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Research in the Wu Laboratory

The presence of homology can have surprising and far-reaching consequences for gene activity and chromosome positioning, the totality of which define the rapidly growing field of homology effects (1-4).  Good examples of the impact of homology can be found among mammals, plants, insects, ciliates, and fungi, and range from gene activation or repression to changes in DNA sequence, epigenetic marking, and interchromosomal interactions, including the phenomena of transvection (1, 3, 5-10), paramutation (11-13), repeat-induced point mutation (14-16), X-inactivation (17-19), parental imprinting (20, 21) random monoallelism (22, 23), and other forms gene silencing (e.g., 24). The implications are significant – a capacity to sense homology may be a common attribute of genes.  Importantly, the critical roles played by homology effects in gene regulation and inheritance make them key players in development. With an eye toward the implications of homology effects for human health, we have worked in the following areas, developing new technologies where we can:

Transvection

Homolog pairing

Structural heterozygosity

Loss-of-heterozygosity (LOH)

Polycomb group (PcG) genes

Chromosome segregation

Ultraconserved elements (UCEs)

Technology development:
RMCE
High-throughput FISH
Oligopaints
 

***Note***

The Wu laboratory also houses the Personal Genetics Education project (pgEd), which promotes conversations about personal genetics and its implications for society. Click here for the pgEd website.
 
 

Transvection

Some of the most potent homology effects arise through transvection, in which the pairing of homologous chromosomal regions leads to changes in the activity of those regions (1, 5-7, 25). First described in Drosophila, where homologous chromosomes are paired throughout development in somatic cells (26, 27), transvection is a striking example of how interchromosomal interactions can influence gene expression. We have studied two forms of transvection at the yellow gene of Drosophila. In one, enhancers of one allele act in trans on the promoter of a paired homolog, with the cis-trans choice being determined by the integrity of the cis-linked promoter (28-34). In the other, pairing-mediated changes in gene topology are believed to enable enhancers to bypass a chromatin insulator (26). In particular, we have proposed that structural heterozygosity of allelic regions can cause lengths of chromatin to be topologically constrained, perhaps even looped out, and then subjected to changes in gene expression (26, 35).

Homolog pairing

Homolog pairing and the impact it has on gene activity is just one example of how chromosome positioning and interchromosomal interactions influence gene expression (36-39). To better understand this level of gene regulation, we have been engaged in a search for the genes that are involved in the positioning of chromosomes, with a primary focus on homolog pairing (40). Thus far, we have found Topoisomerase II to be important for pairing in Drosophila (41). Using a method we developed for high-throughput FISH, we are now conducting whole-genome screens for pairing genes in Drosophila and, when Oligopaints become available, will move on to a screen of the human genome. (Click here to read more about our high-throughput FISH and Oligopaint technologies.)

Structural heterozygosity

How sensitive is the genome to structural heterozygosity? In particular, can the pairing of homologous chromosomes during meiosis detect small structural differences and, if so, how small a difference can the genome detect? Studies in fungi of a phenomenon called meiotic silencing of unpaired DNA (MSUD) indicate that deletions and insertions on the order of a few hundred base pairs can elicit a response of the genome in the germline (42, 43). Is the animal genome as sensitive? These questions are of particular importance because of the hundreds of polymorphic copy number variants (CNVs) in the human population (44-46). We are exploring this issue by examining the impact of heterozygous deletions during meiosis in C. elegans and mice.

Loss-of-heterozygosity (LOH)

Some mechanisms can cause a diploid cell to be functionally haploid. These include X-inactivation (17-19), parental imprinting (20, 21), mononallelism (22, 23), and loss-of-heterozygosity (LOH) through mitotic recombination (47-50), to mention just a few examples. We are exploring these phenomena in mammalian and Drosophila cells. For example, we have revisted current models for random X-inactivation, which is believed to involve a random choice between the maternal and paternal X chromosomes. In particular, we have proposed two alternative explanations for X-inactivation (51). One considers the model of Amar Klar for mating type switching in S. pombe (52), and suggests that random X-inactivation need not be random if there is involvement of an asymmetric strand-specific mark on one of the X chromosomes. The other is consistent with random choice, but not one between two X chromosomes. In either case, our models suggest that the two X chromosomes communicate their status, which is consistent with the X-inactivation centers coming into physical proximity at some point during the process of X-inactivation (53-55).

Polycomb group (PcG) genes

Some members of the Polycomb group (PcG) genes, which encode chromatin proteins, are important for pairing-associated phenotypes (56-58). We have studied two such genes, Psc and Su(z)2, where we have identified functional domains and obtained evidence for intramolecular regulation (35, 59, 60). We are now exploring how Psc and Su(z)2 control gene activity. As Psc is a core component of the PRC1 chromatin complex, we are particularly interested in how Psc and Su(z)2 function within the context of a complex.

Chromosome segregation

We have been interested extending a handful of studies in Drosophila and mice showing that the segregation of sister chromatids can be nonrandom after mitotic recombination in some tissues (61-65). To this end, we collaborated in the development of a new method, called Twin Spot Generator (TSG) (66), which resembles the technology of Mosaic Analysis with Double Markers (MADM) (67). Our goal is to further document these curious observations of nonrandom sister chromatid segregation and then identify the genes that control this type of chromosome behavior.

Ultraconserved elements (UCEs)

The perfect conservation of ultraconserved elements (UCEs) between the reference genomes of human, mouse, and rat presents one of the most enigmatic observations to emerge from genome sequencing (68). In brief, this level of conservation cannot be easily explained by known protein-coding or regulatory functions. Furthermore, although extreme conservation is believed to imply importance in function, it appears that at least some of these elements can be deleted from the mouse without consequence (69), indicating that there are sequences in the human genome that have not been permitted to vary for 300 million years and yet are dispensable. We propose that ultraconservation occurs because the maternal and paternal copies of each UCE undergo a process of sequence comparison in which change in sequence or copy number leads to loss-of-fitness (70, 71). Such a model would drive ultraconservation as well as function to maintain genome integrity. We are now conducting studies to test this model.

Technology development

RMCE using φC31 integrase: Transgene studies are complicated by position effects arising from the chromosomal regions flanking insertion sites.  Such position effects also preclude straightforward comparisons of transgenes located in different genomic regions. The complications are further compounded in studies of homolog pairing, where studies often require transgenes to be placed in allelic positions. To address these issues, we developed a technique that uses φC31 integrase, which is functional in Drosophila (72), to place transgenes into predetermined sites via cassette exchange (73-74). Current work in this area is being spearheaded by Jack Bateman, who is now a faculty member at Bowdoin College (http://www.bowdoin.edu/faculty/j/jbateman/). (Click here for the RMCE User Website.)

High-throughput FISH: We have developed a technology that enables a single individual to conduct thousands of FISH assays per day in 384-well plates. This technology enables FISH-based whole-genome screens for genes that are involved in chromosome positioning and behavior, nuclear organization, and genome structure. Currently, we are using this technology to screen the Drosophila genome for genes involved in somatic homolog pairing.

Oligopaints: We are also developing a technology, called Oligopaints, in order to lower the cost of generating FISH probes. Our goal is to enable high-throughput FISH studies that target single-copy genomic regions as well as improve the capacity of researchers to trace the path of chromosomes through the nucleus. If successful, Oligopaints could also serve to make clinical karyotyping affordable enough to become as routine as a blood test. Our work in this area has benefitted from the contributions of IDT, Olympus, and MYcroarray.

References

  1.   Wu CT, Morris JR. Transvection and other homology effects. Curr Opin Gen Dev. 1999 Dev. 9:237-246 (Review) PMID: 10322135.

  2.   Cogoni C. Unifying homology effects. Nat Genet. 2002 30:245-6 (Review) PMID: 11919555.

  3.   Wu C-t, Dunlap JC.  Homology effects: The difference between 1 and 2.  Adv Gen. 2002; 46: xvii-xxiii (Preface) PMID: 12136788.

  4.   Catalanotto C, Nolan T, Cogoni C. Homology effects in Neurospora crassa. FEMS Microbiol Lett. 2006 254:182-9 (Review) PMID: 16445744.

  5.   Lewis EB. The theory and application of a new method of detecting chromosomal rearrangements in Drosophila melanogaster. Am Nat. 1954 88:225-239.

  6.   Duncan IW. Transvection effects in Drosophila. Annu Rev Genet. 2002 36:521-556 (Review) PMID: 12429702.

  7.   Kennison JA, Southworth JW. Transvection in Drosophila. Adv Genet. 2002 46:399-420 (Review) PMID: 11931232.

  8.   Grant-Downton RT, Dickinson HG. Plants, pairing and phenotypes--two's company? Trends Genet. 2004 20:188-195 (Review) PMID: 15041173.

  9.   McKee BD. Homologous pairing and chromosome dynamics in meiosis and mitosis. Biochim Biophys Acta. 2004 1677:165-180 (Review) PMID: 15020057.

10.   Zickler D. From early homologue recognition to synaptonemal complex formation. Chromosoma. 2006 115:158-174 (Review) PMID: 16570189.

11.   Arteaga-Vazquez MA, Chandler VL. Paramutation in maize: RNA mediated trans-generational gene silencing. Curr Opin Genet Dev. 2010 20:156-63 (Review) PMID: 20153628, PMCID: PMC2859986.

12.   Hollick JB. Paramutation and development. Annu Rev Cell Dev Biol. 2010 26:557-79 (Review) PMID: 19575656.

13.   Stam M. Paramutation: a heritable change in gene expression by allelic interactions in trans. Mol Plant. 2009 2:578-88 (Review) PMID: 19825640.

14.   Selker EU. Repeat-induced gene silencing in fungi. Adv Genet. 2002 46:439-450 (Review) PMID: 11931234.

15.   Galagan JE, Selker EU. RIP: the evolutionary cost of genome defense. Trends Genet. 2004 20:417-23. (Review) PMID: 15313550.

16.   Selker EU. Genome defense and DNA methylation in Neurospora. Cold Spring Harb Symp Quant Biol. 2004 69:119-24 (Review) PMID: 16117640.

17.   Augui S, Nora EP, Heard E. Regulation of X-chromosome inactivation by the X-inactivation centre. Nat Rev Genet. 2011 12:429-42 (Review) PMID: 21587299.

18.   Lee JT. The X as model for RNA's niche in epigenomic regulation. Cold Spring Harb Perspect Biol. 2010 2:a003749 (Review) PMID: 20739414.

19.   Morey C, Avner P. Genetics and epigenetics of the X chromosome. Ann N Y Acad Sci. 2010 1214:E18-33 (Review) PMID: 21382199.

20.   Li Y, Sasaki H. Genomic imprinting in mammals: its life cycle, molecular mechanisms and reprogramming. Cell Res. 2011 21:466-73 (Review) PMID: 21283132.

21.   Raissig MT, Baroux C, Grossniklaus U. Regulation and flexibility of genomic imprinting during seed development. Plant Cell 2011 23:16-26 (Review) PMID: 21278124, PMCID: PMC3051244.

22.   Gimelbrant A, Hutchinson JN, Thompson BR, Chess A. Widespread monoallelic expression on human autosomes. Science. 2007 318:1136-40. PMID: 18006746.

23.   Yang PK, Kuroda MI. Noncoding RNAs and intranuclear positioning in monoallelic gene expression. Cell 2007 128:777-86 (Review) PMID: 17320513.

24.   Normark BB. The evolution of alternative genetic systems in insects. Annu Rev Entomol. 2003 48:397-423 (Review) PMID: 12221039.

25.   Wu C-t.  Transvection, nuclear structure, and chromatin proteins. J Cell Biol. 1993 120:587-90 (Review) PMID: 8425891, PMCID: PMC2119552.

26.   Stevens NM. A study of the germ cells of certain Diptera with reference to the heterchromosomes and the phenomena of synapsis. J Exp Zool. 5:359-74.

27.   Metz, CW. Chromosome studies on the Diptera: II. The paired association of chromosomes in the Diptera, and its significance. J Exp Zool. 21:213-79.

28.   Morris JR, Chen J-l, Geyer PK, Wu C-t.  Two modes of transvection: Enhancer action in trans and bypass of a chromatin insulator in cis.  Proc Natl Acad Sci USA 1998 95:10740-5 PMID: 9724774, PMCID: PMC27965. 

29.   Morris JR, Chen J-l, Filandrinos ST, Dunn RC, Fisk R, Geyer PK, Wu C-t.  An analysis of transvection at the yellow locus of Drosophila melanogaster.  Genetics 1999 151:633-51 PMID: 9927457, PMCID: PMC1460495. 

30.   Morris JR, Geyer PK, Wu C-t.  Core promoter elements can regulate transcription on a separate chromosome in trans.  Genes Dev 1999 13:253-8 PMID: 9990850, PMCID: PMC316431.

31.   Chen J-L, Huisinga KL, Viering MM, Ou SA, Wu C-t, Geyer PK.  Enhancer action in trans is permitted throughout the Drosophila genome.  Proc Natl Acad Sci USA 2002 99:3723-8 PMID: 11904429, PMCID: PMC122591.

32.   Morris JR, Petrov DA, Lee AM, Wu C-t.  Enhancer choice in cis and trans: role of the promoter.  Genetics 2004 167:1739-47 PMID: 15342512, PMCID: PMC1471007.

33.   Lee AM, Wu C-t.  Enhancer-promoter communication at the yellow gene of Drosophila melanogastor: Diverse promoters participate in and regulate trans interactions.  Genetics 2006 174:1867-80 PMID: 17057235, PMCID: PMC1698615.

34.   Ou SA, Chang E, Lee S, So K, Wu C-t, Morris JR. Effects of chromosomal rearrangements on transvection at the yellow gene of Drosophila melanogaster. Genetics 2009; 183:483-96 PMID: 19667134, PMCID: PMC2766311.

35.   Wu C-t, Howe M.  A genetic analysis of the Suppressor 2 of zeste complex of Drosophila melanogaster.  Genetics 1995 140:139-81 PMID: 7635282, PMCID: PMC1206544. 

36.   Phillips JE, Corces VG. CTCF: master weaver of the genome. Cell. 2009 137:1194-211 (Review) PMID: 20462379, PMCID: PMC3040116.

37.   Hübner MR, Spector DL. Chromatin dynamics. Annu Rev Biophys. 2010 39:471-89 (Review) PMID: 20462379, PMCID: PMC2894465.

38.   Schoenfelder S, Clay I, Fraser P. The transcriptional interactome: gene expression in 3D. Curr Opin Genet Dev. 2010 20:127-33 (Review) PMID: 20211559.

39.   Williams A, Spilianakis CG, Flavell RA. Interchromosomal association and gene regulation in trans. Trends Genet. 2010 26:188-97 (Review) PMID: 20236724, PMCID: PMC2865229.

40.   Bateman JR, Wu C-t. A genome-wide survey argues that every zygotic gene product is dispensable for the initiation of somatic homolog pairing in Drosophila. Genetics 2008; 180:1329-42. PMID: 18791221, PMCID: PMC2581938.

41.   Williams BR, Bateman JR, Novikov ND, Wu C-t. Disruption of topoisomerase II perturbs pairing in Drosophila cell culture. Genetics 2007 177:31-46 PMID: 17890361, PMCID: PMC2013714.

42.   Burgoyne PS, Mahadevaiah SK, Turner JM. The consequences of asynapsis for mammalian meiosis. Nat Rev Genet. 2009 10:207-16 (Review) PMID: 19188923.

43.   Kelly WG, Aramayo R. Meiotic silencing and the epigenetics of sex. Chromosome Res. 2007 15:633-51 (Review) PMID: 17674151.

 

44.   Girirajan S, Eichler EE. Phenotypic variability and genetic susceptibility to genomic disorders. Hum Mol Genet. 2010 19(R2):R176-87 (Review) PMID: 20807775, PMCID: PMC2953748.

45.   Lee C, Scherer SW. The clinical context of copy number variation in the human genome. Expert Rev Mol Med. 2010 12:e8 (Review) PMID: 20211047.

46.   Alkan C, Coe BP, Eichler EE. Genome structural variation discovery and genotyping. Nat Rev Genet. 2011 12:363-76 (Review) PMID: 21358748.

47.   Bacolod MD, Schemmann GS, Giardina SF, Paty P, Notterman DA, Barany F. Emerging paradigms in cancer genetics: some important findings from high-density single nucleotide polymorphism array studies. Cancer Res. 2009 69:723-7 (Review) PMID: 19155292.

48.   Lapunzina P, Monk D. The consequences of uniparental disomy and copy number neutral loss-of-heterozygosity during human development and cancer. Biol Cell. 2011 103:303-17 (Review) PMID: 21651501.

49.   Lindstrom DL, Leverich CK, Henderson KA, Gottschling DE. Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae. PLoS Genet. 2011 7:e1002015. PMID: 21436897, PMCID: PMC3060066.

50.   Makishima H, Maciejewski JP. Pathogenesis and consequences of uniparental disomy in cancer. Clin Cancer Res. 2011 17:3913-23 (Review) PMID: 21518781.

51.   Wu C-t, Williams BR.  Does random X-inactivation reflect random choice between two X chromosomes?  Genetics 2004 167:1525-8 PMID: 15280260, PMCID: PMC1470940.

52.   Klar AJ. Lessons learned from studies of fission yeast mating-type switching and silencing. Annu Rev Genet. 2007 41:213-36 (Review) PMID: 17614787.

53.   Bacher CP, Guggiari M, Brors B, Augui S, Clerc P, Avner P, Eils R, Heard E. Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nat Cell Biol. 2006. 8:293-9. PMID: 16434960.

54.   Xu N, Tsai CL, Lee JT. Transient homologous chromosome pairing marks the onset of X inactivation. Science. 2006. 311:1149-52. PMID: 16424298.

55.   Anguera MC, Sun BK, Xu N, Lee JT. X-chromosome kiss and tell: how the Xs go their separate ways. Cold Spring Harb Symp Quant Biol. 2006;71:429-37 (Review) PMID: 17381325.

56.   Wu C-t, Jones RS, Lasko PF, Gelbart WM.  Homeosis and the interaction of zeste and white in Drosophila.  Mol Gen Genet 1989 218:559-64 PMID: 2511424.

57.   Kassis JA. Pairing-sensitive silencing, polycomb group response elements, and transposon homing in Drosophila. Adv Genet. 2002 46:421-38 (Review) PMID: 11931233.

58.   Brown JL, Kassis JA. Spps, a Drosophila Sp1/KLF family member, binds to PREs and is required for PRE activity late in development. Development. 2010 137:2597-602 PMID: 20627963.

59.   King IFG, Emmons RB, Francis NJ, Wild B, Müller J, Kingston RE, Wu C-t.  Analysis of a Polycomb Group protein defines regions that link repressive activity on nucleosomal templates to in vivo function.  Mol Cell Biol 2005 25:6578-91. PMID: 16024794, PMCID: PMC1190323.

60.   Emmons RB, Genetti H, Filandrinos S, Lokere J, Wu C-t. Molecular genetic analysis of Suppressor 2 of zeste identifies key functional domains. Genetics 2009; 182:999-1013. PMID: 19528329, PMCID: PMC2728886.

61.   Pimpinelli S, Ripoll P. Nonrandom segregation of centromeres following mitotic recombination in Drosophila melanogaster. Proc Natl Acad Sci USA. 1986 83:3900-3 PMID: 3086868, PMCID: PMC323632.

62.   Beumer KJ, Pimpinelli S, Golic KG. Induced chromosomal exchange directs the segregation of recombinant chromatids in mitosis of Drosophila. Genetics 1998 150:173-88 PMID: 9725837, PMCID: PMC1460302.

63.   Liu P, Jenkins NA, Copeland NG. Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells. Nat Genet. 2002 30:66-72 PMID: 11740496.

64.   Armakolas A, Klar AJ. Cell type regulates selective segregation of mouse chromosome 7 DNA strands in mitosis. Science. 2006 311:1146-9 PMID: 16497932.

65.   Armakolas A, Klar AJ. Left-right dynein motor implicated in selective chromatid segregation in mouse cells. Science 2007 315:100-1 PMID: 17204651.

66.   Griffin R, Sustar A, Bonvin M, Binari R, del Valle Rodriguez A, Hohl AM, Bateman JR, Villalta C, Heffern E, Grunwald D, Bakal C, Desplan C, Schubiger G, Wu C-t, Perrimon N. The twin spot generator for differential Drosophila lineage analysis. Nat Meth. 2009 6:600-2 PMID: 19633664, PMCID: PMC2720837.

67.   Zong H, Espinosa JS, Su HH, Muzumdar MD, Luo L. Mosaic analysis with double markers in mice. Cell. 2005 121:479-92 PMID: 15882628.

68.   Bejerano G, Pheasant M, Makunin I, Stephen S, Kent WJ, Mattick JS, Haussler D. Ultraconserved elements in the human genome. Science. 2004 304:1321-5 PMID: 15131266.

69.   Ahituv N, Zhu Y, Visel A, Holt A, Afzal V, Pennacchio LA, Rubin EM. Deletion of ultraconserved elements yields viable mice. PLoS Biol. 2007 5:e234 PMID: 17803355, PMCID: PMC1964772.

70.   Derti A, Roth FP, Church GM, and Wu C-t. Mammalian ultraconserved elements are strongly depleted among segmental duplications and copy number variants. Nat Genet. 2006 38:1216-20 PMID: 16998490.

71.   Chiang CWK, Derti A, Schwartz D, Chou MF, Hirschhorn JN, Wu C-t. Ultraconserved elements: Analyses of dosage sensitivity, motifs, and boundaries. Genetics. 2008 180:2277-93. PMID: 18957701, PMCID: PMC2600958.

72.   Groth AC, Fish M, Nusse R, Calos MP. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics. 2004 166:1775-82 PMID:15126397, PMCID: PMC1470814.

73.   Bateman JR, Lee AM, Wu C-t. Site-specific transformation of Drosophila via φC31 integrase-mediated cassette exchange. Genetics. 2006 173:769-777.  PMID: 16547094, PMCID: PMC1526508.

74. Bateman JR, Wu C-t. A simple PCR-based method for the construction of RMCE donor vectors. Genetics. 2008 180:1763-6. PMID: 18791223, PMCID: PMC2581973.