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The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting “enhancer” elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony’s transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer’s activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits., Author Summary One of the greatest challenges in understanding the relationship between genotype and phenotype is to discern how changes in DNA affect the normal functioning of genes. Mutations may generate a new function for a gene, yet it is frequently observed that they inactivate some aspect of a gene’s normal capacity. Investigations focused on understanding the developmental basis for the evolution of anatomical structures has found a prevalent role for mutations that alter developmental gene regulation. In animals, genes are transcriptionally activated in specific tissues during development by regulatory sequences distributed across their expansive non-protein coding regions. Regulatory elements known as silencers act to prevent genes from being expressed in certain tissues, providing a mechanism for precise control. Here, we show how a silencer that prevents expression of a pigment-producing enzyme in certain Drosophila species has repeatedly been subject to inactivating mutations that increased this gene’s expression. This example illustrates how such negative-acting regulatory sequences can represent a convenient target for increasing gene expression through the loss of a genetic element.

Retrotransposition of endogenous retroviruses (ERVs) poses a substantial threat to genome stability. Transcriptional silencing of a subset of these parasitic elements in early mouse embryonic and germ cell development is dependent upon the lysine methyltransferase SETDB1, which deposits H3K9 trimethylation (H3K9me3) and the co-repressor KAP1, which binds SETDB1 when SUMOylated. Here we identified the transcription co-factor hnRNP K as a novel binding partner of the SETDB1/KAP1 complex in mouse embryonic stem cells (mESCs) and show that hnRNP K is required for ERV silencing. RNAi-mediated knockdown of hnRNP K led to depletion of H3K9me3 at ERVs, concomitant with de-repression of proviral reporter constructs and specific ERV subfamilies, as well as a cohort of germline-specific genes directly targeted by SETDB1. While hnRNP K recruitment to ERVs is dependent upon KAP1, SETDB1 binding at these elements requires hnRNP K. Furthermore, an intact SUMO conjugation pathway is necessary for SETDB1 recruitment to proviral chromatin and depletion of hnRNP K resulted in reduced SUMOylation at ERVs. Taken together, these findings reveal a novel regulatory hierarchy governing SETDB1 recruitment and in turn, transcriptional silencing in mESCs., Author Summary Retroelements, including endogenous retroviruses (ERVs), pose a significant threat to genome stability. In mouse embryonic stem (ES) cells, the enzyme SETDB1 safeguards the genome against transcription of specific ERVs by depositing a repressive mark H3K9 trimethylation (H3K9me3). Although SETDB1 is recruited to ERVs by its binding partner KAP1, the molecular basis of this silencing pathway is not clear. Using biochemical and genetic approaches, we identified hnRNP K as a novel component of this silencing pathway that facilitates the recruitment of SETDB1 to ERVs to promote their repression. HnRNP K binds to ERV sequences via KAP1 and subsequently promotes SETDB1 binding. Together, our results reveal a novel function for hnRNP K in transcriptional silencing of ERVs and demonstrate a new regulatory mechanism governing the deposition of H3K9me3 by SETDB1 in ES cells.

Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element.

Active and silenced chromatin domains are often in close juxtaposition to one another, and enhancer and silencer elements operate over large distances to regulate the genes in these domains. The lack of promiscuity in the function of these elements suggests that active mechanisms exist to restrict their activity. Insulators are DNA elements that restrict the effects of long-range regulatory elements. Studies on different insulators from different organisms have identified common themes in their mode of action. Numerous insulators map to promoters of genes or have binding sites for transcription factors and like active chromatin hubs and silenced loci, insulators also cluster in the nucleus. These results bring into focus potential conserved mechanisms by which these elements might function in the nucleus.

The precise control of transgene expression is essential for biopharmaceutical manufacturing, gene therapy and tissue engineering. We have designed a novel conditional transcription technology, which enables reversible induction, repression and adjustment of desired transgene expression using the clinically inert 6-hydroxy-nicotine (6HNic). The 6-hydroxy-nicotine oxidase (6HNO) repressor (HdnoR), which manages nicotine metabolism in Arthrobacter nicotinovorans pAO1 by binding to a specific operator of the 6-hydroxy-nicotine oxidase (O NIC ), was fused to the Krueppel-associated box protein of the human kox-1 gene (KRAB) to create a synthetic 6HNic-dependent transsilencer (NS) that controls chimeric mammalian promoters, which are assembled by cloning tandem O NIC operators 3′ of a constitutive promoter. In the absence of 6HNic, NS binds to O NIC and silences the constitutive promoter, which otherwise drives high-level transgene expression when the NS-O NIC interaction stops in the presence of 6HNic. Generic NICE ON technology was compatible with a variety of constitutive viral and mammalian housekeeping promoters, each of which enabled specific induced, repressed, adjusted and reversible transgene expression profiles in Chinese hamster ovary (CHO-K1), baby hamster kidney (BHK-21) as well as in human fibrosarcoma (HT-1080) cells. NICE ON also proved successful in controlling multicistronic expression units for coordinated transcription of up to three transgenes and in the fine-tuning of transcription–translation networks, in which RNA polymerase II- and III-dependent promoters, engineered for 6HNic responsiveness, drove expression of siRNAs that triggered specific transgene knockdown. NICE ON represents a robust and versatile technology for the precise tuning of transgene expression in mammalian cells.

Huntington's disease (HD) is a late-onset, autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion. The number of repeats on the HD chromosome explains most of the variability in age of onset, but genetic factors other than the HD gene are responsible for part of the residual variance. Based on the role played by the brain derived neurotrophic factor (BDNF) in neurodysfunction and neurodegeneration in HD, we searched for novel polymorphisms in the neuron restrictive silencer element located in the BDNF promoter. Then, the effect of the Val66Met variant in determining age of onset was tested in a large sample of HD carriers by using a multivariate regression approach. The CAG repeat number accounted for 62% of the variance. After correction for the predominant effect of the CAG expansion, no multiple regression model provided evidence of association between the Val66Met genotype and variation in age-at-onset. Additional studies are warranted to further investigate BDNF as genetic modifier of the HD phenotype.

The inhibition of glucose-6-phosphate dehydrogenase (G6PD) expression by arachidonic acid occurs by changes in the rate of pre-mRNA splicing. Here, we have identified a cis-acting RNA element required for regulated splicing of G6PD mRNA. Using transfection of G6PD RNA reporter constructs into rat hepatocytes, the cis-acting RNA element involved in this regulation was localized to nucleotides 43-72 of exon 12 in the G6PD mRNA. In in vitro splicing assays, RNA substrates containing exon 12 were not spliced. In contrast, RNA substrates containing other regions (exons 8 and 9 or exons 10 and 11) of the G6PD mRNA were efficiently spliced. Furthermore, exon 12 can inhibit splicing when substituted for other exons in RNA substrates that are readily spliced. This activity of the exon 12 regulatory element suggests that it is an exonic splicing silencer. Consistent with its activity as a splicing silencer, spliceosome assembly was inhibited on RNA substrates containing exon 12 compared with RNAs representing other regions of the G6PD transcript. Elimination of nucleotides 43-72 of exon 12 did not restore splicing of exon 12-containing RNA; thus, the 30-nucleotide element may not be exclusively a silencer. The binding of heterogeneous nuclear ribonucleoproteins K, L, and A2/B1 from both HeLa and hepatocyte nuclear extracts to the element further supports its activity as a silencer. In addition, SR proteins bind to the element, consistent with the presence of enhancer activity within this sequence. Thus, an exonic splicing silencer is involved in the inhibition of splicing of a constitutively spliced exon in the G6PD mRNA.

Alu family sequences are middle repetitive short interspersed elements (SINEs) dispersed throughout vertebrate genomes that can modulate gene transcription. The human (h) GH locus contains 44 complete and four partial Alu elements. An Sx Alu repeat lies in close proximity to the hGH-1 and hGH-2 genes in the 3′-flanking region. Deletion of the Sx Alu repeat in reporter constructs containing hGH-1 3′-flanking sequences increased reporter activity in transfected pituitary GC cells, suggesting this region contained a repressor element. Analysis of multiple deletion fragments from the 3′-flanking region of the hGH-1 gene revealed a strong orientation- and position-independent silencing activity mapping between nucleotides 2158 and 2572 encompassing the Sx Alu repeat. Refined mapping revealed that the silencer was a complex element comprising four discrete entities, including a core repressor domain (CRD), an antisilencer domain (ASE) that contains elements mediating the orientation-independent silencer activity, and two domains flanking the CRD/ASE that modulate silencer activity in a CRD-dependent manner. The upstream modulator domain is also required for orientation-independent silencer function. EMSA with DNA fragments representing all of the silencer domains yielded a complex pattern of DNA-protein interactions indicating that numerous GC cell nuclear proteins bind specifically to the CRD, ASE, and modulator domains. The silencer is GH promoter dependent and, in turn, its presence decreases the rate of promoter-associated histone acetylation resulting in a significant decrease of RNA polymerase II recruitment to the promoter. The silencer may provide for complex regulatory control of hGH gene expression in pituitary cells.

Precursor messenger RNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles in a stepwise process. The spliceosome's dynamic nature suggests the potential for regulation at numerous points along the assembly pathway; however, thus far, naturally occurring regulation of splicing has only been found to influence a small subset of spliceosomal intermediates. Here we report that the exonic splicing silencer (ESS1) that represses splicing of PTPRC (encoding CD45) exon 4 does not function by the typical mechanism of inhibiting binding of U1 or U2 small nuclear ribonucleoproteins (snRNPs) to the splice sites. Instead, a U1-, U2- and ATP-dependent complex forms across exon 4 that is required for inhibiting progression to the U4-U6-U5 tri-snRNP-containing B complex. Such inhibition represents a new mechanism for splicing regulation and suggests that regulation can probably occur at many of the transitions along the spliceosome assembly pathway.

The AMPA receptor subunit GluR2 is downregulated in neurons following a wide range of neurological insults. Here we report that suppression of GluR2 gene promoter activity is associated with kainate (KA)-induced downregulation of GluR2 subunit levels in primary cultured cortical neurons. RT-PCR and Northern blotting showed a significant decrease in GluR2 mRNA in cultured neurons after KA exposure. Transfection of cultured neurons with an expression vector pGL3-GluR2(-298/+283), where the reporter gene firefly luciferase was driven by the GluR2 promoter, revealed that KA exposure suppressed the transcriptional activation of the GluR2 promoter. Furthermore, the expression of the RE1-silencing transcription factor (REST) was increased in KA-exposed cortical neurons; enhanced binding of REST to RE1-like silencer element in the proximal promoter of the GluR2 subunit gene was evidenced by electrophoresis mobility shift assay. Chromatin immunoprecipitation showed that suppressed activity of the GluR2 promoter in cultured neurons after KA exposure was related to deacetylation of histone H4. These results indicate that REST as a crucial factor binds to RE1-like silencer element in the GluR2 promoter, suppressing transcription of the GluR2 subunit gene during KA exposure. Our data suggest that transcriptional suppression of the GluR2 subunit gene may contribute at least in part to downregulation of GluR2 subunit protein in neurons during KA exposure. Because our experiments showed a reduction of glutamate release in KA-exposed cortical neurons, REST may play a latent role in delayed neuronal death or in seizure-induced tolerance.

We designed a new promoter that drives transgene expression in an exclusively neuron-specific manner. The promoter of superior cervical ganglion10 (SCG10), expressed in neurons, was further modified to enhance its neuron specificity and activity by changing its length and fusing a multiple neuronal restrictive silencer element (NRSE) to its upstream or downstream regions. The promoter, which contained 2 kb original promoter length and two extra NRSEs in its downstream region, eventually exhibited remarkable neuron specificity as well as strong activity. To further amplify the promoter activity, the promoter was introduced into a Cre recombinase (Cre)-expressing adenovirus, and subsequent combination with Cre-inducible enhanced green fluorescence protein (EGFP)-expressing adenovirus vector, which has much stronger general promoter, resulted in a remarkably strong gene expression exclusively in neuronal cells of mixed cultures and in an animal model. This system is also applicable to astrocyte-specific expression; for instance, by changing the Cre promoter cassette to an astrocyte-specific promoter. The present relatively compact promoter combined with Cre/loxP system could be useful for a wide range of transgene experiments in vivo as well as for clinical applications.

Tombusviridae is a large positive-strand RNA virus family. Tomato bushy stunt virus (TBSV), the type virus of this family, has a genome ending with AGCCC(-OH), termed the 3'-complementary silencer sequence (3'CSS). The 3'CSS is able to base pair with a complementary internally-located sequence, 5'GGGCU, called the replication silencer element (RSE). In TBSV, previous compensatory mutational analysis of the RSE-3'CSS interaction showed it to be functionally important for viral RNA synthesis both in vitro and in vivo. However, these investigations also revealed that the RSE and 3'CSS are very sensitive to nucleotide changes, even when base pairing potential between the two elements is maintained. Consequently, an alternative investigative approach was used in this study where the wild-type sequences of these elements were preserved and their surrounding contexts were modified. Results from these analyses, using a TBSV DI RNA, revealed important new structural requirements necessary for the RSE and 3'CSS to operate in vivo. Collectively, the data suggest that accessibility of the elements and their proximity to adjoining stem structures are important functional parameters. Based on these findings, a working structural model for the TBSV RSE-3'CSS interaction is proposed that involves coaxial stacking of adjacent helices at either end of the RSE-3'CSS interaction. Components of this structural model are extendable to potential RSE-3'CSS interactions that were identified throughout Tombusviridae by comparative sequence analysis. This survey also revealed a significant level of diversity and modularity with respect to RSEs, 3'CSSs and their structural contexts and, moreover, suggests that RSE-3'CSS interactions are prevalent in Tombusviridae and related viruses.

Nuclear aggregates of polyglutamine (polyQ)-expanded proteins are associated with a number of neurodegenerative diseases including Huntington's disease (HD) and spinocerebellar ataxias (SCAs). The nuclear deposition of polyQ proteins correlates with rearrangements of nuclear matrix, transcriptional dysregulation, and cell death. To explore the requirement for polyQ tracks in educing such cellular responses, we examined whether a non-polyQ protein can deposit as nuclear aggregates and elicit similar responses. We report that a protein chimera (GFP170*) composed of the green fluorescent protein (GFP) fused to an internal fragment of the Golgi Complex Protein (GCP-170) forms nuclear aggregates analogous to those formed by polyQ proteins. Like the polyQ nuclear aggregates, GFP170* inclusions recruit molecular chaperones and proteasomal components, alter nuclear structures containing the promyelocytic leukemia protein (PML), recruit transcriptional factors such as CREB-binding protein (CBP) and p53, repress p53 transcriptional activity, and induce cell death. Our results indicate that nuclear aggregation and transcriptional effects are not unique to polyQ-containing proteins and may represent a general response to misfolded proteins in the nucleus.

Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease primarily affecting the brainstem, retina and Purkinje cells of the cerebellum. The disease is caused by a polyglutamine expansion in ataxin-7, a protein found in two complexes TFTC and STAGA, involved in transcriptional regulation. Transcriptional dysregulation has been implicated in the pathology of several polyglutamine diseases. In this paper, we analyzed the effect of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP and the Purkinje cell expressed nuclear receptor RORalpha1. We could show that transcription mediated by both CBP and RORalpha1 was repressed by expanded ataxin-7. Interestingly, repression of transcription could also be observed with wild-type full-length ataxin-7, not only on CBP- and RORalpha1-mediated transcription, but also on basal transcription. The repression could be counteracted by inhibition of deacetylation, suggesting that ataxin-7 may act as a repressor of transcription by inhibiting the acetylation activity of TFTC and STAGA.

Mcm10 is an essential protein that participates in both the initiation and the elongation of DNA replication. In this study we demonstrate a role for Mcm10 in the maintenance of heterochromatic silencing at telomeres and HM loci of budding yeast. Two mcm10 mutants drastically reduce silencing of both URA3 and ADE2 reporter genes integrated into these silent loci. When exposed to α-factor, mcm10 mutant cells display a “shmoo-cluster” phenotype associated with a defect in the maintenance of silencing. In addition, when combined with a defect in the establishment of silent chromatin, mcm10 mutants demonstrate a synergistic defect in HML silencing. Consistent with a direct silencing function, Mcm10p shows a two-hybrid interaction with Sir2p and Sir3p that is destroyed by the mcm10-1 mutation and dependent on the C-terminal 108 amino acids. Tethering GBD-MCM10 to a defective HMR-E silencer is not sufficient to restore silencing. Furthermore, mutations in MCM10 inhibit the ability of GBD-SIR3 to restore silencing when tethered to a defective HMR-E. Suppressor mutations in MCM2, which suppress the temperature sensitivity of mcm10-1, fail to overcome the mcm10-1 silencing defect, suggesting that MCM10's role in transcriptional silencing may be separate from its essential functions in DNA replication.

Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78: 8254-8263, 2004). To define cis -acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae , which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3′-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.

The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.

The human aromatase (hCYP19) gene is controlled by tissue-specific promoters that lie upstream of tissue-specific first exons. Placenta-specific exon I.1 lies approximately 100,000 bp upstream of exon II. Previously, we observed that genomic sequences within 501 bp upstream of exon I.1 mediate placenta-specific expression. In the present study, transgenic mice were created carrying hCYP19I.1−246:hGH/hGX, hCYP19I.1−201:hGH, and hCYP19I.1−125:hGH fusion genes to further delineate 5′-flanking sequences within 501 bp of exon I.1 that are required to mediate placenta-specific hCYP19 gene expression. As little as 246 bp of hCYP19 exon I.1 5′-flanking sequence was sufficient to direct placenta-specific expression in transgenic mice. By contrast, transgenes containing 201 or 125 bp of exon I.1 5′-flanking DNA were not expressed in mouse placenta. Furthermore, hCYP19I.1−246:hGX transgene expression was developmentally regulated; expression was observed as early as embryonic d 7.5 (E7.5) in several cells of the trophoblast ectoderm, on E8.5 in some trophoblast giant cells, and by E9.5 in giant cells and the labyrinthine layer. By contrast, expression of the hCYP19I.1−501:hGH transgene was first observed on E10.5 and was restricted to the labyrinthine layer, which is most analogous to the human syncytiotrophoblast. This suggests the presence of regulatory elements between −501 and −246 bp that may bind inhibitory transcription factors expressed in giant cells. These findings from transgenic experiments together with deletion mapping studies using transfected human placental cells indicate that the concerted interaction of strong placenta-specific enhancers and silencers within this 501-bp region mediate labyrinthine and syncytiotrophoblast-specific CYP19 gene expression.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 2 modulates the tau N-terminal domain, which interacts with the axonal membrane. Exon 10 codes for a microtubule binding domain, increasing the affinity of tau for microtubules. Both exons are excluded from fetal brain, but their default behavior is inclusion, suggesting that silencers are involved in their regulation. Exon 2 is significantly reduced in myotonic dystrophy type 1, whose symptoms include dementia. Mutations that affect exon 10 splicing cause frontotemporal dementia (FTDP). In this study, we investigated three regulators of exon 2 and 10 splicing: serine/arginine-rich (SR) proteins SRp55, SRp30c, and htra2beta1. The first two inhibit both exons; htra2beta1 inhibits exon 2 but activates exon 10. By deletion analysis, we identified splicing silencers located at the 5' end of each exon. Furthermore, we demonstrated that SRp30c and SRp55 bind to both silencers and to each other. In exon 2, htra2beta1 binds to the inhibitory heterodimer through its RS1 domain but not to exon 2, whereas in exon 10 the heterodimer may sterically interfere with htra2beta1 binding to a purine-rich enhancer (defined by FTDP mutation E10-Delta5 = Delta280K) directly downstream of the silencer. Increased exon 10 inclusion in FTDP mutant ENH (N279K) may arise from abolishing SRp30c binding. Also, htra2beta3, a naturally occurring variant of htra2beta1, no longer inhibits exon 2 splicing but can partially rescue splicing of exon 10 in FTDP mutation E10-Delta5. This work provides interesting insights into the splicing regulation of the tau gene.

The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.

Regulation of transgene expression is a major goal of gene therapy research. Previously, we have developed a complex adenovirus (Ad) vector with tetracycline-regulated expression of a Fas ligand (FasL)-green fluorescent protein (GFP) fusion protein. This vector delivered high levels of activity that was regulated by doxycycline. However, this regulation was limited by the low but significant background activity of the TRE promoter. Recently, the Tet-regulated transcriptional silencer, tTS, was reported to suppress efficiently basal TRE activity without affecting induced expression levels. Here, we report development of Ad vectors that incorporate tTS in combination with that of reverse transactivator (rtTA) coupled with TRE promoter driving transgene expression. Incorporation of tTS improved control of transgene expression in vitro, so that an induction range of over three orders of magnitude was achieved in some cell lines. Effective regulation of transgene expression was also seen in a mouse model in vivo, following systemic vector delivery. In the case of FasL-GFP expression, significant improvement in the control of apoptotic activity both in vitro and in a mouse hepatotoxicity model was demonstrated when using rtTA-tTS vectors. In conclusion, a highly effective transgene regulation system, deliverable by a single adenoviral vector, is now available.

Floral scent is important to plant reproduction because it attracts pollinators to the sexual organs. Therefore, volatile emission is usually tuned to the foraging activity of the pollinators. In Petunia hybrida, volatile benzenoids determine the floral aroma. Although the pathways for benzenoid biosynthesis have been characterized, the enzymes involved are less well understood. How production and emission are regulated is unknown. By targeted transcriptome analyses, we identified ODORANT1 (ODO1), a member of the R2R3-type MYB family, as a candidate for the regulation of volatile benzenoids in Petunia hybrida cv W115 (Mitchell) flowers. These flowers are only fragrant in the evening and at night. Transcript levels of ODO1 increased before the onset of volatile emission and decreased when volatile emission declined. Downregulation of ODO1 in transgenic P. hybrida Mitchell plants strongly reduced volatile benzenoid levels through decreased synthesis of precursors from the shikimate pathway. The transcript levels of several genes in this pathway were reduced by suppression of ODO1 expression. Moreover, ODO1 could activate the promoter of the 5-enol-pyruvylshikimate-3-phosphate synthase gene. Flower pigmentation, which is furnished from the same shikimate precursors, was not influenced because color and scent biosynthesis occur at different developmental stages. Our studies identify ODO1 as a key regulator of floral scent biosynthesis.

Exonic splicing silencers (ESSs) are cis-regulatory elements that inhibit the use of adjacent splice sites, often contributing to alternative splicing (AS). To systematically identify ESSs, an in vivo splicing reporter system was developed to screen a library of random decanucleotides. The screen yielded 141 ESS decamers, 133 of which were unique. The silencer activity of over a dozen of these sequences was also confirmed in a heterologous exon/intron context and in a second cell type. Of the unique ESS decamers, most could be clustered into groups to yield seven putative ESS motifs, some resembling known motifs bound by hnRNPs H and A1. Potential roles of ESSs in constitutive splicing were explored using an algorithm, ExonScan, which simulates splicing based on known or putative splicing-related motifs. ExonScan and related bioinformatic analyses suggest that these ESS motifs play important roles in suppression of pseudoexons, in splice site definition, and in AS.

The N-terminal alanine residues of the silencing protein Sir3 and of Orc1 are acetylated by the NatA Nα-acetyltransferase. Mutations demonstrate that the N terminus of Sir3 is important for its function. Sir3 and, perhaps, also Orc1 are the NatA substrates whose lack of acetylation in ard1 and nat1 mutants explains the silencing defect of those mutants.

To understand the DNA-methylation mediated gene silencing mechanisms, we analyzed in cell culture of the promoter function of the MAGE-A1 gene, which is frequently demethylated and over-expressed in human hepatocellular carcinoma. We have established the correlation of the DNA methylation of the promoter CpG island with expression status of this gene in a panel of the established liver cancer cell lines. The crucial CpG dinucleotide(s) within the minimal promoter subjected to the control mediated by DNA methylation with profound biological functions was also delineated. Furthermore, a novel sequence-specific DNA-protein interaction at the -30 CpG dinucleotide upstream of the gene was found having a vital part to play in the DNA methylation mediated transcription silencing of the MAGE-A1 gene. Our results would not only provide new insights into the DNA methylation mediated mechanisms over transcription of the MAGE-A1 gene, but also pave the way for further defining the cross-talk among DNA methylation, histone modification and chromatin remodeling in detail.

The fibroblast growth factor receptor 2 (FGFR2) gene exons IIIb and IIIc are alternatively spliced in a mutually exclusive and cell type-specific manner. FGFR2 exon choice depends on both activation and silencing. Exon IIIb silencing requires cis-acting elements upstream and downstream of the exon. To examine the influence of transcription on exon IIIb silencing, the putative RNA polymerase II (RNAPII)-pausing MAZ4 element was inserted at different positions within the FGFR2 minigene construct. MAZ4 insertions 5' to the upstream silencing elements or between exon IIIb and downstream silencing elements result in decreased silencing. An insertion 3' of the downstream silencing elements, however, has no effect on splicing. An RT-PCR elongation assay shows that the MAZ4 site in these constructs is likely to be a RNAPII pause site. Insertion of another RNAPII pause site into the minigene has a similar effect on exon IIIb silencing. Transfection of in vitro transcribed RNA demonstrates that the cell type specificity of FGFR2 alternative splicing requires co-transcriptional splicing. Additionally, changing the promoter alters both FGFR2 minigene splicing and the MAZ4 effect. We propose that RNAPII pauses at the MAZ4 elements resulting in a change in the transcription elongation complex that influences alternative splicing decisions downstream.

The neuronal-specific protein complexin I (CPX I) plays an important role in controlling the Ca(2+)-dependent neurotransmitter release. Since insulin exocytosis and neurotransmitter release rely on similar molecular mechanisms and that pancreatic beta-cells and neuronal cells share the expression of many restricted genes, we investigated the potential role of CPX I in insulin-secreting cells. We found that pancreatic islets and several insulin-secreting cell lines express high levels of CPX I. The beta-cell expression of CPX I is mediated by the presence of a neuron restrictive silencer element located within the regulatory region of the gene. This element bound the transcriptional repressor REST, which is found in most cell types with the exception of mature neuronal cells and beta-cells. Overexpression of CPX I or silencing of the CPX I gene (Cplx1) by RNA interference led to strong impairment in beta-cell secretion in response to nutrients such as glucose, leucine and KCl. This effect was detected both in the early and the sustained secretory phases but was much more pronounced in the early phase. We conclude that CPX I plays a critical role in beta-cells in the control of the stimulated-exocytosis of insulin.

Human pre-mRNAs contain a definite number of exons and several pseudoexons which are located within intronic regions. We applied a computational approach to address the question of how pseudoexons are neglected in favor of exons and to possibly identify sequence elements preventing pseudoexon splicing. A search for possible splicing silencers was carried out on a pseudoexon selection that resembled exons in terms of splice site strength and exon splicing enhancer (ESE) representation; three motifs were retrieved through hexamer composition comparisons. One of these functions as a powerful silencer in transfection-based splicing assays and matches a previously identified silencer sequence with hnRNP H binding ability. The other two motifs are novel and failed to induce skipping of a constitutive exon, indicating that they might act as weak repressors or in synergy with other unidentified elements. All three motifs are enriched in pseudoexons compared with intronic regions and display higher frequencies in intronless gene-coding sequences compared with exons. We consider that a subpopulation of pseudoexons might rely on negative regulators for splicing repression; this hypothesis, if experimentally verified, might improve our understanding of exonic splicing regulatory sequences and provide the identification of a novel mutation target for human genetic diseases.

The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses flowering during embryo and seedling development. As fie alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early flowering and homeotic conversion of leaves, flower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2) and SHOOTMERISTEMLESS (STM), whereas BREVIPEDICELLUS (BP) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our findings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle.

Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded (ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. The NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with NRSF/REST transcriptional machinery, resulting in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST into cells with neuronal identity that can express neuronal genes. The mechanism of action appears to be mediated through a dsRNA/protein interaction, rather than through siRNA or miRNA. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of noncoding RNAs as key regulators of cell behavior at both transcriptional and posttranscriptional levels.

Vimentin exhibits a complex pattern of developmental- and tissue-specific expression and is aberrantly expressed in most metastatic tumors. The human vimentin promoter contains multiple DNA elements, some of which enhance gene expression and one that inhibits. A silencer element (at -319) binds the repressor ZBP-89. Further upstream (at -757) is an element, which acts positively in the presence of the silencer element and, thus, is referred to as an antisilencer (ASE). Previously, we showed that Stat1alpha binds to this element upon induction by IFN-gamma. However, substantial binding and reporter gene activity was still present in nontreated cells. Here, we have found that Stat3 binds to the ASE element in vitro. Transfection experiments in COS-1 cells with various vimentin promoter--reporter constructs show that gene activity is dependent upon the cotransfection and activation of Stat3. Moreover, activated Stat3 can overcome ZBP-89 repression. Coimmunoprecipitation studies demonstrate that Stat3 and ZBP-89 can interact and confocal microscopy detects these factors to be colocalized in the nucleus. Moreover, a correlation exists between the presence of activated Stat3 and vimentin expression in MDA-MB-231 cells, which is lacking in MCF7 cells where vimentin is not expressed. In the light of these results, we propose that the interaction of Stat3 and ZBP-89 may be crucial for overcoming the effects of the repressor ZBP-89, which suggests a novel mode for Stat3 gene activation.

the maternal and the paternal side (McGrath and Solter 1984; Surani et al. 1984). Their genetic nonequivalence is mediated by genomic imprinting, an epigenetic mechanism resulting in a parent-of-origin-dependent expression of a small number of genes (Tilghman 1999). The best studied pair of imprinted genes are H19 and Igf2, two closely linked, oppositely imprinted genes located on mouse chromosome 7. H19 is transcribed from the maternally inherited allele (Bartolomei et al. 1991), whereas Igf2 is paternally expressed (DeChiara et al. 1991). The coordinated expression of H19 and Igf2 is controlled by two regulatory elements: a shared enhancer downstream of the H19 gene (Leighton et al. 1995), and an element referred to as imprinting control region (ICR) located upstream of the H19 gene (Fig. 1). Much attention has focused on the H19 ICR as a key element in the regulation of monoallelic expression of H19 and Igf2, as its deletion results in activation of the normally silent H19 allele and a concomitant reduction in Igf2 expression upon paternal transmission, with a reciprocal effect upon maternal inheritance of the deletion (Thorvaldsen et al. 1998).

DMP1 is an acidic phosphorylated protein with the spatial and temporal expression that is largely restricted to bone and tooth tissues. The biological function of DMP1 is associated with biomineralization of bone, cartilage and tooth development. To study the cell-specific expression of DMP1, a 2,512 bp upstream segment of the human gene was isolated and characterized. A series of progressive deletions of the human DMP1 5' flanking sequence were ligated to the luciferase reporter gene, and their promoter activities examined in transfected human osteoblast-like (MG-63) and dental pulp (HDP-D) cells that express DMP1 and hepatic (HepG2) and uterine (HeLa) cells lacking DMP1 expression. A critical cis-regulatory element located between nt -150 and -63 was found to act as a specific silencer responsible for the negative regulation of DMP1 in HepG2 and HeLa cells. The transcriptional activity of this element in MG-63 and HDP-D cells had a 5-7-fold increase than that observed in HepG2 and HeLa cells. Electrophoretic mobility shift assays (EMSAs) showed that a 6-bp DNA sequence in this element was bound by two nuclear factors that are expressed at high levels in HepG2 and HeLa versus MG-63 and HDP-D cells. Competitive assays by EMSAs suggest that the 6-bp core DNA sequence, AG(T/C)C(A/G)C, is a novel DNA-protein binding site and conserved with high identity in reported DMP1 promoters for all species. Furthermore, point mutations of the core sequence caused a marked increase of DMP1 promoter activity in HepG2 and HeLa cells. We speculate that this silencing cis-element may play a critical role in the regulation of DMP1 cell-specific expression.

The enzyme glutamine synthetase (GS) ranks as one of the most remarkable glucocorticoid-inducible mammalian genes. In many tissues and cell lines, the synthetic glucocorticoid dexamethasone alone increases GS expression several fold. The direct response is mainly mediated by a cellular glucocorticoid receptor that, upon binding of the hormone, interacts with glucocorticoid responsive elements (GREs) of the gene. In cells of hepatocellular origin the response is mediated by a GRE located in the first intron of the gene. Surprisingly, hepatocytes do not respond to glucocorticoids with enhanced GS expression, despite the presence of an intact glucocorticoid receptor, which, in the same cells, stimulates expression of other genes such as tyrosine amino transferase. Reporter gene assays identified a sequence element downstream from the intronic GRE that inhibits the enhancement of expression by glucocorticoids. This silencer was designated GS silencer element of the rat. Gel mobility shift assays demonstrate the binding of a factor in hepatocyte nuclear extract. This yet unknown factor was designated GS silencer-binding protein. It is absent in FAO cells that respond to glucocorticoids with enhanced expression of GS and present in HepG2 cells that do not respond.

The Saccharomyces cerevisiae RRM3 gene encodes a 5' to 3' DNA helicase. While replication of most of the yeast genome was not dependent upon Rrm3p, in its absence, replication forks paused and often broke at an estimated 1400 discrete sites, including tRNA genes, centromeres, inactive replication origins, and transcriptional silencers. These replication defects were associated with activation of the intra-S phase checkpoint. Activation of the checkpoint was critical for viability of rrm3Delta cells, especially at low temperatures. Each site whose replication was affected by Rrm3p is assembled into a nonnucleosomal protein-DNA complex. At tRNA genes and the silent mating type loci, disruption of these complexes eliminated dependence upon Rrm3p. These data indicate that the Rrm3p DNA helicase helps replication forks traverse protein-DNA complexes, naturally occurring impediments that are encountered in each S phase.

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, we previously identified a novel DNA element [14 bp 5′-repressor element (FRE)] of the 5-HT1A receptor gene that mediates repression in neuronal and non-neuronal cells (Ou et al., 2000). We have now cloned a novel DNA binding protein [five′ repressor element under dual repression binding protein-1 (Freud-1)] that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptorsin vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression.

The cyclooxygenase-2 (COX-2) enzyme catalyzes the rate-limiting step of prostaglandin formation in inflammatory states, and COX-2 overexpression plays a key role in carcinogenesis. To understand the mechanisms regulating COX-2 expression, we examined its posttranscriptional regulation mediated through the AU-rich element (ARE) within the COX-2 mRNA 3′-untranslated region (3′UTR). RNA binding studies, performed to identify ARE-binding regulatory factors, demonstrated binding of the translational repressor protein TIA-1 to COX-2 mRNA. The significance of TIA-1-mediated regulation of COX-2 expression was observed in TIA-1 null fibroblasts that produced significantly more COX-2 protein than wild-type fibroblasts. However, TIA-1 deficiency did not alter COX-2 transcription or mRNA turnover. Colon cancer cells demonstrated to overexpress COX-2 through increased polysome association with COX-2 mRNA also showed defective TIA-1 binding both in vitro and in vivo. These findings implicate that TIA-1 functions as a translational silencer of COX-2 expression and support the hypothesis that dysregulated RNA-binding of TIA-1 promotes COX-2 expression in neoplasia.

Huntingtin protein is mutated in Huntington disease. We previously reported that wild-type but not mutant huntingtin stimulates transcription of the gene encoding brain-derived neurotrophic factor (BDNF; ref. 2). Here we show that the neuron restrictive silencer element (NRSE) is the target of wild-type huntingtin activity on BDNF promoter II. Wild-type huntingtin inhibits the silencing activity of NRSE, increasing transcription of BDNF. We show that this effect occurs through cytoplasmic sequestering of repressor element-1 transcription factor/neuron restrictive silencer factor (REST/NRSF), the transcription factor that binds to NRSE. In contrast, aberrant accumulation of REST/NRSF in the nucleus is present in Huntington disease. We show that wild-type huntingtin coimmunoprecipitates with REST/NRSF and that less immunoprecipitated material is found in brain tissue with Huntington disease. We also report that wild-type huntingtin acts as a positive transcriptional regulator for other NRSE-containing genes involved in the maintenance of the neuronal phenotype. Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington disease. We conclude that wild-type huntingtin acts in the cytoplasm of neurons to regulate the availability of REST/NRSF to its nuclear NRSE-binding site and that this control is lost in the pathology of Huntington disease. These data identify a new mechanism by which mutation of huntingtin causes loss of transcription of neuronal genes.

The imprinting of the genes on human chromosome 11p15.5 is thought to be controlled by two imprinting control regions located in two differentially methylated CpG islands upstream of the H19 gene (H19 DMR) and in intron 10 of the KCNQ1 gene (KvDMR). We have examined sequences in the human 11p15.5 genomic imprinted region for the presence of insulators and silencers using a position- and enhancer-dependent stable transfection assay. We have confirmed the existence of insulators in H19 DMR and discovered two novel insulators in the IGF2 gene. We have also found two novel silencer sequences; one is located in KvDMR, a region that is thought to contain the promoter for the KCNQ1OT1 transcript, and another is in the CDKN1C gene. We have demonstrated binding of CTCF protein in vitro to all the insulator and silencer sequences that we have detected. We discuss the differences in the regulation of imprinting controlled by the two imprinting control regions in chromosome 11p.

The retinoid receptors have major roles throughout development, even in the absence of ligand. Here, we summarize an emerging theme whereby gene repression, mediated by unliganded retinoid receptors, can dictate cell fate. In addition to activating transcription, retinoid receptors actively repress gene transcription by recruiting cofactors that promote chromatin compaction. Two developmental processes for which gene silencing by the retinoid receptors is essential are head formation in Xenopus and skeletal development in the mouse. Inappropriate repression, by oncogenic retinoic acid (RA)**Abbreviations used in this paper: APL, acute promyelocytic leukemia; dnRARα, dominant–negative version of the RARα; E, embryonic age; HDAC, histone deacetylase; LCoR, ligand-dependent corepressor; NCoR, nuclear receptor corepressor; RA, retinoic acid; RAR, RA receptor; RARE, RXR homodimer bound to bipartite response element; RXR, retinoid X receptor; TSA, trichostatin A; CYP26, cytochrome p450, 26; TR, thyroid hormone receptor. receptor (RAR) fusion proteins, blocks myeloid differentiation leading to a rare form of leukemia. Our current understanding of the developmental role of retinoid repression and future perspectives in this field are discussed.

Fission yeast centromeres are composed of two domains: the central core and the outer repeats. Although both regions are required for full centromere function, the central core has a distinct chromatin structure and is likely to underlie the kinetochore itself, as it is associated with centromere-specific proteins. Genes placed within either region are transcriptionally silenced, reflecting the formation of a functional kinetochore complex and flanking centromeric heterochromatin. Here, transcriptional silencing was exploited to identify components involved in central core silencing and kinetochore assembly or structure. The resulting sim (silencing in the middle of the centromere) mutants display severe chromosome segregation defects. sim2+ encodes a known kinetochore protein, the centromere-specific histone H3 variant Cnp1CENP-A. sim4+ encodes a novel essential coiled-coil protein, which is specifically associated with the central core region and is required for the unusual chromatin structure of this region. Sim4 coimmunoprecipitates with the central core component Mis6 and, like Mis6, affects Cnp1CENP-A association with the central domain. Functional Mis6 is required for Sim4 localization at the kinetochore. Our analyses illustrate the fundamental link between silencing, chromatin structure, and kinetochore function, and establish defective silencing as a powerful approach for identifying proteins required to build a functional kinetochore.

We demonstrate that the tendons associated with the axial skeleton derive from a heretofore unappreciated, fourth compartment of the somites. Scleraxis (Scx), a bHLH transcription factor, marks this somitic tendon progenitor population at its inception, and is continuously expressed through differentiation into the mature tendons. Two earlier-formed somitic compartments, the sclerotome and myotome, interact to establish this fourth Scx-positive compartment. The tendon progenitors are induced at the sclerotome's edge, at the expense of skeletogenic Pax1 positive cells and in response to FGF signaling in the adjacent myotome. The tendon primordia thus form in a location abutting the two tissues that the mature tendons must ultimately connect. Tendon progenitor formation may reveal a general mechanism for the specification of other somitic subcompartments.

Expression of the POU transcription factor Brn-3a is regulated spatially and temporally during embryogenesis and can be detected in a particular subset of neurons and neuronal tumors. In the present study, we investigated the transcriptional regulation of Brn-3a expression. The human promoter is TATA-less and contains two bona fide mRNA start sites spread over 500 nucleotides 5′ of the ATG translation initiation codon. Furthermore, a second TATA-less promoter was detected in the first intron of the Brn-3a gene and our data suggest that this promoter regulates expression of the shorter isoform of the Brn-3a gene. Transcriptional activity from this promoter was found in neuronal cell lines but not in epithelial cells. The activity of this promoter was strongly enhanced upon deletion of a 24 base pair (bp) DNA element downstream of the transcription initiation sites. In addition, this DNA element was able to repress transcription from a heterologous promoter suggesting a function as a transcriptional silencer. The more distal upstream Brn-3a promoter directing the expression of the longer Brn-3a isoform is not affected by the presence of this putative 24 bp DNA element. We propose therefore that the existence of two promoters in the Brn-3a gene offers a possibility for the observed differential and cell type-specific expression of the gene.

Overexpression of vascular endothelial growth factor (VEGF) is implicated in a number of diseases. It is therefore critical that mechanisms exist to strictly regulate VEGF expression. A hypoxia-responsive (HR) region of the VEGF promoter which binds the HIF-1 transcription factor is a target for many signals that up-regulate VEGF transcription. Repressors targeting the HIF-1 transcription factor have been identified but no repressors directly binding the HR promoter region had been reported. We now report a novel mechanism of repression of the VEGF HR region involving DNA binding. We find that single strand DNA-specific cold shock domain (CSD or Y-box) proteins repress the HR region via a binding site downstream of the HIF-1 site. The repressor site is functional in unstimulated, normoxic fibroblasts and represents a novel means to prevent expression of VEGF in the absence of appropriate stimuli. We characterized complexes forming on the VEGF repressor site and identified a previously unreported nuclear CSD protein complex containing dbpA. Nuclear dbpA appears to bind as a dimer and we determined a means by which nuclear CSD proteins may enter double strand DNA to bind to their single strand sites to bring about repression of the VEGF HR region.

The G protein-coupled receptor GPR10 is highly localized to areas of the brain. In an effort to reveal transcriptional determinants of this tissue specificity, we recognized a putative NRSE (neuron-restrictive silencer element) located in the 5′ promoter region of the gene. The cognate NRSE binding protein NRSF (neuron-restrictive silencer factor) restricts gene expression to mature neurons and endocrine cells by repressing their transcription in non-neuronal/-endocrine cells. In cell lines where NRSF-mediated gene repression has been functionally established, the activity of the GPR10 promoter was repressed in a manner consistent with NRSE-dependent regulation. A specific point mutation to confer non-functionality of the NRSE revealed a 10-fold de-repression of reporter gene expression. In contrast, in the GPR10-expressing cell line GH3, mRNA transcripts of NRSF were undetectable and suppression of promoter activity was not observed. However, transfection of a rat NRSF expression vector resulted in significant repression of transcription, which was reversed by mutation of the NRSE. In conclusion, we demonstrate that the GPR10 gene is specifically regulated by NRSF, and suggest this to be a contributory factor in the tissue-specific distribution of GPR10 in vivo.

Background: Metazoan centromeres are generally composed of large repetitive DNA structures packaged in heterochromatin. Similarly, fission yeast centromeres contain large inverted repeats and two distinct silenced domains that are both required for centromere function. The central domain is flanked by outer repetitive elements coated in histone H3 methylated on lysine 9 and bound by conserved heterochromatin proteins. This centromeric heterochromatin is required for cohesion between sister centromeres. Defective heterochromatin causes premature sister chromatid separation and chromosome missegregation. The role of cis-acting DNA sequences in the formation of centromeric heterochromatin has not been established.Results: A deletion strategy was used to identify centromeric sequences that allow heterochromatin formation in fission yeast. Fragments from the outer repeats are sufficient to cause silencing of an adjacent gene when inserted at a euchromatic chromosomal locus. This silencing is accompanied by the local de novo methylation of histone H3 on lysine 9, recruitment of known heterochromatin components, Swi6 and Chp1, and the provision of a new strong cohesin binding site. In addition, we demonstrate that the chromodomain of Chp1 binds to MeK9-H3 and that Chp1 itself is required for methylation of histone H3 on lysine 9.Conclusions: A short sequence, reiterated at fission yeast centromeres, can direct silent chromatin assembly and cohesin recruitment in a dominant manner. The heterochromatin formed at the euchromatic locus is indistinguishable from that found at endogenous centromeres. Recruitment of Rad21-cohesin underscores the link between heterochromatin and chromatid cohesion and indicates that these centromeric elements act independently of kinetochore activity to recruit cohesin.

Background: Eukaryotic DNA replication starts at many origins. Some origins are used early in S phase, while others are programmed to fire later. In general, late replication is correlated with transcriptional inactivity and with location near the nuclear periphery. However, the mechanisms that determine replication timing are unclear, and the cause-and-effect relationship between late replication, transcriptional inactivity, and location at the nuclear periphery is unknown. Results: Using budding yeast, we show that a transcriptional silencer, HMR-E , can reset the time of initiation of ARS305 from early to late. This resetting requires Sir proteins, which are silencers of transcription. Resetting can also be achieved by targeting Sir4 to ARS305 . HMR-E sequences and targeted Sir4, both of which cause late replication of ARS305 , also cause transcriptional silencing of the nearby APA1 gene. Conclusions: Sir proteins are sufficient to reprogram an origin from early to late; that is, Sir proteins are a cause of late replication. Presumably, the tight chromatin structure promoted by Sir proteins favors both transcriptional inactivity and late replication.

Neural crest cells (NCC) are a transient migratory cell population that generates diverse cell types such as neurons and glia of the enteric nervous system (ENS). Via an insertional mutation screen for loci affecting NCC development in mice, we identified one line—named TashT—that displays a partially penetrant aganglionic megacolon phenotype in a strong male-biased manner. Interestingly, this phenotype is highly reminiscent of human Hirschsprung’s disease, a neurocristopathy with a still unexplained male sex bias. In contrast to the megacolon phenotype, colonic aganglionosis is almost fully penetrant in homozygous TashT animals. The sex bias in megacolon expressivity can be explained by the fact that the male ENS ends, on average, around a “tipping point” of minimal colonic ganglionosis while the female ENS ends, on average, just beyond it. Detailed analysis of embryonic intestines revealed that aganglionosis in homozygous TashT animals is due to slower migration of enteric NCC. The TashT insertional mutation is localized in a gene desert containing multiple highly conserved elements that exhibit repressive activity in reporter assays. RNAseq analyses and 3C assays revealed that the TashT insertion results, at least in part, in NCC-specific relief of repression of the uncharacterized gene Fam162b; an outcome independently confirmed via transient transgenesis. The transcriptional signature of enteric NCC from homozygous TashT embryos is also characterized by the deregulation of genes encoding members of the most important signaling pathways for ENS formation—Gdnf/Ret and Edn3/Ednrb—and, intriguingly, the downregulation of specific subsets of X-linked genes. In conclusion, this study not only allowed the identification of Fam162b coding and regulatory sequences as novel candidate loci for Hirschsprung’s disease but also provides important new insights into its male sex bias., Author Summary Hirschsprung’s disease (also known as aganglionic megacolon) is a severe congenital defect of the enteric nervous system (ENS) resulting in complete failure to pass stools. It is characterized by the absence of neural ganglia (aganglionosis) in the distal gut due to incomplete colonization of the embryonic intestines by neural crest cells (NCC), the ENS precursors. Hirschsprung’s disease has an incidence of 1 in 5000 newborns and a 4:1 male sex bias. Although many genes have been associated with this complex genetic disease, most of its heritability as well as its male sex bias remain unexplained. Here, we describe an insertional mutant mouse line (“TashT”) in which virtually all homozygotes display colonic aganglionosis due to defective migration of enteric NCC, but in which only a subset of homozygotes develops megacolon. Surprisingly, this group is almost exclusively male. The TashT ENS defect stems, at least in part, from the disruption of long-range interactions between evolutionarily conserved elements with silencer activity and Fam162b, resulting in NCC-specific upregulation of this uncharacterized protein coding gene. Global analysis of gene expression further revealed that several hundreds of genes are significantly deregulated in TashT enteric NCC. Interestingly, this dataset includes multiple X-linked candidate genes potentially underlying the male sex bias. Taken together, our data pave the way for a clearer understanding of the intriguing male sex bias of Hirschsprung’s disease.