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1.  A Forward Genetic Screen Identifies Eukaryotic Translation Initiation Factor 3, Subunit H (eIF3h), as an Enhancer of Variegation in the Mouse 
G3: Genes|Genomes|Genetics  2012;2(11):1393-1396.
We have used a forward genetic screen to identify genes required for transgene silencing in the mouse. Previously these genes were found using candidate-based sequencing, a slow and labor-intensive process. Recently, whole-exome deep sequencing has accelerated our ability to find the causative point mutations, resulting in the discovery of novel and sometimes unexpected genes. Here we report the identification of translation initiation factor 3, subunit H (eIF3h) in two modifier of murine metastable epialleles (Mommes) lines. Mice carrying mutations in this gene have not been reported previously, and a possible involvement of eIF3h in transcription or epigenetic regulation has not been considered.
doi:10.1534/g3.112.004036
PMCID: PMC3484669  PMID: 23173090
mouse; epigenetics; forward genetic screen; eIF3h
2.  Reduced dosage of the modifiers of epigenetic reprogramming Dnmt1, Dnmt3L, SmcHD1 and Foxo3a has no detectable effect on mouse telomere length in vivo 
Chromosoma  2011;120(4):377-385.
Studies carried out in cultured cells have implicated modifiers of epigenetic reprogramming in the regulation of telomere length, reporting elongation in cells that were null for DNA methyltransferase DNA methyltransferase 1 (Dnmt1), both de novo DNA methyltransferases, Dnmt3a and Dnmt3b or various histone methyltransferases. To investigate this further, we assayed telomere length in whole embryos or adult tissue from mice carrying mutations in four different modifiers of epigenetic reprogramming: Dnmt1, DNA methyltransferase 3-like, structural maintenance of chromosomes hinge domain containing 1, and forkhead box O3a. Terminal restriction fragment analysis was used to compare telomere length in homozygous mutants, heterozygous mutants and wild-type littermates. Contrary to expectation, we did not detect overall lengthening in the mutants, raising questions about the role of epigenetic processes in telomere length in vivo.
Electronic supplementary material
The online version of this article (doi:10.1007/s00412-011-0318-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s00412-011-0318-9
PMCID: PMC3140923  PMID: 21553025
3.  Divergent Evolution of CHD3 Proteins Resulted in MOM1 Refining Epigenetic Control in Vascular Plants 
PLoS Genetics  2008;4(8):e1000165.
Arabidopsis MOM1 is required for the heritable maintenance of transcriptional gene silencing (TGS). Unlike many other silencing factors, depletion of MOM1 evokes transcription at selected loci without major changes in DNA methylation or histone modification. These loci retain unusual, bivalent chromatin properties, intermediate to both euchromatin and heterochromatin. The structure of MOM1 previously suggested an integral nuclear membrane protein with chromatin-remodeling and actin-binding activities. Unexpected results presented here challenge these presumed MOM1 activities and demonstrate that less than 13% of MOM1 sequence is necessary and sufficient for TGS maintenance. This active sequence encompasses a novel Conserved MOM1 Motif 2 (CMM2). The high conservation suggests that CMM2 has been the subject of strong evolutionary pressure. The replacement of Arabidopsis CMM2 by a poplar motif reveals its functional conservation. Interspecies comparison suggests that MOM1 proteins emerged at the origin of vascular plants through neo-functionalization of the ubiquitous eukaryotic CHD3 chromatin remodeling factors. Interestingly, despite the divergent evolution of CHD3 and MOM1, we observed functional cooperation in epigenetic control involving unrelated protein motifs and thus probably diverse mechanisms.
Author Summary
Epigenetic regulation of transcription usually involves changes in histone modifications, as well as DNA methylation changes in plants and mammals. Previously, we found an exceptional epigenetic regulator in Arabidopsis, MOM1, acting independently of these epigenetic marks. Interestingly, MOM1 controls loci associated with bivalent chromatin marks, intermediate to active euchromatin and silent heterochromatin. Such bivalent marks are often associated with newly inserted and/or potentially active transposons, silent transgenes, and certain chromosomal loci. Notably, bivalent chromatin seems to be characteristic for embryonic stem cells, where such loci change their activity and determination of epigenetic marks during cell differentiation. Here, we provide evidence that in vascular plants, the MOM1-like proteins evolved from the ubiquitous eukaryotic chromatin remodeling factor CHD3. The domains necessary for CHD3 function degenerated in MOM1, became dispensable for its gene silencing activity, and were replaced by a novel, unrelated domain providing silencing function. Therefore, MOM1-like proteins use a different silencing mechanism compared to the ancestral CHD3s. In spite of this divergent evolution, CHD3 and MOM1 seem to retain a functional cooperation in control of transcriptionally silent loci. Our results provide an unprecedented example of an evolutionary path for epigenetic components resulting in increased complexity of an epigenetic regulatory network characteristic for multicellular eukaryotes.
doi:10.1371/journal.pgen.1000165
PMCID: PMC2507757  PMID: 18725928
4.  Smchd1-Dependent and -Independent Pathways Determine Developmental Dynamics of CpG Island Methylation on the Inactive X Chromosome 
Developmental Cell  2012;23(2):265-279.
Summary
X chromosome inactivation involves multiple levels of chromatin modification, established progressively and in a stepwise manner during early development. The chromosomal protein Smchd1 was recently shown to play an important role in DNA methylation of CpG islands (CGIs), a late step in the X inactivation pathway that is required for long-term maintenance of gene silencing. Here we show that inactive X chromosome (Xi) CGI methylation can occur via either Smchd1-dependent or -independent pathways. Smchd1-dependent CGI methylation, the primary pathway, is acquired gradually over an extended period, whereas Smchd1-independent CGI methylation occurs rapidly after the onset of X inactivation. The de novo methyltransferase Dnmt3b is required for methylation of both classes of CGI, whereas Dnmt3a and Dnmt3L are dispensable. Xi CGIs methylated by these distinct pathways differ with respect to their sequence characteristics and immediate chromosomal environment. We discuss the implications of these results for understanding CGI methylation during development.
Graphical Abstract
Highlights
► Xi CGIs exhibit either fast or slow DNA methylation kinetics ► Xi CGI methylation requires Dnmt3b but not Dnmt3a or Dnmt3L ► Recruitment of the chromosomal protein Smchd1 is a late step in X inactivation ► Slow, but not fast, Xi CGI methylation requires the chromosomal protein Smchd1
What mechanisms contribute to developmental gene regulation and long-term silencing? Gendrel et al. uncover on the inactive X chromosome parallel pathways of slow and fast CpG-island methylation kinetics associated with different chromosomal contexts. Only slow methylation needs the chromosomal protein Smchd1, but both pathways require the de novo methyltransferase Dnmt3b.
doi:10.1016/j.devcel.2012.06.011
PMCID: PMC3437444  PMID: 22841499
5.  Silencing of toxic gene expression by Fis 
Nucleic Acids Research  2012;40(10):4358-4367.
Bacteria and bacteriophages have evolved DNA modification as a strategy to protect their genomes. Mom protein of bacteriophage Mu modifies the phage DNA, rendering it refractile to numerous restriction enzymes and in turn enabling the phage to successfully invade a variety of hosts. A strong fortification, a combined activity of the phage and host factors, prevents untimely expression of mom and associated toxic effects. Here, we identify the bacterial chromatin architectural protein Fis as an additional player in this crowded regulatory cascade. Both in vivo and in vitro studies described here indicate that Fis acts as a transcriptional repressor of mom promoter. Further, our data shows that Fis mediates its repressive effect by denying access to RNA polymerase at mom promoter. We propose that a combined repressive effect of Fis and previously characterized negative regulatory factors could be responsible to keep the gene silenced most of the time. We thus present a new facet of Fis function in Mu biology. In addition to bringing about overall downregulation of Mu genome, it also ensures silencing of the advantageous but potentially lethal mom gene.
doi:10.1093/nar/gks037
PMCID: PMC3378877  PMID: 22287621
6.  Smchd1 regulates a subset of autosomal genes subject to monoallelic expression in addition to being critical for X inactivation 
Background
Smchd1 is an epigenetic modifier essential for X chromosome inactivation: female embryos lacking Smchd1 fail during midgestational development. Male mice are less affected by Smchd1-loss, with some (but not all) surviving to become fertile adults on the FVB/n genetic background. On other genetic backgrounds, all males lacking Smchd1 die perinatally. This suggests that, in addition to being critical for X inactivation, Smchd1 functions to control the expression of essential autosomal genes.
Results
Using genome-wide microarray expression profiling and RNA-seq, we have identified additional genes that fail X inactivation in female Smchd1 mutants and have identified autosomal genes in male mice where the normal expression pattern depends upon Smchd1. A subset of genes in the Snrpn imprinted gene cluster show an epigenetic signature and biallelic expression consistent with loss of imprinting in the absence of Smchd1. In addition, single nucleotide polymorphism analysis of expressed genes in the placenta shows that the Igf2r imprinted gene cluster is also disrupted, with Slc22a3 showing biallelic expression in the absence of Smchd1. In both cases, the disruption was not due to loss of the differential methylation that marks the imprint control region, but affected genes remote from this primary imprint controlling element. The clustered protocadherins (Pcdhα, Pcdhβ, and Pcdhγ) also show altered expression levels, suggesting that their unique pattern of random combinatorial monoallelic expression might also be disrupted.
Conclusions
Smchd1 has a role in the expression of several autosomal gene clusters that are subject to monoallelic expression, rather than being restricted to functioning uniquely in X inactivation. Our findings, combined with the recent report implicating heterozygous mutations of SMCHD1 as a causal factor in the digenically inherited muscular weakness syndrome facioscapulohumeral muscular dystrophy-2, highlight the potential importance of Smchd1 in the etiology of diverse human diseases.
doi:10.1186/1756-8935-6-19
PMCID: PMC3707822  PMID: 23819640
Clustered protocadherins; Genomic imprinting; Monoallelic expression; Smchd1; X inactivation
7.  Structural Basis of Transcriptional Gene Silencing Mediated by Arabidopsis MOM1 
PLoS Genetics  2012;8(2):e1002484.
Shifts between epigenetic states of transcriptional activity are typically correlated with changes in epigenetic marks. However, exceptions to this rule suggest the existence of additional, as yet uncharacterized, layers of epigenetic regulation. MOM1, a protein of 2,001 amino acids that acts as a transcriptional silencer, represents such an exception. Here we define the 82 amino acid domain called CMM2 (Conserved MOM1 Motif 2) as a minimal MOM1 fragment capable of transcriptional regulation. As determined by X-ray crystallography, this motif folds into an unusual hendecad-based coiled-coil. Structure-based mutagenesis followed by transgenic complementation tests in plants demonstrate that CMM2 and its dimerization are effective for transcriptional suppression at chromosomal loci co-regulated by MOM1 and the siRNA pathway but not at loci controlled by MOM1 in an siRNA–independent fashion. These results reveal a surprising separation of epigenetic activities that enable the single, large MOM1 protein to coordinate cooperating mechanisms of epigenetic regulation.
Author Summary
Epigenetic shifts in transcriptional activities are usually correlated with changes in chromatin properties and covalent modification of DNA and/or histones. There are, however, exceptional regulators that are able to switch epigenetic states without the apparent involvement of changes in chromatin or DNA modifications. MOM1 protein, derived from CHD3 chromatin remodelers, belongs to this group. Here we defined a very small domain of MOM1 (less than 5% of its total sequence) that is sufficient for epigenetic regulation. We solved the structure of this domain and found that it forms a dimer with each monomer consisting of unusual consecutive 11 amino-acid hendecad repeats folding into an antiparallel coiled-coil. In vivo experiments demonstrated that the formation of this coiled-coil is essential for silencing activity; however, it is effective only at loci co-silenced by MOM1 and small RNAs. At loci not controlled by small RNAs, the entire MOM1 protein is required. Our results demonstrate that a single epigenetic regulator is able to differentially use its domains to control diverse chromosomal targets. The acquisition of the coiled-coil domain of MOM1 reflects a neofunctionalization of CHD3 proteins, which allowed MOM1 to broaden its activity and to provide input into multiple epigenetic pathways.
doi:10.1371/journal.pgen.1002484
PMCID: PMC3276543  PMID: 22346760
8.  Expression, crystallization and preliminary X-ray diffraction analysis of the CMM2 region of the Arabidopsis thaliana Morpheus’ molecule 1 protein 
In order to investigate its function in transcriptional gene silencing, the highly conserved motif 2 from A. thaliana Morpheus’ molecule 1 protein was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 3.2 Å.
Of the known epigenetic control regulators found in plants, the Morpheus’ molecule 1 (MOM1) protein is atypical in that the deletion of MOM1 does not affect the level of epigenetic marks controlling the transcriptional status of the genome. A short 197-amino-acid fragment of the MOM1 protein sequence can complement MOM1 deletion when coupled to a nuclear localization signal, suggesting that this region contains a functional domain that compensates for the loss of the full-length protein. Numerous constructs centred on the highly conserved MOM1 motif 2 (CMM2) present in these 197 residues have been generated and expressed in Escherichia coli. Following purification and crystallization screening, diamond-shaped single crystals were obtained that diffracted to ∼3.2 Å resolution. They belonged to the trigonal space group P3121 (or P3221), with unit-cell parameters a = 85.64, c = 292.74 Å. Structure determination is ongoing.
doi:10.1107/S1744309110021068
PMCID: PMC2917290  PMID: 20693667
Morpheus’ molecule 1; conserved MOM1 motif 2; coiled-coil domain; epigenetic; transcriptional gene silencing
9.  Mutation in a locus linked to penB-nmp causes suppression of the Mtr phenotype of Neisseria gonorrhoeae. 
The chromosomal locus mtr, which encodes low-level resistance to multiple antibacterial agents in Neisseria gonorrhoeae, is subject to phenotypic suppression by env mutations that increase the permeability of the envelope. We have identified a new locus, mom (for modifier of Mtr), which is located on the chromosome very close to penB and nmp, loci known to be linked to each other and to spc. Phenotypic suppression of Mtr was recognized by reductions of resistance to benzylpenicillin and also to oxacillin and the hydrophobic agents novobiocin and erythromycin. The resistance to each of these antibiotics returned to the Mtr levels in mom+ transformants isolated by selection for increased resistance to either novobiocin or erythromycin; the accompanying change of the outer membrane protein I seroreactions confirmed the proximity of nmp and mom. Thus, some mutant gonococci display wild-type antibiotic susceptibilities but can express multiple resistance following a mom+ mutation that releases the suppressed Mtr phenotype.
PMCID: PMC172327  PMID: 3142343
10.  Epigenetic Functions of Smchd1 Repress Gene Clusters on the Inactive X Chromosome and on Autosomes 
Molecular and Cellular Biology  2013;33(16):3150-3165.
The Smchd1 gene encodes a large protein with homology to the SMC family of proteins involved in chromosome condensation and cohesion. Previous studies have found that Smchd1 has an important role in CpG island (CGI) methylation on the inactive X chromosome (Xi) and in stable silencing of some Xi genes. In this study, using genome-wide expression analysis, we showed that Smchd1 is required for the silencing of around 10% of the genes on Xi, apparently independent of CGI hypomethylation, and, moreover, that these genes nonrandomly occur in clusters. Additionally, we found that Smchd1 is required for CpG island methylation and silencing at a cluster of four imprinted genes in the Prader-Willi syndrome (PWS) locus on chromosome 7 and genes from the protocadherin-alpha and -beta clusters. All of the affected autosomal loci display developmentally regulated brain-specific methylation patterns which are lost in Smchd1 homozygous mutants. We discuss the implications of these findings for understanding the function of Smchd1 in epigenetic regulation of gene expression.
doi:10.1128/MCB.00145-13
PMCID: PMC3753908  PMID: 23754746
11.  Digenic inheritance of an SMCHD1 mutation and an FSHD-permissive D4Z4 allele causes facioscapulohumeral muscular dystrophy type 2 
Nature genetics  2012;44(12):1370-1374.
Facioscapulohumeral dystrophy (FSHD) is characterized by chromatin relaxation of the D4Z4 macrosatellite array on chromosome 4 and expression of the D4Z4-encoded DUX4 gene in skeletal muscle. The more common form, autosomal dominant FSHD1, is caused by a contraction of the D4Z4 array, whereas the genetic determinants and inheritance of D4Z4 array contraction-independent FSHD2 are unclear. Here we show that mutations in SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18 reduce SMCHD1 protein levels and segregate with genome-wide D4Z4 CpG hypomethylation in human kindreds. FSHD2 occurs in individuals who inherited both the SMCHD1 mutation and a normal-sized D4Z4 array on a chromosome 4 haplotype permissive for DUX4 expression. Reducing SMCHD1 levels in skeletal muscle results in contraction-independent DUX4 expression. Our study identifies SMCHD1 as an epigenetic modifier of the D4Z4 metastable epiallele and as a causal genetic determinant of FSHD2 and possibly other human diseases subject to epigenetic regulation.
doi:10.1038/ng.2454
PMCID: PMC3671095  PMID: 23143600
12.  Mutations in CG8878, a Novel Putative Protein Kinase, Enhance P Element Dependent Silencing (PDS) and Position Effect Variegation (PEV) in Drosophila melanogaster 
PLoS ONE  2014;9(3):e71695.
Genes in multicellular organisms are expressed as part of a developmental program that is largely dependent on self-perpetuating higher-order chromatin states. The mechanism of establishing and maintaining these epigenetic events is well studied in Drosophila. The first known example of an epigenetic effect was that of (PEV) in Drosophila, which has been shown to be due to gene silencing via heterochromatin formation. We are investigating a process similar to Position Effect Variegation (PEV) using a mini-w transgene, called Pci, inserted in the upstream regulatory region of ci. The mini-white+ transgene in Pci is expressed throughout the adult eye; however, when other P or KP elements are present, a variegated eye phenotype results indicating random w+ silencing during development. This P element dependent silencing (PDS) can be modified by the haplo-suppressors/triplo-enhancers, Su(var)205 and Su(var)3–7, indicating that these heterochromatic modifiers also act dose dependently in PDS. Here we use a spontaneous derivative mutation of Pci called PciE1 (E1) that variegates like PDS in the absence of P elements, presumably due to an adjacent gypsy element insertion, to screen for second-site modifier mutations that enhance variable silencing of white+ in E1. We isolated 7 mutations in CG8878, an essential gene, that enhance the E1 variegated phenotype. CG8878, a previously uncharacterized gene, potentially encodes a serine/threonine kinase whose closest Drosophila paralogue, ballchen (nhk-1), phosphorylates histones. These mutant alleles enhance both PDS at E1 and Position Effect Variegation (PEV) at wm4, indicating a previously unknown common silencing mechanism between the two.
doi:10.1371/journal.pone.0071695
PMCID: PMC3948951  PMID: 24614804
13.  Import of ADP/ATP carrier into mitochondria: two receptors act in parallel 
The Journal of Cell Biology  1990;111(6):2353-2363.
We have identified the yeast homologue of Neurospora crassa MOM72, the mitochondrial import receptor for the ADP/ATP carrier (AAC), by functional studies and by cDNA sequencing. Mitochondria of a yeast mutant in which the gene for MOM72 was disrupted were impaired in specific binding and import of AAC. Unexpectedly, we found a residual, yet significant import of AAC into mitochondria lacking MOM72 that occurred via the receptor MOM19. We conclude that both MOM72 and MOM19 can direct AAC into mitochondria, albeit with different efficiency. Moreover, the precursor of MOM72 apparently does not require a positively charged sequence at the extreme amino terminus for targeting to mitochondria.
PMCID: PMC2116357  PMID: 2177474
14.  Identification of five novel modifier loci of ApcMin harbored in the BXH14 recombinant inbred strain 
Carcinogenesis  2012;33(8):1589-1597.
Every year thousands of people in the USA are diagnosed with small intestine and colorectal cancers (CRC). Although environmental factors affect disease etiology, uncovering underlying genetic factors is imperative for risk assessment and developing preventative therapies. Familial adenomatous polyposis is a heritable genetic disorder in which individuals carry germ-line mutations in the adenomatous polyposis coli (APC) gene that predisposes them to CRC. The Apc Min mouse model carries a point mutation in the Apc gene and develops polyps along the intestinal tract. Inbred strain background influences polyp phenotypes in Apc Min mice. Several Modifier of Min (Mom) loci that alter tumor phenotypes associated with the Apc Min mutation have been identified to date. We screened BXH recombinant inbred (RI) strains by crossing BXH RI females with C57BL/6J (B6) Apc Min males and quantitating tumor phenotypes in backcross progeny. We found that the BXH14 RI strain harbors five modifier loci that decrease polyp multiplicity. Furthermore, we show that resistance is determined by varying combinations of these modifier loci. Gene interaction network analysis shows that there are multiple networks with proven gene–gene interactions, which contain genes from all five modifier loci. We discuss the implications of this result for studies that define susceptibility loci, namely that multiple networks may be acting concurrently to alter tumor phenotypes. Thus, the significance of this work resides not only with the modifier loci we identified but also with the combinations of loci needed to get maximal protection against polyposis and the impact of this finding on human disease studies.
Abbreviations:APCadenomatous polyposis coliGWASgenome-wide association studiesQTLquantitative trait lociSNPsingle-nucleotide polymorphism.
doi:10.1093/carcin/bgs185
PMCID: PMC3499057  PMID: 22637734
15.  Novel non-specific DNA adenine methyltransferases 
Nucleic Acids Research  2011;40(5):2119-2130.
The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.
doi:10.1093/nar/gkr1039
PMCID: PMC3299994  PMID: 22102579
16.  Complex Alternative Splicing of the Smarca2 Gene Suggests the Importance of Smarca2-B Variants 
Journal of Cancer  2011;2:386-400.
BRM is an ATPase component of the SWI/SNF complex that regulates chromatin remodeling and cell proliferation and is considered a tumor suppressor. In this study we characterized transcripts from the Smarca2 gene that encodes the BRM protein. We found that the human Smarca2 gene (hSmarca2), like its mouse counterpart (mSmarca2), also initiated a short transcript from intron 27 of the long transcript. We name the long and short transcripts as Smarca2-a and Smarca2-b, respectively. Like its human counterpart, mSmarca2-a also underwent alternative splicing at the 54-bp exon 29. The hSmarca2-b had two alternative initiation sites and underwent alternative splicing at three different 3' sites of exon 1 and at exons 2, 3 and/or 5. We identified nine hSmarca2-b mRNA variants that might produce five different proteins. mSmarca2-b also underwent alternative splicing at exon 3 and/or exon 5, besides alternatively retaining part of intron 1 in exon 1. Smarca2-b was expressed more abundantly than Smarca2-a in many cell lines and was more sensitive to serum starvation. Moreover, cyclin D1 also regulated the expression of both Smarca2-a and Smarca2-b in a complex manner. These data suggest that the functions of the Smarca2 gene may be very complex, not just simply inhibiting cell proliferation, and in certain situations may be elicited mainly by expressing the much less known Smarca2-b, not the better studied Smarca2-a and its products BRM proteins.
PMCID: PMC3148773  PMID: 21811517
Alternative splicing; Tumor suppressor gene; BRM; Smarca2; SWI/SNF complex; Cancer; Cyclin D1
17.  Olig2 Targets Chromatin Remodelers To Enhancers To Initiate Oligodendrocyte Differentiation 
Cell  2013;152(1-2):248-261.
Summary
Establishment of oligodendrocyte identity is crucial for subsequent events of myelination in the central nervous system (CNS). Here, we demonstrate that activation of ATP-dependent SWI/SNF chromatin-remodeling enzyme Smarca4/Brg1 at the differentiation onset is necessary and sufficient to initiate and promote oligodendrocyte lineage progression and maturation. Genome-wide multistage studies by ChIP-seq reveal that oligodendrocyte-lineage determination factor Olig2 functions as a pre-patterning factor to direct Smarca4/Brg1 to oligodendrocyte-specific enhancers. Recruitment of Smarca4/Brg1 to distinct subsets of myelination regulatory genes is developmentally regulated. Functional analyses of Smarca4/Brg1 and Olig2 co-occupancy relative to chromatin epigenetic marking uncover novel stage-specific cis-regulatory elements that predict sets of transcriptional regulators controlling oligodendrocyte differentiation. Together, our results demonstrate that regulation of the functional specificity and activity of a Smarca4/Brg1-dependent chromatin-remodeling complex by Olig2, coupled with transcriptionally-linked chromatin modifications, is critical to precisely initiate and establish the transcriptional program that promotes oligodendrocyte differentiation and subsequent myelination of the CNS.
doi:10.1016/j.cell.2012.12.006
PMCID: PMC3553550  PMID: 23332759
18.  An N-ethyl-N-nitrosourea mutagenesis recessive screen identifies two candidate regions for murine cardiomyopathy that map to chromosomes 1 and 15 
N-ethyl-N-nitrosourea (ENU) mutagenesis screens have been successful for identifying genes that affect important biological processes and diseases. However, for heart-related phenotypes, these screens have been employed exclusively for developmental phenotypes, and to date no adult cardiomyopathy-causing genes have been discovered through a mutagenesis screen. To identify novel disease-causing and disease-modifying genes for cardiomyopathy, we performed an ENU recessive mutagenesis screen in adult mice. Using noninvasive echocardiography to screen for abnormalities in cardiac function, we identified a heritable cardiomyopathic phenotype in two families. To identify the chromosomal regions where the mutations are localized, we used a single nucleotide polymorphism (SNP) panel for genetic mapping of mouse mutations. This panel provided whole-genome linkage information and identified the mutagenized candidate regions at the proximal end of chromosome 1 (family EN1), and at the distal end of chromosome 15 (family EN25). We have identified 94 affected mice in family EN1 and have narrowed the candidate interval to 1 Mb. We have identified 20 affected mice in family EN25 and have narrowed the candidate interval to 12 Mb. The identification of the genes responsible for the observed phenotype in these families will be strong candidates for disease-causing or disease-modifying genes in patients with heart failure.
doi:10.1007/s00335-009-9184-7
PMCID: PMC2743897  PMID: 19387734
19.  A Position Effect on the Heritability of Epigenetic Silencing 
PLoS Genetics  2008;4(10):e1000216.
In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks.
Author Summary
Epigenetics involves the heritable alteration of gene activity without changes in DNA sequence. Although clearly a repository for heritable information, what makes epigenetic states distinct is that they are far more labile than those associated with DNA sequence. The epigenetic landscape of eukaryotic genomes is far from uniform. Vast stretches of them are effectively epigenetically silenced, while other regions are largely active. The experiments described here suggest that the propensity to maintain heritable epigenetic states can vary depending on position within the genome. Because transposable elements, or transposons, move from place to place within the genome, they make an ideal probe for differences in epigenetic states at various positions. Our model system uses a single transposon, MuDR in maize, and a variant of MuDR, Mu killer (Muk). When MuDR and Muk are combined genetically, MuDR elements become epigenetically silenced, and they generally remain so even after Muk is lost in subsequent generations. However, we have identified a particular position at which the MuDR element reactivates after Muk is lost. These data show that there are some parts of the maize genome that are either competent to erase epigenetic silencing or are incapable of maintaining it. These results suggest that erasure of heritable information may be an important component of epigenetic regulation.
doi:10.1371/journal.pgen.1000216
PMCID: PMC2563033  PMID: 18846225
20.  The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster. 
Molecular and Cellular Biology  1996;16(10):5717-5725.
A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging. Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene was constructed with the cDNA and found to have the effect opposite from that of the mutation, namely, to suppress variegation. Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins. We discuss the link established here between a deubiquitinating enzyme and epigenetic silencing processes.
PMCID: PMC231572  PMID: 8816485
21.  COMPREHENSIVE MOLECULAR CHARACTERIZATION OF CLEAR CELL RENAL CELL CARCINOMA 
Nature  2013;499(7456):43-49.
Genetic changes underlying clear cell renal cell carcinoma (ccRCC) include alterations in genes controlling cellular oxygen sensing (e.g. VHL) and the maintenance of chromatin states (e.g. PBRM1). We surveyed more than 400 tumors using different genomic platforms and identified 19 significantly mutated genes. The PI3K/Akt pathway was recurrently mutated, suggesting this pathway as a potential therapeutic target. Widespread DNA hypomethylation was associated with mutation of the H3K36 methyltransferase SETD2, and integrative analysis suggested that mutations involving the SWI/SNF chromatin remodeling complex (PBRM1, ARID1A, SMARCA4) could have far-reaching effects on other pathways. Aggressive cancers demonstrated evidence of a metabolic shift, involving down-regulation of genes involved in the TCA cycle, decreased AMPK and PTEN protein levels, up-regulation of the pentose phosphate pathway and the glutamine transporter genes, increased acetyl-CoA carboxylase protein, and altered promoter methylation of miR-21 and GRB10. Remodeling cellular metabolism thus constitutes a recurrent pattern in ccRCC that correlates with tumor stage and severity and offers new views on the opportunities for disease treatment.
doi:10.1038/nature12222
PMCID: PMC3771322  PMID: 23792563
22.  Unlocking the Bottleneck in Forward Genetics Using Whole-Genome Sequencing and Identity by Descent to Isolate Causative Mutations 
PLoS Genetics  2013;9(1):e1003219.
Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.
Author Summary
Damaging mutations in single genes are an important source of information about the causes of disease, including more complex genetic disease; but these single gene disorders are typically rare in humans. An important strategy for identifying new disease mechanisms is to introduce multiple random mutations in mice and test the mice for biological differences; these mice act as models of human disease. However, discovering the disease-causing mutation is time-consuming and complex, requiring further generations of breeding. In this study we demonstrate a method that overcomes these problems by sequencing the entire genomes of multiple mice that have inherited a disease-causing mutation from a common ancestor. We use an algorithm that uses knowledge of all the mutations carried by the sequenced mice to identify the regions of the genome and mutations that are common to all the mice. Using this method we can rapidly link biological traits to genetic mutations. In contrast to current approaches, our strategy does not require large amounts of breeding, and it permits more accurate measurement of a wider range of traits; consequently its introduction will significantly reduce the number of mice required in the future, increase the number of traits that can be detected, and accelerate the discovery of new pathways and gene functions relevant to human diseases.
doi:10.1371/journal.pgen.1003219
PMCID: PMC3561070  PMID: 23382690
23.  A modifying locus for familial adenomatous polyposis may be present on chromosome 1p35-p36. 
Journal of Medical Genetics  1996;33(4):268-273.
Mutations of the APC gene cause familial adenomatous polyposis (FAP) in humans and multiple intestinal neoplasia (Min) in laboratory mouse strains. A dominant modifying gene (Mom1), which partially suppresses the min phenotype, has been mapped to mouse chromosome 4. This region is syntenic with human chromosome 1p35-p36. The phospholipase A2 (Pla2s) locus is an excellent candidate for Mom1 and the equivalent human locus PLA2G2A is found on chromosome 1p35. It does not necessarily follow, however, than any modifier of mouse polyposis also influences human disease. In order to test whether a locus on 1p modifies FAP, subjects from 28 FAP families have been typed at microsatellite loci on this chromosome arm. The severity of their duodenal polyposis has also been assessed by endoscopy. Pedigree (lod score) linkage analysis found no evidence of a simple, dominant modifying gene, comparable with the action of Mom1 in inbred mouse strains. Given the more complex genetic and environmental interactions likely to exist in outbred human populations, it is probably more appropriate to use tests which do not specify a mode of inheritance. Using these methods of analysis, the data suggest that a locus on chromosome 1p35-p36 may influence the severity of duodenal FAP.
PMCID: PMC1050573  PMID: 8730279
24.  A Functional Screen for Regulatory Elements that Improve Retroviral Vector Gene Expression 
Blood cells, molecules & diseases  2010;45(4):343-350.
Recombinant retroviruses constitute the most common class of gene delivery vectors used in hematopoietic cell-based gene therapy. However, the use of these vectors can be limited by inadequate levels of transgene expression, often mediated by expression variegation and vector silencing due to chromosomal position effects. Toward the goal of addressing this problem, we sought to identify cis-regulatory elements from the human genome that can improve the level and stability of retroviral vector gene expression. Libraries of size-selected fragments from the human genome were cloned into the “double-copy” position of the gammaretroviral reporter vector MGPN2, and the resulting vectors underwent several rounds of transduction and selection for high-level vector GFP expression. From this screen we identified both enhancer-like elements and vector mutations associated with increased vector expression. One element, H-11, exhibited enhancer activity in a mouse bone marrow progenitor colony assay, a human promoter trap assay, and a long-term mouse bone marrow transplant assay. This element seems to be an orientation-dependent, tissue-independent enhancer.
doi:10.1016/j.bcmd.2010.08.005
PMCID: PMC2993826  PMID: 20846887
enhancer; insulator; retrovirus; regulatory element; screen
25.  Characterization of ENU-induced Mutations in Red Blood Cell Structural Proteins 
Murine models with modified gene function as a result of N-ethyl-N-nitrosourea (ENU) mutagenesis have been used to study phenotypes resulting from genetic change. This study investigated genetic factors associated with red blood cell (RBC) physiology and structural integrity that may impact on blood component storage and transfusion outcome. Forward and reverse genetic approaches were employed with pedigrees of ENU-treated mice using a homozygous recessive breeding strategy. In a “forward genetic” approach, pedigree selection was based upon identification of an altered phenotype followed by exome sequencing to identify a causative mutation. In a second strategy, a “reverse genetic” approach based on selection of pedigrees with mutations in genes of interest was utilised and, following breeding to homozygosity, phenotype assessed. Thirty-three pedigrees were screened by the forward genetic approach. One pedigree demonstrated reticulocytosis, microcytic anaemia and thrombocytosis. Exome sequencing revealed a novel single nucleotide variation (SNV) in Ank1 encoding the RBC structural protein ankyrin-1 and the pedigree was designated Ank1EX34. The reticulocytosis and microcytic anaemia observed in the Ank1EX34 pedigree were similar to clinical features of hereditary spherocytosis in humans. For the reverse genetic approach three pedigrees with different point mutations in Spnb1 encoding RBC protein spectrin-1β, and one pedigree with a mutation in Epb4.1, encoding band 4.1 were selected for study. When bred to homozygosity two of the spectrin-1β pedigrees (a, b) demonstrated increased RBC count, haemoglobin (Hb) and haematocrit (HCT). The third Spnb1 mutation (spectrin-1β c) and mutation in Epb4.1 (band 4.1) did not significantly affect the haematological phenotype, despite these two mutations having a PolyPhen score predicting the mutation may be damaging. Exome sequencing allows rapid identification of causative mutations and development of databases of mutations predicted to be disruptive. These tools require further refinement but provide new approaches to the study of genetically defined changes that may impact on blood component storage and transfusion outcome.
doi:10.5936/csbj.201303012
PMCID: PMC3962129
Red Blood Cell; N-ethyl-N-nitrosourea (ENU); Ankyrin; Spectrin-1β; Band 4.1; Missense Library

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