Terry Magnuson, PhD

TMagnuson Sarah Graham Kenan Professor
Chair, Department of Genetics

Vice Dean for Research, School of Medicine
Program Director, Cancer Genetics, Lineberger Comprehensive Cancer Center

 

Research Interests

Key words: Mammalian Genetics/Genomics/Development/Mouse Models of Human Disease

A new model for random X chromosome inactivation: X chromosome inactivation (XCI) reduces the number of    actively transcribed X chromosomes to one per diploid set of autosomes, allowing for dosage equality between the sexes. In eutherians, the inactive X chromosome in XX female embryos is randomly selected. The mechanisms for determining both how many X chromosomes are present and which to inactivate are unknown. To understand these mechanisms, researchers have developed models based on data collected from experiments using X chromosome mutations and transgenes. However, considerable new data have been generated and no one model can account for all of the data generated. Thus, we have developed a novel model, called the feedback model, which combines aspects of older models with the implications of the recent XCI data. The feedback model consists of three major components: a signaling feedback loop, a mechanism for choice, and a description of X-linked loci that directly affect XCI initiation.

EED is required for PRC2 complex stability and function: The PRC2 polycomb group complex is composed of several proteins including the histone methyltransferase (HMTase) EZH2, the WD-repeat protein EED, and the Zn-finger protein SUZ12.  EZH2 is the catalytic subunit that methylates histone H3 on lysine 27 (H3K27), which serves as an epigenetic mark mediating silencing of gene transcription.  H3K27 can be mono-, di-, or trimethylated (H3K27me1, H3K27me2, and H3K27me3, respectively).  Hence, either PRC2 must be regulated so as to add one methyl group to certain nucleosomes but two or three to others, or distinct complexes must be responsible for H3K27me1, me2, and me3.  Consistent with the latter possibility, H3K27me2 and me3, but not me1, are absent in Suz12-/- embryos, which lack both SUZ12 and EZH2 protein.  Mammalian proteins required for H3K27me1 have not been identified.  We showed that in the absence of EED, EZH2 and SUZ12 are degraded and the PRC2 complex is destabilized. Furthermore, unlike SUZ12 and EZH2, EED is required not only for H3K27me2 and me3 but also for global H3K27me1.  These results indicated that EED is essential for PRC2 function.

X inactivationPRC2 Complex is Dispensable for Initiation of Random X-Chromosome Inactivation: The Polycomb group (PcG) proteins are thought to silence gene expression by modifying chromatin.  The Polycomb Repressive Complex 2 (PRC2) plays an essential role in mammalian X-chromosome inactivation (XCI), a model system to investigate heritable gene silencing.  In the mouse, two different forms of XCI occur.  In the pre-implantation embryo, all cells undergo imprinted inactivation of the paternal X-chromosome.  During the peri-implantation period, cells destined to give rise to the embryo proper erase the imprint and randomly inactivate either the maternal or the paternal X-chromosome (Xp); extra-embryonic cells, on the other hand, maintain imprinted XCI of the Xp.  PRC2 proteins are enriched on the inactive-X during early stages of both imprinted and random XCI.  It is therefore thought that PRC2 contributes to the initiation of XCI.  Mouse embryos lacking the essential PRC2 component EED harbor defects in the maintenance of imprinted XCI in differentiating trophoblast cells.  Assessment of PRC2 requirement in the initiation of XCI, however, has been hindered by the presence of maternally-derived proteins in the early embryo. Here we show that Eed-/- embryos initiate and maintain random XCI despite lacking any functional EED protein prior to the initiation of random XCI.  Thus, despite being enriched on the Xi, PcGs appear to be dispensable for the initiation and maintenance of random XCI.  These results highlight the lineage- and differentiation state-specific requirements for PcGs in XCI and argue against a function for PcGs in the formation of the facultative heterochromatin of the inactive X-chromosome.

PRC2PRC2 Protects the inactive X chromosome from differentiation-induced reactivation: The Polycomb group (PcG) encodes an evolutionarily conserved set of chromatin-modifying proteins that are thought to maintain cellular transcriptional memory by stably silencing gene expression.  In mouse embryos mutated for the PcG protein Eed, X-chromosome inactivation (XCI) is not stably maintained in extra-embryonic tissues.  Eed is a component of a histone-methyltransferase complex that is thought to contribute to stable silencing in undifferentiated cells due to its enrichment on the inactive X-chromosome (Xi) in cells of the early mouse embryo and in stem cells of the extra-embryonic trophectoderm lineage.  We have demonstrate that the Xi in Eed-/- trophoblast stem (TS) cells and in cells of the trophectoderm-derived extra-embryonic ectoderm in Eed-/- embryos remains transcriptionally silent, despite lacking the PcG-mediated histone modifications that normally characterize the facultative heterochromatin of the Xi.  While undifferentiated Eed-/- TS cells maintained XCI, reactivation of the Xi occurred when these cells were differentiated.  These results indicate that PcG complexes are not necessary to maintain transcriptional silencing of the Xi in undifferentiated stem cells.  Instead, PcG proteins appear to propagate cellular memory by preventing transcriptional activation of facultative heterochromatin during differentiation.

PRC2 is dispensable for maintenance of embryonic stem cell pluripotency: The three core components of PRC2, Eed, Ezh2, and Suz12, are highly expressed in embryonic stem (ES) cells where they are have been postulated to repress developmental regulators and thereby prevent differentiation to maintain the pluripotent state. We performed gene expression and chimera analyses on low and high passage Eednull ES cells to determine whether PRC2 is required for the maintenance of pluripotencym.  We found that, although developmental regulators are overexpressed in Eednull ES cells, both low and high passage cells are functionally pluripotent as determined by their ability to contribute to differentiated derivatives of all three germ layers in chimeric embryos.  We hypothesize that they are pluripotent because they maintain expression of critical pluripotency factors.  Given that EED is required for stability of EZH2, the catalytic subunit of the complex, these data suggest that PRC2 is not necessary for the maintenance of the pluripotent state in ES cells.  We propose a positive-only model of embryonic stem cell maintenance, where positive regulation of pluripotency factors is sufficient to mediate stem cell pluripotency.

Regulation of Mammalian Autosomal Imprinting: Studies of imprinted gene clusters have served as a useful paradigm in unraveling the complex mechanisms of gene regulation that occur during development and disease. While covalent modifications to histones and DNA continue to be a subject of intense study, a growing number of observations suggest that higher order chromatin structure and positioning within the nucleus play vital roles in the regulation of gene expression. Since the differential expression patterns and epigenetic modifications of most imprinted clusters are known, these genes provide a convenient system to study the relationship between epigenetic gene regulation and positioning within the nucleus. We have analyzed the localization of a number of imprinted gene clusters in trophoblast stem (TS) cell nuclei, and found that in many cases, single alleles are associated with either the nuclear or nucleolar peripheries. Moreover, some of these regions undergo dramatic repositioning during the course of TS cell differentiation. Intriguingly, undifferentiated TS cells that lack the Polycomb Repressive Complex 2 component responsible for maintaining the repressed state of these alleles, (Eed), recapitulate their pattern of localization observed in differentiated wild-type TS cells. Our data not only demonstrate that the nucleus undergoes an ordered reorganization during differentiation, but that the positioning of a region is closely tied with its epigenetic modifications.

The Chromatin-Remodeling Enzyme BRG1 Plays an Essential Role in Primitive Erythropoiesis and Vascular Development: ATP-dependent chromatin-remodeling complexes contribute to the proper temporal and spatial patterns of gene expression in mammalian embryos and therefore play important roles in a number of developmental processes.  SWI/SNF-like chromatin-remodeling complexes utilize one of two different ATPases as their catalytic subunits: brahma (BRM, also known as Smarca2) and brahma-related gene 1 (BRG1, also known as Smarca4).  We have conditionally deleted a floxed Brg1 allele with a Tie2-Cre transgene, which is expressed in developing hematopoietic and endothelial cells.  Brg1mutant embryos die at midgestation from anemia since mutant primitive erythrocytes fail to transcribe embryonic a- and b-globins and subsequently undergo apoptosis.  Additionally, vascular remodeling of the extraembryonic yolk sac is abnormal in Brg1mutant embryos.  Importantly, Brm deficiency does not exacerbate the erythropoietic or vascular abnormalities found in Brg1 mutant embryos, implying that Brg1-containing SWI/SNF-like complexes rather than Brm-containing complexes play a critical role in primitive erythropoiesis and in early vascular development.

Drosophila CTCF is required for Fab-8 enhancer blocking activity in S2 cells: CTCF is a conserved transcriptional regulator with binding sites in DNA insulators identified in vertebrates and invertebrates.  The Drosophila Abdominal-B locus contains CTCF binding sites in the Fab-8 DNA insulator.  Previous reports have shown that Fab-8 has enhancer blocking activity in Drosophila transgenic assays.  We now confirm the enhancer blocking capability of the Fab-8 insulator in stably transfected Drosophila S2 cells and show this activity depends on the Fab-8 CTCF binding sites.  Furthermore, knockdown of Drosophila CTCF by RNAi in our stable cell lines demonstrates that CTCF itself is critical for Fab-8 enhancer blocking.

Gene Expression Profiling in the Mouse Brain to Model Polygenic Heritable Autistm: Autism is a highly heritable neurodevelopmental disorder with no consistent neuropathological hallmarks, diagnosed through behavioral inventory rather than a biomarker. Monogenic inheritance appears to underlie only a fraction of cases, leaving the vast majority explained by complex polygenic inheritance. To investigate the molecular and genetic nature of autism, we have modeled a core symptom of autism, social deficit, in inbred strains of mice. Using gene expression profiles in the brain and behavioral phenotyping relevant to social deficits, we have identified basic cellular functions that may be dysregulated in the brain in autism. Correlations between these two complex datasets indicate that cell proliferation, morphology, and signaling may be perturbed in disorders with altered social behavior. Furthermore, networks of gene interaction revealed by expression profiling illustrate underlying molecular biology common to monogenic and polygenic forms of autism.

 


Publications

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Lab Members

  • Dominic Ciavatta
Research Assistant Professor Email
  • Mauro Calabrese
Postdoctoral Fellow Email
  • Ron Chandler
Postdoctoral Fellow Email
  • Andrew Fedoriw
Postdoctoral Fellow Email
  • Yuna Kim
Postdoctoral Fellow Email
  • Weipeng Mu
Postdoctoral Fellow Email
  • Joshua Mugford
Postdoctoral Fellow Email
  • Nicholas Osborne
Postdoctoral Fellow Email
  • Michael Pohlers
Postdoctoral Fellow Email
  • Karl Shpargel
Postdoctoral Fellow Email
  • Joshua Starmer
Postdoctoral Fellow Email
  • Jeff Gray
Graduate Student Email
  • Rex Williams
Graduate Student Email
  • Zhihong Lin
Technician Email
  • Della Yee
Technician Email