Anguera Laboratory

XIST FISH, Anguera Lab, Penn VetOur laboratory investigates X-chromosome Inactivation, and how this epigenetic process contributes to female-biased autoimmunity.

We are investigating how female lymphocytes maintain X-chromosome Inactivation, which is an epigenetic process responsible for equalizing gene expression between sexes.  X-chromosome Inactivation silences one X-chromosome in female cells, and this process is initiated and maintained by the long noncoding RNA Xist.

Lab Mission:

  • To understand the female bias underlying autoimmune disorders such as lupus
  • To investigate how X-Chromosome Inactivation is maintained in female lymphocytes and becomes mis-regulated in female-biased autoimmune disorders
  • To investigate the function and mechanisms for novel X-linked long noncoding RNAs important for early human development
  • To identify therapeutic opportunities for correcting X-linked dosage imbalances in autoimmune disorders

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Interested in Working With Us?

We are always seeking highly motivated students and post-doctoral fellows with an interest in:

  • Epigenetics, X-chromosome Inactivation, Imprinting
  • Immunology and female-biased autoimmune disorders
  • Pluripotent stem cell biology
  • Long noncoding RNAs
  • Genetics
  • Bioinformatics

Interested post-doctoral candidates should inquire by sending e-mail to anguera@vet.upenn.edu

Interested graduate students should visit the Department of Cellular and Molecular Biology (CAMB) at UPenn or inquire by sending e-mail to: anguera@vet.upenn.edu

Contact Information

The Anguera Lab
University of Pennsylvania
School of Veterinary Medicine
Department of Biomedical Studies
Rosenthal Building, Room 304
Philadelphia, PA 19104

3800 Spruce St., Rm 390EB
Office Phone: 215.898.0567
Lab Phone: 215.898.9541

FAX: 215.573.6810

anguera@vet.upenn.edu

X-Chromosome Inactivation (XCI) is one of theCalico cats demonstrate x-mosaicism 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.

The X-chromosome contains about 1000 genes with roles in development, reproduction, metabolism, and immunity. Mammalian females (including humans) are healthier and have longer life-spans than males. Females have better survival rates than males from illnesses caused by infectious diseases, injury, or sepsis. The X-chromosome plays an important role in the hyper-responsive female immune system because this chromosome has the highest density of immune-related genes. Females are also more susceptible to a number of autoimmune diseases, suggesting that the enhanced immune function may be related to the loss of self-tolerance.

The X-chromosome contains about 1000 genes with roles in development, reproduction, metabolism, and immunity. Mammalian females (including humans) are healthier and have longer life-spans than males. Females have better survival rates than males from illnesses caused by infectious diseases, injury, or sepsis. The X-chromosome plays an important role in the hyper-responsive female immune system because this chromosome has the highest density of immune-related genes. Females are also more susceptible to a number of autoimmune diseases, suggesting that the enhanced immune function may be related to the loss of self-tolerance.

XCI occurs during early female embryonic development, and is initiated by expression of Xist RNA which recruits heterochromatic complexes for transcriptional silencing.  Xist RNA and heterochromatin modifications associate with the inactive X chromosome with each cell division, and can be visualized using fluorescence microscopy. Xist RNA also functions to maintain XCI in somatic cells.

We recently discovered that XCI is maintained differently in female lymphocytes. Naïve T and B cells, which are quiescent, lack Xist RNA and heterochromatin marks on the inactive X. After lymphocyte stimulation, these epigenetic modifications re-localize back to the inactive X, as the cells re-enter the cell cycle and actively proliferate. This dynamic mechanism for XCI maintenance is unique to immune cells, and our lab investigates the molecular details of this process. The major areas of investigation in our lab are: (1) to understand how XCI is maintained in female lymphocytes, and how this contributes to female-biased autoimmunity; (2) to investigate epigenetic mechanisms involving long noncoding RNAs during early human development and placental progenitors.

Penn Vet banner MCA 2016 PNAS

Research Techniques

  • 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


Mouse Sic region contains chromatin modifications

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.

naive vs activated human T Cells


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.

 XIST RNA

 

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.

  Anguera - EVT cyto lncRHOXF1 RNA

Syrett CM, Sindhava V, Hodawadekar S, Myles A, Liang G, Zhang Y, Nandi S, Cancro M, Atchison M, Anguera MC. Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells. PLoS Genet. 2017 Oct 9;13(10):e1007050. doi: 10.1371/journal.pgen.1007050. [Epub ahead of print] PMID: 28991910

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.


  • Camille’s paper was accepted in Plos Genetics.
  • Camille's NRSA predoctoral fellowship (F31) was awarded from the NIH! (August 2017).
  • Summer lab axe throwing competition: Anguera lab against the Lengner lab (Aug 2017).
  • Camille received a travel award to attend the EMBO Workshop on B cells in health and disease in Girona, Spain (August 2017).
  • The Anguera Lab welcomes its newest member, Axel Rosa the axolotl (August 2017).
  • Welcome to the Lab, Bam Paneru! (July 2017).
  • Camille receives a Goldie Simon Preceptorship award from the Lupus Foundation of America (June 2017).
  • Welcome to the Lab, Isabel Sierra! Isabel is a CAMB-DSRB PhD Student (June 2017).
  • Welcome Donavon Sandoval-Heglund! Don is a Penn Undergraduate and is a part of the PURM summer research program (May 2017).
  • Erin Achilles (Vet ’19) received an NIH-Merial Summer Research grant to work in the lab (May 2017).
  • Steve received the Morris Animal Foundation summer research fellowship, Congrats Steve! (May 2017).
  • Welcome to the Anguera Lab, Anna Martin! Anna received an HHMI career fellowship grant! She will be taking a year off of Vet school to do research full-time in the Anguera & Atchison labs (April 2017).
  • Jianle’s JOVE methods paper is now in press (April 2017).
  • Camille was appointed to the Developmental Biology Training Grant (T32) at Penn (June 2016).
  • 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

  • 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).

Team Anguera

In the Anguera Lab, life isn't always about work. Here are some examples of how the Anguera team unwind, too.

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 anguera party 6
Anguera Lab Members
  • Assistant Professor, Biomedical Sciences
  • Head of Anguera Lab
  • 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.
  • Isabel Sierra, Penn VetIsabel Sierra, BS
  • GraduateStudent
  • Isabel, born and raised in Philadelphia, is a PhD student in the Developmental, Stem Cell, and Regenerative Biology graduate group. After graduating from Drexel University in 2013, she took a research assistant position at the Wistar Institute in a lab studying the histone H3.3 deposition pathway and it's involvement in pediatric glioblastoma. After three years, she applied to graduate school with the hopes of pursuing her two favorite topics: developmental biology and epigenetics. Outside of the lab Isabel likes to go to shows and explore the food scene around Philly.
  • Bam Dev Paneru, PhD, Penn VetBam Dev Paneru, PhD
  • Post-doctoral Fellow
  • Bam Paneru holds BS and MS degree in Microbiology, and PhD in Molecular Biosciences. During his PhD, he studied the role of non-coding RNAs (long non-coding RNAs and microRNAs) in immune response, growth and fillet quality traits in commercial rainbow trout strain. As a postdoctoral researcher, he is currently investigating Xi-maintenance in human B cell and its association with lupus.  
  • CamilleSyrettCamille 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 Martin, BS
  • Penn Vet student
  •  Anna is a Penn VMD student currently working in the Anguera lab.  She is taking a year off of Vet school to work on a research project investigating autoimmunity and the inactive X chromosome.

  • Steve Hanes, BS
  • Penn Vet student
  • Steve is a Penn Vet VMD student, who has worked two summers in the Anguera lab.
  • Don Sandoval-Anguera lab 160Donovan Sandoval
  • Penn Undergraduate Student
  • Don is a Penn undergraduate hailing from Southern California, and studies biology and nutrition. In the lab he's currently investigating X-inactivation in quiescent cell populations. When he finds the time, he enjoys going on long meandering runs, swimming, and baking.
Former Anguera Lab Members
Jianle Wang, PhD, Penn Vet            

Jianle Wang, PhD

 Anna Jones, Anguera Lab, Penn Vet

Anna Jones, BS

Ian Penkala, Anguera Lab 

Ian Penkala, BS

MarianneKrammer

Marianne Krammer, BS

TaylorBryant

TaylorBryant, BS