Enhancer Of Position Effect Variegation Research Articles

BICRA, a SWI/SNF Complex Member, Is Associated with BAF-Disorder Related Phenotypes in Humans and Model Organisms

SWI/SNF-related intellectual disability disorders (SSRIDDs) are rare neurodevelopmental disorders characterized by developmental disability, coarse facial features, and fifth digit/nail hypoplasia that are caused by pathogenic variants in genes that encode for members of the SWI/SNF (or BAF) family of chromatin remodeling complexes. We have identified 12 individuals with rare variants (10 loss-of-function, 2 missense) in the BICRA (BRD4 interacting chromatin remodeling complex-associated protein) gene, also known as GLTSCR1, which encodes a subunit of the non-canonical BAF (ncBAF) complex. These individuals exhibited neurodevelopmental phenotypes that include developmental delay, intellectual disability, autism spectrum disorder, and behavioral abnormalities as well as dysmorphic features. Notably, the majority of individuals lack the fifth digit/nail hypoplasia phenotype, a hallmark of most SSRIDDs. To confirm the role of BICRA in the development of these phenotypes, we performed functional characterization of the zebrafish and Drosophila orthologs of BICRA. In zebrafish, a mutation of bicra that mimics one of the loss-of-function variants leads to craniofacial defects possibly akin to the dysmorphic facial features seen in individuals harboring putatively pathogenic BICRA variants. We further show that Bicra physically binds to other non-canonical ncBAF complex members, including the BRD9/7 ortholog, CG7154, and is the defining member of the ncBAF complex in flies. Like other SWI/SNF complex members, loss of Bicra function in flies acts as a dominant enhancer of position effect variegation but in a more context-specific manner. We conclude that haploinsufficiency of BICRA leads to a unique SSRIDD in humans whose phenotypes overlap with those previously reported.

Variation in Position Effect Variegation Within a Natural Population.

Changes in chromatin state may drive changes in gene expression, and it is of growing interest to understand the population genetic forces that drive differences in chromatin state. Here, we use the phenomenon of position effect variegation (PEV), a well-studied proxy for chromatin state, to survey variation in PEV among a naturally derived population. Further, we explore the genetic architecture of natural variation in factors that modify PEV. While previous mutation screens have identified over 150 suppressors and enhancers of PEV, it remains unknown to what extent allelic variation in these modifiers mediate interindividual variation in PEV. Is natural variation in PEV mediated by segregating genetic variation in known Su(var) and E(var) genes, or is the trait polygenic, with many variants mapping elsewhere in the genome? We designed a dominant mapping study that directly answers this question and suggests that the bulk of the variance in PEV does not map to genes with prior annotated impact to PEV. Instead, we find enrichment of top P-value ranked associations that suggest impact to active promoter and transcription start site proximal regions. This work highlights extensive variation in PEV within a population, and provides a quantitative view of the role naturally segregating autosomal variants play in modifying PEV-a phenomenon that continues to shape our understanding of chromatin state and epigenetics.

A TFTC/STAGA Module Mediates Histone H2A and H2B Deubiquitination, Coactivates Nuclear Receptors, and Counteracts Heterochromatin Silencing

Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

Loss-of-Function Alleles of the JIL-1 Histone H3S10 Kinase Enhance Position-Effect Variegation at Pericentric Sites in Drosophila Heterochromatin

In this study we show that loss-of-function alleles of the JIL-1 histone H3S10 kinase act as enhancers of position-effect variegation at pericentric sites whereas the gain-of-function JIL-1(Su(var)3-1[3]) allele acts as a suppressor strongly supporting a functional role for JIL-1 in maintaining euchromatic chromatin and counteracting heterochromatic spreading and gene silencing.

The lamin Dm0 allele Ari3 acts as an enhancer of position effect variegation of the w m4 allele in Drosophila

The association of lamin and lamin binding proteins with peripheral heterochromatin suggests the possibility that lamins may influence gene expression by participating in the epigenetic regulation of chromatin stucture. To test this hypothesis we have examined the effect of a recently generated partial loss-of-function lamin Dm0 allele Ari3 on PEV of the wm4 allele in the Drosophila eye. The Lam ( Ari3 ) allele is characterized by a truncation of the COOH-terminal domain and lacks the CaaX box that localizes lamin to the inner nuclear membrane. We show that the Lam ( Ari3 ) allele strongly increased silencing of wm4 expression, thus acting as an enhancer of PEV. These results indicate that lamins may be involved in regulating gene silencing and heterochromatic spreading at the wm4 locus and provide evidence that lamins may contribute to the regulation of higher-order chromatin organization.

A high proportion of genes involved in position effect variegation also affect chromosome inheritance.

Suppressors and enhancers of position effect variegation (PEV) have been linked to the establishment and maintenance of heterochromatin. The presence of centromeres and other inheritance elements in heterochromatic regions suggests that suppressors and enhancers of PEV, Su(var) s and E(var)s [collectively termed Mod(var)s], may be required for chromosome inheritance. In order to test this hypothesis, we screened 59 ethyl methanesulfonate-generated Drosophila Mod(var)s for dominant effects on the partially compromised inheritance of a minichromosome ( J21A) missing a portion of the genetically defined centromere. Nearly half of these Mod(var)s significantly increased or decreased the transmission of J21A. Analyses of homozygous mutant larval neuroblasts suggest that these mutations affect cell cycle progression and native chromosome morphology. Five out of six complementation groups tested displayed mitotic abnormalities, including phenotypes such as telomere fusions, overcondensed chromosomes, and low mitotic index. We conclude that Mod(var)s as a group are highly enriched for genes that encode essential inheritance functions. We propose that a primary function of Mod(var)s is to promote chromosome inheritance, and that the gene silencing phenotype associated with PEV may be a secondary consequence of the heterochromatic structures required to carry out these functions.

Polycomb Group Suppressor of Zeste 12 Links Heterochromatin Protein 1α and Enhancer of Zeste 2

Drosophila suppressor of zeste 12 (Su(z)12) is a Polycomb group (PcG) transcriptional repressor and is present in E(z)-ESC, a multiprotein complex with methylation activity specific for lysine 9 and 27 of histone H3. Although PcG- and heterochromatin-mediated gene silencing have been considered distinct, mutant flies of Su(z)12 showed not only homeotic transformation but also position effect variegation. We now report that the mammalian SU(Z)12 directly interacts with heterochromatin protein 1alpha (HP1alpha) and PcG enhancer of zeste 2 (EZH2), the mammalian counterpart of E(z), in vitro and in vivo. Two distinct domains in SU(Z)12 are involved in these interactions, the region between the zinc finger motif and the VEFS (VRN2-EMF2-FIS2-Su(z)12) box for HP1alpha (amino acid residues 479-536) and the VEFS box for EZH2 (amino acid residues 600-639), which are not mutually exclusive. Interestingly this region of the VEFS box has been shown to be critical for the phenotype of the Su(z)12 mutant fly. In addition SU(Z)12 represses transcription activity in the presence of HP1alpha in a reporter assay. These results provide a molecular explanation for the functional link of these epigenetic silencing processes mediated by Su(z)12.

Pitkin(D), a novel gain-of-function enhancer of position-effect variegation, affects chromatin regulation during oogenesis and early embryogenesis in Drosophila.

The vast majority of the >100 modifier genes of position-effect variegation (PEV) in Drosophila have been identified genetically as haplo-insufficient loci. Here, we describe pitkin(Dominant) (ptn(D)), a gain-of-function enhancer mutation of PEV. Its exceptionally strong enhancer effect is evident as elevated spreading of heterochromatin-induced gene silencing along euchromatic regions in variegating rearrangements. The ptn(D) mutation causes ectopic binding of the SU(VAR)3-9 heterochromatin protein at many euchromatic sites and, unlike other modifiers of PEV, it also affects stable position effects. Specifically, it induces silencing of white+ transgenes inserted at a wide variety of euchromatic sites. ptn(D) is associated with dominant female sterility. +/+ embryos produced by ptn(D)/+ females mated with wild-type males die at the end of embryogenesis, whereas the ptn(D)/+ sibling embryos arrest development at cleavage cycle 1-3, due to a combined effect of maternally provided mutant product and an early zygotic lethal effect of ptn(D). This is the earliest zygotic effect of a mutation so far reported in Drosophila. Germ-line mosaics show that ptn+ function is required for normal development in the female germ line. These results, together with effects on PEV and white+ transgenes, are consistent with the hypothesis that the ptn gene plays an important role in chromatin regulation during development of the female germ line and in early embryogenesis.

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.

The lawc gene is a new member of the trithorax-group that affects the function of the gypsy insulator of Drosophila.

Mutations in the lawc gene result in a pleiotropic phenotype that includes homeotic transformation of the arista into leg. lawc mutations enhance the phenotype of trx-G mutations and suppress the phenotype of Pc mutations. Mutations in lawc affect homeotic gene transcription, causing ectopic expression of Antennapedia in the eye-antenna imaginal disc. These results suggest that lawc is a new member of the trithorax family. The lawc gene behaves as an enhancer of position-effect variegation and interacts genetically with mod(mdg4), which is a component of the gypsy insulator. In addition, mutations in the lawc gene cause alterations in the punctated distribution of mod(mdg4) protein within the nucleus. These results suggest that the lawc protein is involved in regulating the higher-order organization of chromatin.

The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes.

The Additional sex combs (Asx) gene of Drosophila is a member of the Polycomb group of genes, which are required for maintenance of stable repression of homeotic and other loci. Asx is unusual among the Polycomb group because: (1) one Asx allele exhibits both anterior and posterior transformations; (2) Asx mutations enhance anterior transformations of trx mutations; (3) Asx mutations exhibit segmentation phenotypes in addition to homeotic phenotypes; (4) Asx is an Enhancer of position-effect variegation and (5) Asx displays tissue-specific derepression of target genes. Asx was cloned by transposon tagging and encodes a protein of 1668 amino acids containing an unusual cysteine cluster at the carboxy terminus. The protein is ubiquitously expressed during development. We show that Asx is required in the central nervous system to regulate Ultrabithorax. ASX binds to multiple sites on polytene chromosomes, 70% of which overlap those of Polycomb, polyhomeotic and Polycomblike, and 30% of which are unique. The differences in target site recognition may account for some of the differences in Asx phenotypes relative to other members of the Polycomb group.

Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.

A New Enhancer of Position-Effect Variegation in Drosophila melanogaster Encodes a Putative RNA Helicase That Binds Chromosomes and Is Regulated by the Cell Cycle

In Drosophila melanogaster, position-effect variegation of the white gene has been a useful phenomenon by which to study chromosome structure and the genes that modify it. We have identified a new enhancer of variegation locus, Dmrnahel (hel). Deletion of mutation of hel enhances white variegation, and this can be reversed by a transformed copy of hel+. In the presence of two endogenous copies, the transformed hel+ behaves as a suppressor of variegation. hel is an essential gene and functions both maternally and zygotically. The HEL protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential HEL homologues have been found in mammals, yeast and worms. HEL protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. We propose that the HEL protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that HEL is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis.

Dosage-dependent modifiers of position effect variegation in Drosophila and a mass action model that explains their effect.

Twelve dominant enhancers of position effect variegation, representing four loci on the second and third chromosomes of Drosophila melanogaster, have been induced by P-element mutagenesis. Instead of simple transposon insertions, seven of these mutations are cytologically visible duplications and three are deficiencies. The duplications define two distinct regions, each coinciding with a locus that also behaves as a dominant haplo-dependent suppressor of variegation. Conversely, two of the deficiencies overlap with a region that contains a haplo-dependent enhancer of variegation while duplications of this same region act to suppress variegation. The third deficiency defines another haplo-dependent enhancer. These data indicate that loci capable of modifying variegation do so in an antipodal fashion through changes in the wild-type gene copy number and may be divided into two reciprocally acting classes. Class I modifiers enhance variegation when duplicated or suppress variegation when deficient. Class II modifiers enhance when deficient but suppress when duplicated. From our data, and those of others, we propose that in Drosophila there are about 20 to 30 dominant loci that modify variegation. Most appear to be of the class I type whereas only two class II modifiers have been identified so far. From these observations we put forth a model, based on the law of mass action, for understanding how such suppressor-enhancer loci function. We propose that each class I modifier codes for a structural protein component of heterochromatin and their effects on variegation are a consequence of their dosage dependent influence on the extent of the assembly of heterochromatin at the chromosomal site of the position effect. It is further proposed that class II modifiers may inhibit the class I products directly, bind to hypothetical termination sites that define heterochromatin boundaries or promote euchromatin formation. Consistent with our mass action model we find that combining two enhancers together produce additive and not epistatic effects. Also, since different enhancers have different relative strengths on different variegating mutants, we suggest that heterochromatic domains are constructed by a combinatorial association of proteins. The mass action model proposed here is of general significance for any assembly driven reaction and has implications for understanding a wide variety of biological phenomena.(ABSTRACT TRUNCATED AT 400 WORDS)

Genes which suppress position-effect variegation in Drosophila melanogaster are clustered

We have isolated more than 50 dominant suppressors and 3 dominant enhancers of position effect variegation in Drosophila melanogaster. To our surprise, genetic mapping studies revealed that the Su(var) mutations are not randomly dispersed throughout the genome of this organism. Rather they fall into very discrete clusters. Moreover, all of the induced Su(var) mutations which map to a major second chromosome cluster are recessive lethal, whereas all of those which map within any of several third chromosome clusters are viable. Complementation analysis involving Su(var) mutations located within the 2L cluster suggests that several different loci occupy this site. Thus, these clusters may represent groupings of functionally related but distinct Su(var) loci.