X-Chromosome Inactivation (XCI) is one of the best-characterized epigenetic phenomena where long noncoding RNAs are key players that regulate gene expression. Female mammals (XX) have two X-chromosomes, and one X is randomly chosen for transcriptional silencing in order to equalize the expression of X-linked genes compared to males (XY). Thus females are mosaic for X-chromosome expression, where a cell will express the maternal or paternal X. One great example of female X-mosaicism are calico cats.
XCI is regulated by a variety of long noncoding RNA genes located at a special region on the X-chromosome: the X-Inactivation Center (XIC). The master regulator of XCI is the long noncoding RNA Xist, required for initiating and maintaining XCI. XIST RNA is a spliced 17kb transcript exclusively expressed from the inactive X and the RNA coats the entire chromosome, which can be visualized using fluorescence in situ hybridization (FISH).
Long noncoding RNAs are epigenetic regulators, making heritable changes to gene expression without changing DNA sequence. One of the remarkable findings of the human genome sequencing project is that just 2% of the genome is protein coding, yet 70-90% of the genome is transcribed. The explosion of next-generation sequencing experiments has sparked investigation into this ‘dark matter’ of the genome, and recent estimates suggest that there are 10,000-20,000 long noncoding RNAs. These transcripts (defined as >200nt in length) exhibit cell and tissue specific expression, yet the majority lack functional characterization.
Our lab is interested in the function and molecular mechanisms of known and novel long noncoding RNAs residing on the X-chromosome. We use XCI as a paradigm for understanding how a long noncoding RNA (XIST) initiates and maintains transcriptional silencing. We use a variety of model systems, including mouse and human pluripotent stem cells, primary mammalian cells, transformed cells, and mice. Our goal is to understand how misregulated expression of long noncoding RNAs contributes to disease and compromises early development.
- Human and mouse embryonic stem cell culture
- Reprogramming mammalian somatic cells to induced Pluripotent Stem Cells (iPSCs)
- Microscopy: RNA and DNA fluorescence in situ hybridization (FISH), immunofluorescence detection of proteins and chromatin modifications in mammalian pluripotent stem cells, somatic, and mouse germ cells
- Genome editing using TALENs and CRISPRs for human somatic cells and iPSCs/ESCs
- Molecular biology: PCR and real-time PCR, cloning (restriction enzyme, Gateway, In-Fusion), Southern blotting, Northern blotting
Maintenance of X-Chromosome Inactivation
Our understanding of XCI is mostly from mouse models, yet it’s still unclear how XCI is initiated and maintained in humans. The long noncoding RNA XIST is the master regulator of XCI, and initiates chromosome-wide silencing during early embryonic development. This silencing pattern is maintained by numerous epigenetic modifications into adulthood. We are examining how XIST RNA triggers the transformation of the human active X-chromosome into a heterochromatic inactive X. We are also investigating how abnormal expression from the inactive X contributes to disease susceptibility and progression.
Genetic analysis of the human X-Inactivation Regulatory Center
The X-Inactivation Center (XIC) contains all the necessary genes for silencing an X-chromosome, and these regions are quite different between mouse and human. We are investigating how this region is regulated during early human development using human pluripotent stem cells to model the preimplantation embryo. Using genome editing technologies (TALENs and CRISPRs), we are introducing mutations for investigating how these long noncoding RNA genes function.
Novel long noncoding RNAs important for early human development
Next-generation sequencing experiments have found that there are thousands of long noncoding RNAs exhibiting cell and tissue-specific expression, yet the function of these transcripts is largely unknown. We are examining the transcriptional profile of human female pluripotent stem cells and in vitro differentiated cells to determine the predominant transcripts expressed in these cell types. We use genetic approaches to determine how these transcripts function in vivo.
Luo M, Zhou J, Leu NA, Abreu CM, Wang J, Anguera MC, de Rooij DG, Jasin M, Wang PJ. Polycomb protein SCML2 associates with USP7 and counteracts histone H2A ubiquitination in the XY chromatin during male meiosis. PLoS Genet. 2015 Jan 29;11(1).
Lessing D, Anguera MC, Lee JT. X chromosome inactivation and epigenetic responses to cellular reprogramming. Annu Rev Genomics Hum Genet. 14: 85-110, 2013.
Anguera Montserrat C, Sadreyev Ruslan, Zhang Zhaoqing, Szanto Attila, Payer Bernhard, Sheridan Steven D, Kwok Showming, Haggarty Stephen J, Sur Mriganka, Alvarez Jason, Gimelbrant Alexander, Mitalipova Maisam, Kirby James E, Lee Jeannie T Molecular signatures of human induced pluripotent stem cells highlight sex differences and cancer genes. Cell stem cell 11: 75-90, 2012.
Anguera Montserrat C, Ma Weiyuan, Clift Danielle, Namekawa Satoshi, Kelleher Raymond J, Lee Jeannie T Tsx produces a long noncoding RNA and has general functions in the germline, stem cells, and brain. PLoS genetics 7: e1002248, 2011.
Kim Daniel H, Jeon Yesu, Anguera Montserrat C, Lee Jeannie T X-chromosome epigenetic reprogramming in pluripotent stem cells via noncoding genes. Seminars in cell & developmental biology 22: 336-42, 2011.
Field Martha S, Anguera Montserrat C, Page Rodney, Stover Patrick J 5,10-Methenyltetrahydrofolate synthetase activity is increased in tumors and modifies the efficacy of antipurine LY309887. Archives of biochemistry and biophysics 481: 145-50, 2009.
Anguera Montserrat C, Liu Matthew, Avruch Joseph, Lee Jeannie T Characterization of two Mst1-deficient mouse models. Developmental dynamics : an official publication of the American Association of Anatomists 237: 3424-34, 2008.
Anguera Montserrat C, Stover Patrick J Methenyltetrahydrofolate synthetase is a high-affinity catecholamine-binding protein. Archives of biochemistry and biophysics 455: 175-87, 2006.
Anguera Montserrat C, Field Martha S, Perry Cheryll, Ghandour Haifa, Chiang En-Pei, Selhub Jacob, Shane Barry, Stover Patrick J Regulation of folate-mediated one-carbon metabolism by 10-formyltetrahydrofolate dehydrogenase. The Journal of biological chemistry 281: 18335-42, 2006.
Anguera M C, Sun B K, Xu N, Lee J T X-chromosome kiss and tell: how the Xs go their separate ways. Cold Spring Harbor symposia on quantitative biology 71: 429-37, 2006.
Anguera Montserrat C, Liu Xiaowen, Stover Patrick J Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus. Protein expression and purification 35: 276-83, 2004.
Anguera Montserrat C, Suh Jae Rin, Ghandour Haifa, Nasrallah Ilya M, Selhub Jacob, Stover Patrick J Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation. The Journal of biological chemistry 278: 29856-62, 2003.
Montserrat C. Anguera, PhD
Montserrat grew up in San Diego, CA and attended UC San Diego as an undergraduate, where she studied environmental chemistry. She received her PhD in Biochemistry from Cornell University in Ithaca, NY, where she studied folate metabolism using cell culture and mouse models with Dr. Patrick Stover. She became interested in epigenetics, and joined Jeannie Lee’s lab at the Massachusetts General Hospital and she received a Ruth Kirschstein NRSA. She worked on a variety of projects in the Lee lab investigating long noncoding RNAs from the X-chromosome in mouse and human pluripotent stem cells.
Jianle Wang, PhD
Jianle is studying the function of long noncoding RNAs in human embryonic stem cells and in vitro differentiated trophoblast cells. She is using TALENs to generate various GFP and mCherry reporter human pluripotent stem cells lines and transgenes to investigate functions of specific long noncoding transcripts expressed in human cells. She is using human in vitro differentiated trophoblast cells to investigate novel long noncoding RNAs specific to these cells.
Ian Penkala, BS
Ian attended the University of Pennsylvania where he studied chemical engineering. During his undergraduate studies, he participated in research opportunities across campus, most notably with Dr. Kendra Bence. In the Bence lab, he developed a passion for research and an interest in veterinary medicine. Ian is currently pursuing his VMD/PhD at Penn.
Camille Syrett, BS
Camille is a graduate student in the Developmental, Stem Cell, and Regenerative Biology program at Penn. Originally from State College, PA, Camille attended Allegheny College where she worked with Dr. Ann Kleinschmidt for three years and completed her senior thesis studying peroxidase sequence conservation in Arabidopsis thaliana. After graduation she was a research assistant in the lab of Dr. Michael Granato in the Perelman School of Medicine at UPenn where she studied motor axon guidance cues using zebrafish as a model. In the Anguera lab Camille is interested in investigating the role of novel lncRNAs in human development and disease.
Louis Taylor, BA, MA
Louis attended Wesleyan University where he double-majored in Molecular Biology and Neuroscience. There, he worked with Dr. Amy MacQueen and completed his Master’s thesis studying the role of SUMOylation in the formation of the synaptonemal complex in meiotic S. cerevisiae. Presently he is a research assistant in our lab, and he is investigating novel X-linked noncoding RNAs in mouse and human pluripotent stem cells.
Anna is a senior at Temple University studying Biochemistry. She is currently volunteering in the lab to determine the role of long noncoding RNAs in chromosome-wide transcriptional silencing. Anna hopes to attend veterinary school in Fall 2017.
Former Lab Members:
Marianne Krammer, BS
Marianne is a CAMB predoctoral student who rotated in the lab (Fall 2014).
- Camille received a travel award to present her research at the 2016 Keystone Conference on Long noncoding RNAs (October 2015).
- Jianle was selected to give a seminar on her research at the Penn Postdoctoral Research Symposium (October 2015).
- Camille received a grant from the American Lupus Foundation (June 2015).
In the Anguera Lab, life isn't always about work. Here are some examples of how the Anguera team unwind, too.