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 long noncoding RNAs expressed during early human development. 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
Human-specific mechanisms of X-Chromosome Inactivation
Our understanding of XCI is mostly from mouse models, yet it’s still unclear how XCI is regulated in humans. The XIST gene, the master regulator of XCI, is well conserved between mice and humans, unlike its neighboring long noncoding RNAs. We seek to determine how XIST RNA cooperates with chromatin modifying complexes to convert the human active X-chromosome into a heterochromatic inactive X.
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, and determining how these transcripts function in vivo.
X-linked long noncoding RNAs expressed in male germ cells
We are investigating the function and mechanism of one X-linked long noncoding RNA Tsx (located within the X-Inactivation Center) using mouse models. The Tsx gene is predominantly expressed in testes, escapes meiotic sex chromosome inactivation in pachytene spermatocytes, and exists in various splice forms that lack protein coding potential. Deletion of the Tsx transcript in mice impacts spermatocytes, the proliferation and differentiation of mouse embryonic stem cells, and alters expression of Xist and Tsix RNAs during in vitro differentiation. We are investigating the underlying mechanisms that result in these phenotypes and the protein binding partners that interact with Tsx RNA.
Pachtene spermatocyte with synapsed autosomes (stained in green with Sycp-1). The unsynapsed X and Y chromosomes (XY body) display exclusive g-H2Ax localization (red). Tsx deletion results in apoptosis of pachytene spermatocytes. Testes section of Tsx null mice stained with Sycp 1 (red) and apoptotic cells (TUNEL; green).
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 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.
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. He wants to pursue DVM/PhD program and study tissue engineering in the future. He is currently studying human long noncoding RNAs expressed in human pluripotent stem cells and in vitro derived trophoblast progenitors.
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.