt signaling and compromised proliferation of breast stem/progenitor cells, suggesting that it may serve as a new molecular therapeutic target for interfering with breast stem cell activities.

In this project we will study the role of Pygo2 in chromatin remodeling of mammary epithelial cells. My preliminary studies implicate Pygo2 as a potential regulator of methylation and acetylation of histones, a group of small proteins that pack the long DNA molecules into repetitive units inside the cell nucleus. Epigenetic events such as histone modifications and DNA methylation have been linked to breast cancer. It has been proposed that cancer-associated epigenetic changes inactivate tumor suppressor genes. Drugs that may correct these epigenetic changes are being tested in clinical trials for cancer therapy. Before such epigenetic drugs become widely used for breast cancers, it is essential for us to understand how normal epigenetic patterns are established/maintained and how these alterations occur in breast cancer.

Specifically, we will investigate how Pygo2 recognizes and binds to a specific type of histone modification (H3K4me3) on target chromatin, and in turn recruits protein complexes that have other histone modifying activities (e.g., acetylation). For these experiments, we will make specific changes (mutations) to the Pygo2 protein and test if they interfere with its ability to bind to modified histone (H3K4me3) or to the target chromatin. I will determine the functional consequences (e.g., histone modifications, target gene activation) of these mutations, as well as the mammary progenitor cell activities after knocking down Pygo2 protein (using a technique called RNA interference). Furthermore, I will purify and characterize the components of the Pygo2-containing protein complex using mass spectrometry analysis.

We anticipate that successful completion of the proposed research will provide key molecular insights of how Wnt signaling controls breast stem/progenitor cell activities and may uncover a novel link between chromatin regulation, transcriptional control, and breast cancer stem cell activation.




Final Report (2011)

Breast stem/progenitor cells have extensive self-renewal and differentiation activities. Aberrant activation of these stem cells may produce malignant counterparts that can escape conventional chemo-radiotherapy and serve as the root cause of metastatic breast cancer. Canonical Wntl/beta-catenin signaling pathway plays an important role in the maintenance and expansion of breast stem cells and over-production of Wnt results in breast cancer. Therefore, a thorough understanding of the particular molecular control of breast stem cells will provide new strategy to detect, target and kill these cells to benefit breast cancer patients.

In this project, we studied Pygo2, a newly identified transcriptional co-activator ofWnt signaling, in chromatin regulation of mammary stem/progenitor cells. Ablation of Pygo2 gene in the mouse genome reduces expansion of breast stem/progenitor cells. However, the molecular mechanism underlying this function was unknown. Our study initially revealed that Pygo2 is a chromatin effector that can modulate Wnt target gene expression. Specifically, we showed that Pygo2 can physically interact and functionally enhance an important histone modification (histone H3 lysine 4 tri-methylation) that promotes gene transcription. Furthermore, we also identified histone genes as new targets of Pygo2, and histone H3 lysine 56 acetylation as an additional form of histone modification that is regulated by Pygo2.

Our study, which resulted in 3 publications, thus laid the ground work for future investigation to design and examine small molecules or other types of agents to disrupt the Wnt-Pygo2 connection, thereby blocking the link between Wnt signaling and chromatin regulation in mammary stem/progenitor cells. Such strategy holds the potential to prevent these cells from receiving over-produced signaling inputs and becoming breast cancer cells.

Publications:
Akt Phosphorylates Wnt Coactivator and Chromatin Effector Pygo2 at Serine 48 to Antagonize Its Ubiquitin/Proteasome-Mediated Degradation




Symposium Abstract (2010)

The recently identified breast cancer stem cells (BCSCs) are a minor pool of transformed stem cells that can escape from available therapeutic agents and initiate new breast tumors. A better understanding of the molecular control of BCSCs and their physiological counterparts may provide new targets for more effective therapies. Wnt signaling pathway plays an important role in regulating breast stem/progenitor cells. Aberrant activation of Wnt signaling results in breast cancers with an expanded stem cell pool. Pygopus 2 (Pygo2) is an evolutionarily conserved activator of the Wnt signaling pathway and is over-expressed in breast cancer. Deletion of Pygo2 from the mouse genome leads to reduced Wnt signaling and compromised proliferation of breast stem/progenitor cells, suggesting that it may serve as a new molecular therapeutic target for interfering with breast stem cell activities. However, the underlying molecular mechanism by which Pygo2 regulates the activity of these cells is largely unknown.

In this project we are studying the role of Pygo2 in chromatin remodeling of mammary epithelial cells. My current data implicate that Pygo2 is a novel regulator of methylation and acetylation of histones, a group of small proteins that pack the long DNA molecules into repetitive units inside the cell nucleus and regulate gene expression. Specifically, we are investigating how Pygo2 recognizes and binds to a specific type of histone modification (H3K4me3) on target chromatin, and in turn recruits protein complexes that have other histone modifying activities (e.g., acetylation). By making changes to the Pygo2 PHD domain and knocking down Pygo2 protein by RNA interference, we have successfully established the functional requirement of the Pygo2-H3K4me3 interaction in the expansion of mammary progenitor cells. We showed that the binding of Pygo2 PHD domain to H3K4me3 is enhanced by additional binding with BCL9 protein. The ternary complex formation is likely required for the proliferation of mammary progenitor cells in colony formation assays. Furthermore, we have demonstrated that by association with histone H3K4me3, Pygo2 also plays a positive role in facilitating the methylation of H3K4, which promotes the activation of Wnt/β-catenin target gene expression. To further understand the molecular function of Pygo2 in chromatin regulation, we are performing purification and characterization of the Pygo2-containing protein complex using mass spectrometry analysis.

Epigenetic events such as histone modifications and DNA methylation have been linked to breast cancer. It has been proposed that cancer-associated epigenetic changes inactivate tumor suppressor genes. Drugs that may correct these epigenetic changes are being tested in clinical trials for cancer therapy. Before such epigenetic drugs become widely used for breast cancers, it is essential for us to understand how normal epigenetic patterns are established/maintained and how these alterations occur in breast cancer. We anticipate that successful completion of the proposed research will provide key molecular insights of how Wnt signaling controls breast stem/progenitor cell activities and may uncover a novel link between chromatin regulation, transcriptional control, and breast cancer stem cell activation.



Chromatin effector Pygo2 mediates Wnt-Notch crosstalk to suppress luminal/alveolar potential of mammary stem and basal cells.
Periodical:Cell Stem Cell
Index Medicus: C Stem Cell
Authors: Gu B, Watanabe K, Sun P, Fallahi M, Dai X
Yr: 2013 Vol: Nbr: Abs: Pg:

Pygo2 expands mammary progenitor cells by facilitating histone H3 K4 methylation.
Periodical:Journal of Cell Biology
Index Medicus: J Cell Biol
Authors: Gu B, Sun P, Yuan Y, et al, and Dai X
Yr: 2009 Vol: 185 Nbr: 5 Abs: Pg:811-26

Epithelial stem cells: An epigenetic and wnt-centric perspective.
Periodical:Journal of Cellular Biochemistry
Index Medicus: J Cell Biochem.
Authors: Gu B, Watanabe K, Dai X
Yr: 2010 Vol: 110 Nbr: Abs: Pg:1279-1287

Pygo2 regulates histone gene expression and H3 K56 acetylation in human mammary epithelial cells.
Periodical:Cell Cycle
Index Medicus: Cell Cycle
Authors: Gu B, Watanabe K, Dai X
Yr: 2012 Vol: 11 Nbr: 1 Abs: Pg:79-87