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.