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Qing-Jun Meng Lab

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PhD projects available

Circadian clocks, mammary stem cells and breast cancer

Funding: Project available for individuals with self arranged funding.

Description

Circadian (24 hourly) rhythms generated by our intrinsic biological clocks play critical roles in regulating physiology and behaviour.  Cell autonomous circadian clocks are involved in governing cell division, differentiation, DNA damage repair and cell metabolism. Recent epidemiological studies have suggested links between disruption to circadian rhythms (for example women shift workers) and susceptibility to breast cancer. But the underlying molecular and cellular mechanisms are largely unknown.

In collaboration with the Streuli group, we have recently characterized the circadian clocks in mammary epithelial cells and stem cells.  By time series microarrays, we have revealed several clock controlled pathways that have been previously implicated in stem cell function and in the development of breast cancer.   This project aims to focus on a few of these key genes and pathways, in order to gain in depth understanding of the role of clock genes in the self-renewal, differentiation and malignant transformation of mammary stem cell.   

In this project, we are going to use primary cell culture of mammary epithelial cells and stem cells from transgenic animals with clock gene mutations, mammosphere assays, immunocytochemistry, in vitro gene knockdown studies, real-time clock reporter techniques and lentiviral transduction to address the functional significance of circadian clock genes in regulating the “stemness” and tumorigenesis of mammary cells.  This new knowledge will help understand the functional links between clock disruption and breast cancer risks.

 

Related Publications

  • Janich P, Meng QJ, Benitah SA (2014). Circadian control of tissue homeostasis and adult stem cells. Curr Opin Cell Biol. 31:8-15.
  • Dudek M, Gossan N, Nan Yang N, Im H-J, Ruckshanthi JPD, Yoshitane H, Li X, Jin D, Wang P, Boudiffa M, Bellantuono I, Fukada Y, Boot-Handford RP, Meng QJ (2016). The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J Clin. Invest. 126 (1), 365-376.
  • Yagita K, Horie K, Koinuma S, Nakamura W, Yamanaka I, Urasaki A, Shigeyoshi Y, Kawakami K, Shimada S, Takeda J, Uchiyama Y. (2010). Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro. PNAS. 107(8):3846.

 

Mapping the circadian clock-dependent regulation of the secretory pathway

Funding: Project available for individuals with self arranged funding.

Description

The protein secretory pathway in cells comprises a series of membranous compartments and transport vesicles that the cell uses to synthesise, post-translationally modify, and move proteins for delivery to the plasma membrane or for secretion to the extracellular matrix.  The conventional pathway describes protein synthesis on the luminal face of the endoplasmic reticulum, transport of proteins in 60 nm-diameter COPII vesicles to the Golgi apparatus, and subsequent transport to the plasma membrane in Golgi-to-plasma membrane carriers (GPCs).  Ground-breaking studies on the secretory pathway (which led to the Nobel Prize in Physiology and Medicine 2013) have been carried out using fibroblasts in which small viral proteins have been used as the cargo.  However, the most abundant cargo in fibroblasts is procollagen (which is ~450,000 kDa and 350 nm in length), which is not expected to fit into 60 nm-diameter transport vesicles. 

Using time-series microarray analysis of gene transcription, proteomics, electron microscopy, and immunofluorescence for proteins in the secretory pathway during 24 hours, we have shown that the secretory pathway is under the control of the circadian clock (manuscript in preparation). Our data show that the circadian clock exercises transcriptional and translational control of key proteins that are chaperones of protein folding, interaction partners for COPII-vesicle formation, and regulators of retrograde Golgi-to-ER transport. 

The project is to use state-of-the-art fluorescence and super resolution imaging using a variety of fusion proteins, electron microscopy, cells from specific knockout mice, and targeted knockdown and knockout of novel gene targets including the use of CRISPR/Cas9.  We will: generate a complete three-dimensional map of the protein secretory pathway in fibroblasts at 20 nm resolution, understand how the arrangement and size of compartments changes during 24 hours, locate the positions of key proteins in the pathway, and identify mechanisms of transporting large cargo through the cell. To obtain a deeper understanding of the function of the circadian clock in maintaining tissue homeostasis, we will use mass spectrometry approaches to determine which extracellular matrix proteins are present in the extracellular matrix and in the secretory pathway at 4-hour intervals during 24 hours, in mice that have had specific genes edited by CRISPR/Cas9.

The project will shed new light on how fibroblasts transport large cargo, such as procollagen, and how this process is perturbed in fibrosis and ageing.

Related Publications

  1. Canty, E. G., Lu, Y., Meadows, R. S., Shaw, M. K., Holmes, D. F. and Kadler, K. E. (2004) Co-alignment of plasma membrane channels and protrusions (fibripositors) specifies the parallelism of tendon. Journal of Cell Biology 165: 553-563
  2. Yeung, C-Y C, Gossan N, Lu Y, Hughes A, Hensman J, Bayer M, Kjær M, Kadler KE and Meng QJ (2014). Gremlin-2 is a BMP antagonist that is regulated by the circadian clock. Scientific Reports. 4, 5183; DOI:10.1038/srep05183.
  3. Dudek M, Gossan N, Nan Yang N, Im H-J, Ruckshanthi JPD, Yoshitane H, Li X, Jin D, Wang P, Boudiffa M, Bellantuono I, Fukada Y, Boot-Handford RP, and Meng QJ (2016). The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity. J Clin. Invest. 126 (1), 365-376.
  4. Canty, E. G. and Kadler, K. E. (2005) Procollagen trafficking, processing and fibrillogenesis. Journal of Cell Science 118: 1341-1353
  5. Taylor, S. H., Yeung, C-Y., C., Kalson, N. S., Lu, Y., Zigrino, P., Starborg, T., Warwood, S., Holmes, D. F., Canty-Laird, E. G., Mauch, C. and Kadler, K. E. (2015) MMP14 is required for fibrous tissue expansion. eLIFE doi: 10.7554/eLife.09345.
  6. Starborg, T., Kalson, N. S., Lu, Y., Mironov, A., Cootes, T. F., Holmes, D. F. and Kadler, K. E. (2013) Using transmission electron microscopy and 3View® to determine collagen fibril size and three-dimensional organization. Nature Protocols 8: 1433-1448
  7. Kalson, N. S., Starborg, T., Lu, Y., Mironov, A., Humphries, S. M., Holmes, D. F. and Kadler, K. E. (2013) Non-muscle myosin II powered transport of newly-formed collagen fibrils at the plasma membrane. Proceedings of the National Academy of Sciences U. S. A. 110: E4743-E4752. doi: 10.1073/pnas.1314348110

The role of circadian clock genes in regulating the chondrogenic potential of human Embryonic Stem Cells

Funding: Project available for individuals with self arranged funding.

Description

Osteoarthritis (OA) is one of the most common joint diseases, affecting ~6 million people in UK, causing severe pain, deformity and a loss of mobility. A common feature of OA is the degeneration and loss of articular cartilage and chondrocytes, the only cell type in this tissue. The current clinical treatment for OA is restricted to symptomatic pain relief. The Meng group has recently identified cell autonomous circadian clocks in cartilage tissue, which controls ~600 rhythmic cartilage target genes, many of these genes have previously been implicated in diseases such as OA (Gossan et al., 2013, Arthritis and Rheumatism). hESC, due to their unlimited capacity for self renewal and pluripotency, provide a potential source of chondrocytes to be used in regenerative medicine towards joint diseases such as OA.  The Kimber group has developed a novel three stage chemically defined protocol to generate chondrogenic cells from a number of hESC lines and induced pluripotent cells (Oldershaw et al 2010 Nature Biotech 28,117 and Cheng et al Tiss Eng 2013). hESC can be induced to generate a 94-97% chondrogenic population expressing SOX9, collagen type IIα1 (COL2A1) and aggrecan in vitro within 14 days.

 

In collaboration with the Kimber group, we have recently identified profound changes of circadian clock genes during the chondrogenic differentiation of hESC.  We are particularly interested in the role of clock genes in chondrogenesis.  Such understanding may help identify key niche factors, which may be harnessed to manipulate differentiation of hESC towards regenerative medicine as well as control clock function. 

 

In this project, we are going to use genetic manipulation of hESC, in vitro chondrogenic differentiation, gene expression studies, real-time clock reporter techniques, lentiviral transduction and RNAseq approaches to address the functional significance of circadian clock genes in hESC differentiation. Exploiting the knowledge of circadian clock control will allow us to generate functioning and native-like cartilage tissue for therapeutic use as well as suggesting new disease models and drug targets for improving in vivo and in vitro matrix formation.

Related Publications

  1. Gossan N, Zeef L, Hensman J, Hughes A, Bateman JF, Rowley L, Little, CB, Piggins HD, Rattray M, Boot-Handford RP, Meng QJ, (2013). The circadian clock in chondrocytes regulates genes controlling key aspects of cartilage homeostasis. Arthritis and Rheumatism; Epub ahead of print.
  2. Meng QJ, Maywood E, Bechtold D, Lu WQ, Li J, Gibbs J, Dupré S, Chesham J, Rajamohan F, Knafels J, Sneed B, Zawadzke L, Ohren J, Walton K, Wager T, Hastings M, Loudon A (2010) Entrainment of disrupted circadian behavior through inhibition of Casein Kinase 1 (CK1) enzymes. Proceedings of the National Academy of Sciences of the United States of America, 107 (34), 15240-15245.
  1. Oldershaw RA,  Baxter MA, Lowe ET, Bates N, Grady LN,  Brison DR, Hardingham TE & Kimber SJ. 2010. The directed differentiation of human embryonic stem cells towards chondrocytes Nature Biotech 28, 1187-1193
  2. Soteriou D, Iskender B,  Byron A,  Borg-Bartolo S, Haddock M-C, Baxter M, Humphries, JD, Knight D,  Humphries MJ, and Kimber SJ. (2013) Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Proteins for Human Embryonic Stem Cell Maintenance J Biol Chem 288, 18716-18731