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 X-Chromosome Inactivation, a fundamental biological event that occurs in female mammal for equalizing expression of X-linked genes between genders. The major lines of investigation in my laboratory are: (1) to understand how XCI is maintained in female lymphocyte lineages, and how this contributes to the female-biased susceptibility of lupus; (2) X-linked long noncoding RNAs important for early human development during early lineage specification. Understanding the mechanisms that control the expression and function of genes encoded on the X-chromosome will provide critical new insights into pathogenesis and, perhaps treatment of female-biased disease.
- Microscopy: RNA and DNA fluorescence in situ hybridization (FISH), immunofluorescence detection of proteins and chromatin modifications
- Cell culture of human and mouse lymphocytes, pluripotent stem cells, transformed cells
- Mouse models of autoimmunity
- Genome editing using TALENs and CRISPRs for introducing mutations of coding and noncoding genes
- Molecular biology: PCR and real-time PCR, cloning, Southern blotting, Northern blotting
XCI maintenance is different in mammalian female lymphocytes
We have discovered that the inactive X chromosome lacks the typical heterochromatic modifications in female mature naïve T and B cells. This is the first example of physiologically-relevant female somatic cells that don’t have XIST/Xist RNA, H3K27me3, macroH2A, H2A-ubiquitin, and H4K20me modifications enriched on the inactive X. We also observed that in vitro activation of T and B cells stimulates the return of XIST/Xist RNA and some heterochromatic modifications back to the inactive X.
Using loss of function assays, we identified protein factors responsible for localizing XIST/Xist RNA back to the inactive X during lymphocyte activation. YY1 and hnRNP U, known XIST RNA binding proteins, return the XIST/Xist transcripts back to the inactive X. Elucidating the molecular mechanism of XIST/Xist RNA relocalization back to the inactive X, and how this transcript initiates the return of heterochromatin modifications, will reveal how the memory of transcriptional silencing is maintained after cell division in lymphocytes.
XIST/Xist RNA localization on the inactive X is perturbed in female lupus patients
Remarkably, we have recently found that female lupus patients exhibit differences with XCI compared to healthy controls, which may explain the female bias of autoimmune disorders and why female lupus patients express higher levels of X-linked autoimmunity related genes compared to male patients. SLE-patient B cells have fewer typical XIST RNA clouds and more cells with dispersed patterns of XIST RNA or completely lacking XIST localization. Abnormal Xist RNA localization suggests partial reactivation of the inactive X-chromosome, which could increase gene expression. Using RNA FISH, we demonstrated that female lupus T and B cells frequently exhibit biallelic expression of CD40LG, CXCR3 and TLR7, which correlates with elevated expression of these genes. Moving forward, we are investigating causality of partial X-reactivation for lupus pathogenesis using mouse models. These will be the first experiments that directly connect transcription from the inactive X to lupus pathology.
LncRHOXF1: a novel X-linked long noncoding RNA that regulates the viral response during development of the human placenta
Using single cell RNA-sequencing data from human blastocyst-stage embryos, we identified a highly abundant, human-specific X-linked long noncoding RNA, called lncRHOXF1. We found that lncRHOXF1 is specifically expressed in the trophectoderm and primitive endoderm cells of human embryos and in vitro differentiated human trophectoderm progenitors. This novel long noncoding RNA, when overexpressed in human embryonic stem cells, reduces cell proliferation and increases cellular differentiation. Reducing lncRHOXF1 RNA using siRNAs increased expression of viral response genes, and resulted in higher Sendai virus replication.
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.
Penkala I, Wang J, Syrett CM, Goetzl L, López CB, Anguera MC. LNCRHOXF1: a long noncoding RNA from the X-chromosome that supporesses viral response genes during development of the early human placenta. Mol Cell Biol 2016 Apr 11 [Epub ahead of print].
Wang J, Syrett CM, Kramer MC, Basu A, Atchison ML, Anguera MC. Unusual maintenance of X chromosome inactivation predisposes female lymphocytes for increased expression from the inactive X. Proc Natl Acad Sci U S A. 2016 Mar 21.
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.
- First lab publication to make the cover! Read more...
- "LNCRHOXF1: a long noncoding RNA from the X-chromosome that suppresses viral response genes during development of the early human placenta" is now in press in Molecular Cell Biology (May 2016).
- Jianle was selected to give a talk at the 10th annual meeting of the Organization for the Society of Sex Differences (May 2016).
- Camile was selected to attend the 2016 Van Andel Research Institute summer course on Epigenomics (May 2016).
- Steve Hanes received an NIH-Merial Summer Research grant (2016).
- First publication from the Anguera Lab now in press at PNAS (March 2016).
- Montserrat received an NIH-BIRCWH Scholarship (January 2016).
- Ian Penkala placed 3rd in the 2015 Penn Vet Student Research Day presentations (March 2015).
- 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.
||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.
|| 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.
||Anna Jones, BS
|| 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.