Chromatin Boundary Elements Research Articles

SHIELD: a platform for high-throughput screening of barrier-type DNA elements in human cells

Chromatin boundary elements contribute to the partitioning of mammalian genomes into topological domains to regulate gene expression. Certain boundary elements are adopted as DNA insulators for safe and stable transgene expression in mammalian cells. These elements, however, are ill-defined and less characterized in the non-coding genome, partially due to the lack of a platform to readily evaluate boundary-associated activities of putative DNA sequences. Here we report SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains), a platform tailored for the high-throughput screening of barrier-type DNA elements in human cells. SHIELD takes advantage of the high specificity of serine integrase at heterochromatin, and exploits the natural heterochromatin spreading inside lamina-associated domains (LADs) for the discovery of potent barrier elements. We adopt SHIELD to evaluate the barrier activity of 1000 DNA elements in a high-throughput manner and identify 8 candidates with barrier activities comparable to the core region of cHS4 element in human HCT116 cells. We anticipate SHIELD could facilitate the discovery of novel barrier DNA elements from the non-coding genome in human cells.

Disruption of CTCF Boundary Remodels Chromatin Domain and Reprograms HOX Gene Transcription in Acute Myeloid Leukemia

The organization of the mammalian genome into separate domains either facilitates or restricts interactions between regulatory elements, e.g. enhancers and promoters, for normal gene regulation. CTCF insulator constrains the temporal HOX gene expression patterns within the confined chromatin domain for normal development. However, dysregulation of HOX genes is also a common feature of acute myeloid leukemia (AML). The molecular mechanisms of aberrant HOX expression and associated AML pathogenesis remain unclear. Here, we found that CTCF binding site located between HOXA7 and HOXA9 genes (CBS7/9) acts as a chromatin boundary to establish and maintain AML specific aberrant HOX gene expression patterns and is essential for pathogenesis of myeloid malignancies.To unbiasedly and systematically investigate the mechanism underlying HOX gene regulation in AML, we employed a pooled CRISPR-Cas9 genetic knockout library screening to interrogate intergenic regulatory and chromatin boundary elements in all 4 HOX gene loci. Through this screening, we identified the CBS7/9 chromatin boundary located between HOXA7 and HOXA9 genes that is critical for maintaining aberrant expression of posterior HOXA genes including oncogene HOXA9 and HOXA10 in primary AML patients and cells. To further confirm the role of CBS7/9 in HOX gene regulation, two single-guide RNAs (sgRNAs) flanking 47 base pairs (bps) of the core CTCF motif located between HOXA7 and HOXA9 geneswere employed to guild Cas9 for deletion of the CBS7/9 boundary in 3 different subtypes of AML cells containing MLL rearrangements or NPM1C+ mutation. The defected CBS7/9 resulted in a decrease in CTCF binding to the caudal HOXA domain and an inhibition of posterior HOXA gene expression.To further delineate the role of CBS7/9 chromatin boundary in HOX gene organization and regulation, we then carried out genome wide ChIP-seq, ATAC-seq, 4C, and RNA-seq analysis to examine the changes in chromatin domain organization and corresponding gene expression patterns compared wild-type control and the CBS7/9 KO AML cells. Disruption of CBS7/9 boundary resulted in a spreading of repressive H3K27me3 domain into the caudal HOXA domain that subsequently impaired AML associated active TAD domain by altering enhancer/promoter chromatin accessibility and reducing ectopic interactions among the posterior HOXA genes. Consistent with the role of CBS7/9 boundary in HOX locus chromatin structure and organization, impairment of CBS7/9 boundary function reduced posterior HOXA gene expression and altered myeloid specific transcriptome profiles important for pathogenesis of myeloid malignancies suggesting that the CBS7/9 boundary mediated ectopic HOX expression patterns might be essential for pathogenesis of myeloid malignancies. To further test this possibility, the CBS 7/9 boundary was deleted in both MLL rearranged AML cells and primary patient samples by the CRISPR-Cas9 mediated genome editing and the edited cells were transplanted into NSG mice for the development of human AML in mice. Disruption of CBS chromatin boundary in the HOXA locus reduced human leukemic blast development and invasion as well as enhanced survival of both transplanted AML cell xenograft and patient derived xenograft (PDX) mouse models. Thus, our finding demonstrated that CTCF boundary not only constrains normal gene expression program for memory of cell identity, but also plays a role in maintaining oncogenic transcription program for leukemic transformation. Alteration of CTCF mediated topological boundaries in the oncogene loci provides therapeutic approaches for targeting oncogenic expression program and leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Insulator foci distance correlates with cellular and nuclear morphology in early Drosophila embryos

The three-dimensional (3D) organization of the genome is highly dynamic, changing during development and varying across different tissues and cell types. Recent studies indicate that these changes alter regulatory interactions, leading to changes in gene expression. Despite its importance, the mechanisms that influence genomic organization remain poorly understood. We have previously identified a network of chromatin boundary elements, or insulators, in the Drosophila Antennapedia homeotic complex (ANT-C). These genomic elements interact with one another to tether chromatin loops that could block or promote enhancer-promoter interactions. To understand the function of these insulators, we assessed their interactions by measuring their 3D nuclear distance in developing animal tissues. Our data suggest that the ANT-C Hox complex might be in a folded or looped configuration rather than in a random or extended form. The architecture of the ANT-C complex, as read out by the pair-wise distance between insulators, undergoes a strong compression during late embryogenesis, coinciding with the reduction of cell and nuclear diameters due to continued cell divisions in post-cleavage cells. Our results suggest that genomic architecture and gene regulation may be influenced by cellular morphology and movement during development.

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.

The β-NAD+ salvage pathway and PKC-mediated signaling influence localized PARP-1 activity and CTCF Poly(ADP)ribosylation.

Poly(ADP)ribosylation (PARylation) of the chromatin architectural protein CTCF is critical for CTCF-dependent regulation of chromatin boundary and insulator elements. Loss of CTCF PARylation results in epigenetic silencing of certain tumor suppressor genes through destabilization of nearby chromatin boundaries. We investigated the metabolic and mechanistic processes that regulate PARP-1-mediated CTCF PARylation in human cancer cell lines and discovered a key role for the expression and activity of β-NAD+ salvage enzymes, NAMPT and NMNAT-1. These enzymes are downregulated in cells that exhibit reduced CTCF PARylation, resulting in a decreased concentration of nuclear β-NAD+. In these cells, decreased NMNAT-1 expression is enforced by a proteasome-mediated feedback loop resulting in degradation of NMNAT-1, transcriptional repression of NAMPT, and suppression of PARP-1 activity. Interestingly, dePARylated CTCF is associated in a stable protein complex with PARP-1 and NMNAT-1 in cancer cells harboring silenced tumor suppressor genes. Although the metabolic context in these cells favors suppression of PARP-1 activity, CTCF PARylation can be restored by Protein Kinase C (PKC) signaling. PKC induces dissociation of the catalytically inactive PARP-1/NMNAT-1/CTCF protein complex and phosphorylation of NMNAT-1, which stimulates its proteasome-mediated degradation. Our findings suggest that CTCF PARylation is underpinned by a cellular metabolic context engendered by regulation of the β-NAD+ salvage pathway in which NMNAT-1 acts as a rheostat to control localized β-NAD+ synthesis at CTCF/PARP-1 complexes.

Different Evolutionary Strategies To Conserve Chromatin Boundary Function in the Bithorax Complex.

Chromatin boundary elements subdivide chromosomes in multicellular organisms into physically independent domains. In addition to this architectural function, these elements also play a critical role in gene regulation. Here we investigated the evolution of a Drosophila Bithorax complex boundary element called Fab-7, which is required for the proper parasegment specific expression of the homeotic Abd-B gene. Using a “gene” replacement strategy, we show that Fab-7 boundaries from two closely related species, D. erecta and D. yakuba, and a more distant species, D. pseudoobscura, are able to substitute for the melanogaster boundary. Consistent with this functional conservation, the two known Fab-7 boundary factors, Elba and LBC, have recognition sequences in the boundaries from all species. However, the strategies used for maintaining binding and function in the face of sequence divergence is different. The first is conventional, and depends upon conservation of the 8 bp Elba recognition sequence. The second is unconventional, and takes advantage of the unusually large and flexible sequence recognition properties of the LBC boundary factor, and the deployment of multiple LBC recognition elements in each boundary. In the former case, binding is lost when the recognition sequence is altered. In the latter case, sequence divergence is accompanied by changes in the number, relative affinity, and location of the LBC recognition elements.

Chromatin boundary elements organize genomic architecture and developmental gene regulation in Drosophila Hox clusters.

The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and "delivering" remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development.

Specific combinations of boundary element and Polycomb response element are required for the regulation of the Hox genes in Drosophila melanogaster

In the bithorax complex of Drosophila melanogaster, the chromatin boundary elements (BE) demarcate cis-regulatory domains that regulate Hox genes along the anteroposterior body axis. These elements are closely associated with the Polycomb Response Elements (PREs) and restrict the ectopic activation of cis-regulatory domains during development. The relevance of such specific genomic arrangements of regulatory elements remains unclear. Deletions of individual BE–PRE combination result in distinct homeotic phenotypes. In this study, we show that deletion of two such BE–PRE combinations in cis leads to new genetic interactions, which manifests as dorsal closure defect phenotype in adult abdominal epithelia. We further demonstrate that dorsal closure phenotype results from enhanced and ectopic expression of Hox gene Abd-B in the larval epithelial cells. This suggests a specific role of multiple BE–PRE combinations in the larval epithelial cells for regulation of Abd-B. Using chromosome conformation capture experiments, we show that genetic interactions correlate with direct physical interactions among the BE–PRE combinations. Our results demonstrate the functional relevance of the closely associated BE and PRE combinations in regulation of Hox genes.

Supervised learning method for predicting chromatin boundary associated insulator elements

In eukaryotic cells, the DNA material is densely packed inside the nucleus in the form of a DNA-protein complex structure called chromatin. Since the actual conformation of the chromatin fiber defines the possible regulatory interactions between genes and their regulatory elements, it is very important to understand the mechanisms governing folding of chromatin. In this paper, we show that supervised methods for predicting chromatin boundary elements are much more effective than the currently popular unsupervised methods. Using boundary locations from published Hi-C experiments and modEncode tracks as features, we can tell the insulator elements from randomly selected background sequences with great accuracy. In addition to accurate predictions of the training boundary elements, our classifiers make new predictions. Many of them correspond to the locations of known insulator elements. The key features used for predicting boundary elements do not depend on the prediction method. Because of its miniscule size, chromatin state cannot be measured directly, we need to rely on indirect measurements, such as ChIP-Seq and fill in the gaps with computational models. Our results show that currently, at least in the model organisms, where we have many measurements including ChIP-Seq and Hi-C, we can make accurate predictions of insulator positions.

A ChIP-on-chip tiling array approach detects functional histone-free regions associated with boundaries at vertebrate HOX genes.

Hox genes impart segment identity to body structures along the anterior–posterior axis and are crucial for proper development. A unique feature of the Hox loci is the collinearity between the gene position within the cluster and its spatial expression pattern along the body axis. However, the mechanisms that regulate collinear patterns of Hox gene expression remain unclear, especially in higher vertebrates. We recently identified novel histone-free regions (HFRs) that can act as chromatin boundary elements demarcating successive murine Hox genes and help regulate their precise expression domains (Srivastava et al., 2013). In this report, we describe in detail the ChIP-chip analysis strategy associated with the identification of these HFRs. We also provide the Perl scripts for HFR extraction and quality control analysis for this custom designed tiling array dataset.

Something silent this way forms: the functional organization of the repressive nuclear compartment.

The repressive compartment of the nucleus is comprised primarily of telomeric and centromeric regions, the silent portion of ribosomal RNA genes, the majority of transposable element repeats, and facultatively repressed genes specific to different cell types. This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Both chromatin remodeling proteins and transcription of noncoding RNAs are involved in maintenance of repression in these compartments. Global reorganization of the repressive compartment occurs at each cell division, during early development, and during terminal differentiation. Differential action of chromatin remodeling complexes and boundary element looping activities are involved in mediating these organizational changes. We discuss the evidence that heterochromatin formation and compartmentalization may drive nuclear organization.

Direct Interplay among Histones, Histone Chaperones, and a Chromatin Boundary Protein in the Control of Histone Gene Expression

In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.

Epigenetic Regulation of the Latency-Associated Region of Marek's Disease Virus in Tumor-Derived T-Cell Lines and Primary Lymphoma

Meq is the major Marek's disease virus (MDV)-encoded oncoprotein and is essential for T-cell lymphomagenesis. Meq and several noncoding RNAs, including three microRNA (MiR) clusters, are expressed from the repeats of the MDV genome during latent infection of T cells. To investigate the state of the chromatin in this and flanking regions, we carried out chromatin immunoprecipitation (ChIP) analysis of covalent histone modifications and associated bound proteins. T-cell lines and a lymphoma were compared. The chromatin around the promoters for Meq and the noncoding RNAs in both cell lines and the lymphoma were associated with H3K9 acetylation and H3K4 trimethylation, which are marks of transcriptionally active chromatin. These correlated with bound Meq-c-Jun heterodimers. The only binding site for Meq homodimers is located at the lytic origin of replication (OriLyt), next to the lytic gene pp38. This region lacked active marks and was associated with repressive histone modifications (H3K27 and H3K9 trimethylation). DNA CpG methylation was investigated using methylated DNA precipitation (MeDP). In cell lines, DNA methylation was abundant across the repeats but noticeably reduced or absent around the active promoters. In primary tumors, CpG methylation occurred less than 2 months after infection, focused within the ICP4 gene. These data suggest that nonrandom de novo DNA methylation occurs early in lymphomagenesis. In addition, the histone data indicate a role for Meq in the epigenetic regulation of the MDV genome repeats in transformed T cells and suggest that the OriLyt region and the Meq/MiR region might be separated by chromatin boundary elements, and preliminary data on CTCF binding are consistent with this.

Genome-wide prediction and analysis of human chromatin boundary elements

Boundary elements partition eukaryotic chromatin into active and repressive domains, and can also block regulatory interactions between domains. Boundary elements act via diverse mechanisms making accurate feature-based computational predictions difficult. Therefore, we developed an unbiased algorithm that predicts the locations of human boundary elements based on the genomic distributions of chromatin and transcriptional states, as opposed to any intrinsic characteristics that they may possess. Application of our algorithm to ChIP-seq data for histone modifications and RNA Pol II-binding data in human CD4+ T cells resulted in the prediction of 2542 putative chromatin boundary elements genome wide. Predicted boundary elements display two distinct features: first, position-specific open chromatin and histone acetylation that is coincident with the recruitment of sequence-specific DNA-binding factors such as CTCF, EVI1 and YYI, and second, a directional and gradual increase in histone lysine methylation across predicted boundaries coincident with a gain of expression of non-coding RNAs, including examples of boundaries encoded by tRNA and other non-coding RNA genes. Accordingly, a number of the predicted human boundaries may function via the synergistic action of sequence-specific recruitment of transcription factors leading to non-coding RNA transcriptional interference and the blocking of facultative heterochromatin propagation by transcription-associated chromatin remodeling complexes.

A Single Heterochromatin Boundary Element Imposes Position-Independent Antisilencing Activity in Saccharomyces cerevisiae Minichromosomes

Chromatin boundary elements serve as cis-acting regulatory DNA signals required to protect genes from the effects of the neighboring heterochromatin. In the yeast genome, boundary elements act by establishing barriers for heterochromatin spreading and are sufficient to protect a reporter gene from transcriptional silencing when inserted between the silencer and the reporter gene. Here we dissected functional topography of silencers and boundary elements within circular minichromosomes in Saccharomyces cerevisiae. We found that both HML-E and HML-I silencers can efficiently repress the URA3 reporter on a multi-copy yeast minichromosome and we further showed that two distinct heterochromatin boundary elements STAR and TEF2-UASrpg are able to limit the heterochromatin spreading in circular minichromosomes. In surprising contrast to what had been observed in the yeast genome, we found that in minichromosomes the heterochromatin boundary elements inhibit silencing of the reporter gene even when just one boundary element is positioned at the distal end of the URA3 reporter or upstream of the silencer elements. Thus the STAR and TEF2-UASrpg boundary elements inhibit chromatin silencing through an antisilencing activity independently of their position or orientation in S. cerevisiae minichromosomes rather than by creating a position-specific barrier as seen in the genome. We propose that the circular DNA topology facilitates interactions between the boundary and silencing elements in the minichromosomes.

Epigenetic control of expression of the human L‐ and M‐ pigment genes

Epigenetics alters gene expression by chromatin modification without changing the sequence of DNA. DNA methylation is an essential signal for epigenetic gene regulation. Methylation of cytosine bases at CpG dinucleotides in DNA results in chromatin condensation resulting in suppression of gene expression. DNA methylation has been shown to play important roles in cell differentiation, genomic imprinting and X-chromosome inactivation. We compared the CpG methylation patterns of the promoters of the L-opsin gene (OPN1LW) and the M-opsin gene (OPN1MW), plus a DNase I hypersensitive (DHS) site located about 8 kb (kilobases) upstream of the OPN1LW gene. Comparisons were made using the human retinoblastoma cell line WERI, which expresses the L and M opsin genes when treated with thyroid hormone (T3), and a lymphoblastoid cell line GM06990 that does not express these genes. The results showed that the great majority of the 14 CpGs located within the proximal 200 bp (base pairs) of each promoter, plus 20 bp of the 5'-untranslated region, were hypo-methylated in WERI-Rb-1 cells, whether or not treated with T3, but almost totally methylated in the lymphoblastoid cell line. Three of the CpGs that are located beyond 200 bp from the transcription start site of OPN1LW were hyper-methylated in both WERI and lymphoblastoid cells. Significant differential methylation was also observed within the DHS region (24 CpGs). This DHS region contains a highly conserved motif that binds CCCTC-binding factor (CTCF), referred to as a 'chromatin insulator or boundary element', that has been shown to regulate gene expression at several genome locations. The results suggest that DNA methylation is likely to contribute to regulation of expression of the L- and M-opsin genes during differentiation, as well as to the retinal L:M cone ratio. In addition, thyroid hormone induction of the opsin genes does not appear to alter DNA methylation.

Histone deacetylase Rpd3 antagonizes Sir2-dependent silent chromatin propagation

In the eukaryotic genome, transcriptionally silent chromatin tends to propagate along a chromosome and encroach upon adjacent active chromatin. The silencing machinery can be stopped by chromatin boundary elements. We performed a screen in Saccharomyces cerevisiae for proteins that may contribute to the establishment of a chromatin boundary. We found that disruption of histone deacetylase Rpd3p results in defective boundary activity, leading to a Sir-dependent local propagation of transcriptional repression. In rpd3Δ cells, the amount of Sir2p that was normally found in the nucleolus decreased and the amount of Sir2p found at telomeres and at HM and its adjacent loci increased, leading to an extension of silent chromatin in those areas. In addition, Rpd3p interacted directly with chromatin at boundary regions to deacetylate histone H4 at lysine 5 and at lysine 12. Either the mutation of histone H4 at lysine 5 or a decrease in the histone acetyltransferase (HAT) activity of Esa1p abrogated the silencing phenotype associated with rpd3 mutation, suggesting a novel role for the H4 amino terminus in Rpd3p-mediated heterochromatin boundary regulation. Together, these data provide insight into the molecular mechanisms for the anti-silencing functions of Rpd3p during the formation of heterochromatin boundaries.

Genome wide ChIP-chip analyses reveal important roles for CTCF in Drosophila genome organization

Insulators or chromatin boundary elements are defined by their ability to block transcriptional activation by an enhancer and to prevent the spread of active or silenced chromatin. Recent studies have increasingly suggested that insulator proteins play a role in large-scale genome organization. To better understand insulator function on the global scale, we conducted a genome-wide analysis of the binding sites for the insulator protein CTCF in Drosophila by Chromatin Immunoprecipitation (ChIP) followed by a tiling-array analysis. The analysis revealed CTCF binding to many known domain boundaries within the Abd-B gene of the BX-C including previously characterized Fab-8 and MCP insulators, and the Fab-6 region. Based on this finding, we characterized the Fab-6 insulator element. In genome-wide analysis, we found that dCTCF-binding sites are often situated between closely positioned gene promoters, consistent with the role of CTCF as an insulator protein. Importantly, CTCF tends to bind gene promoters just upstream of transcription start sites, in contrast to the predicted binding sites of the insulator protein Su(Hw). These findings suggest that CTCF plays more active roles in regulating gene activity and it functions differently from other insulator proteins in organizing the Drosophila genome.

A genetic screen in Saccharomyces cerevisiae identifies new genes that interact with mex67-5, a temperature-sensitive allele of the gene encoding the mRNA export receptor

The Mex67p protein, together with Mtr2p, functions as the mRNA export receptor in Saccharomyces cerevisiae by interacting with both mRNA and nuclear pore complexes. To identify genes that interact functionally with MEX67, we used transposon insertion to search for mutations that suppressed the temperature-sensitive mex67-5 allele. Four suppressors are described here. The screen revealed that mutant Mex67-5p, but not wild-type Mex67p, is a target of the nuclear protein quality control mediated by San1p, a ubiquitin-protein ligase that participates in degradation of aberrant chromatin-associated proteins. Our finding that overexpression of the SPT6 gene alleviates the growth defects of the mex67-5 strain, together with the impairment of poly(A)(+) RNA export caused by depletion of Spt6p or the related protein Iws1p/Spn1p, supports the mechanism proposed in mammalian cells for Spt6-mediated co-transcriptional loading of mRNA export factors during transcription elongation. Finally, our results also uncovered genetic connections between Mex67p and the poly(A) nuclease complex and with components of chromatin boundary elements.

Role of an Insulator in Testis‐specific Gene Transcription

Testis-specific promoters are unique in that relatively short proximal promoters of several genes have been shown to be capable of directing tissue- and cell-type-specific expression in transgenic mice. How such small promoter fragments perform the dual functions of maintaining a silenced state in somatic tissues and activating gene expression in the correct germ-cell type in testis remains poorly understood. Studies from our laboratory using the round spermatid-specific SP-10 gene as an experimental model have provided some insights into the mechanisms involved. It was found that the proximal promoter of the SP-10 gene acts as a chromatin insulator or boundary element in somatic tissues and prevents transcription of the SP-10 gene. In round spermatids, the insulator function is relieved, thus facilitating the SP-10 gene transcription. Insulators act as enhancer blockers and/or barriers to heterochromatin to protect the programmed expression of a gene. Typically, insulators are separable from promoters. In the case of the SP-10 gene, however, the insulator overlaps the promoter and operates in a facultative manner. We hypothesize that the proximal promoters of some testis-specific genes have adapted the insulator function to maintain transcriptional silence in the somatic tissues.