The Myc-associated zinc finger protein epigenetically controls expression of interferon-γ-stimulated genes by recruiting STAT1 to chromatin.

The MYC-Associated Zinc Finger Protein (MAZ) plays important roles in chromatin organization and gene transcription regulation. Dysregulated expression of MAZ causes diseases, such as glioblastoma, breast cancer, prostate cancer, and liposarcoma. Previously, it has been reported that MAZ controls the proinflammatory response in colitis and colon cancer via STAT3 signaling, suggesting that MAZ is involved in regulating immunity-related pathways. However, the molecular mechanism underlying this regulation remains elusive. Here, we investigate the regulatory effect of MAZ on interferon-gamma (IFN-γ)-stimulated genes via STAT1, a protein that plays an essential role in immune responses to viral, fungal, and mycobacterial pathogens. We demonstrate that about 80% of occupied STAT1-binding sites colocalize with occupied MAZ-binding sites in HAP1/K562 cells after IFN-γ stimulation. MAZ depletion significantly reduces STAT1 binding in the genome. By analyzing genome-wide gene expression profiles in the RNA-Seq data, we show that MAZ depletion significantly suppresses a subset of the immune response genes, which include the IFN-stimulated genes IRF8 and Absent in Melanoma 2. Furthermore, we find that MAZ controls expression of the immunity-related genes by changing the epigenetic landscape in chromatin. Our study reveals an important role for MAZ in regulating immune-related gene expression.

The Myc-associated zinc finger protein (MAZ) works together with CTCF to control cohesin positioning and genome organization.

The Myc-associated zinc finger protein (MAZ) is often found at genomic binding sites adjacent to CTCF, a protein which affects large-scale genome organization through its interaction with cohesin. We show here that, like CTCF, MAZ physically interacts with a cohesin subunit and can arrest cohesin sliding independently of CTCF. It also shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and consequently affects RNA alternative splicing. CTCF/MAZ double sites are more effective at sequestering cohesin than sites occupied only by CTCF. Furthermore, depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ. In an assay for insulation activity like that used for CTCF, binding of MAZ to sites between an enhancer and promoter results in down-regulation of reporter gene expression, supporting a role for MAZ as an insulator protein. Hi-C analysis of the effect of MAZ depletion on genome organization shows that local interactions within topologically associated domains (TADs) are disrupted, as well as contacts that establish the boundaries of individual TADs. We conclude that MAZ augments the action of CTCF in organizing the genome, but also shares properties with CTCF that allow it to act independently.

CTCF: making the right connections.

The role of the zinc finger protein CTCF in organizing the genome within the nucleus is now well established. Widely separated sites on DNA, occupied by both CTCF and the cohesin complex, make physical contacts that create large loop domains. Additional contacts between loci within those domains, often also mediated by CTCF, tend to be favored over contacts between loci in different domains. A large number of studies during the past 2 years have addressed the questions: How are these loops generated? What are the effects of disrupting them? Are there rules governing large-scale genome organization? It now appears that the strongest and evolutionarily most conserved of these CTCF interactions have specific rules for the orientation of the paired CTCF sites, implying the existence of a nonequilibrium mechanism of generation. Recent experiments that invert, delete, or inactivate one of a mating CTCF pair result in major changes in patterns of organization and gene expression in the surrounding regions. What remain to be determined are the detailed molecular mechanisms for re-establishing loop domains and maintaining them after replication and mitosis. As recently published data show, some mechanisms may involve interactions with noncoding RNAs as well as protein cofactors. Many CTCF sites are also involved in other functions such as modulation of RNA splicing and specific regulation of gene expression, and the relationship between these activities and loop formation is another unanswered question that should keep investigators occupied for some time.

DNA·RNA triple helix formation can function as a cis-acting regulatory mechanism at the human ß-globin locus.

We have identified regulatory mechanisms in which an RNA transcript forms a DNA duplex·RNA triple helix with a gene or one of its regulatory elements, suggesting potential auto-regulatory mechanisms in vivo. We describe an interaction at the human ß-globin locus, in which an RNA segment embedded in the second intron of the ß-globin gene forms a DNA·RNA triplex with the HS2 sequence within the ß-globin locus control region, a major regulator of globin expression. We show in human K562 cells that the triplex is stable in vivo. Its formation causes displacement from HS2 of major transcription factors and RNA Polymerase II, and consequently in loss of factors and polymerase that bind to the human ε- and γ-globin promoters, which are activated by HS2 in K562 cells. This results in reduced expression of these genes. These effects are observed when a small length of triplex-forming RNA is introduced into cells, or when a full-length intron-containing human ß-globin transcript is expressed. Related results are obtained in human umbilical cord blood-derived erythroid progenitor-2 cells, in which ß-globin expression is similarly affected by triplex formation. These results suggest a model in which RNAs conforming to the strict sequence rules for DNA·RNA triplex formation may participate in feedback regulation of genes in cis.

Insulin promoter in human pancreatic ß cells contacts diabetes susceptibility loci and regulates genes affecting insulin metabolism.

Both type 1 and type 2 diabetes involve a complex interplay between genetic, epigenetic, and environmental factors. Our laboratory has been interested in the physical interactions, in nuclei of human pancreatic ß cells, between the insulin (INS) gene and other genes that are involved in insulin metabolism. We have identified, using Circularized Chromosome Conformation Capture (4C), many physical contacts in a human pancreatic ß cell line between the INS promoter on chromosome 11 and sites on most other chromosomes. Many of these contacts are associated with type 1 or type 2 diabetes susceptibility loci. To determine whether physical contact is correlated with an ability of the INS locus to affect expression of these genes, we knock down INS expression by targeting the promoter; 259 genes are either up or down-regulated. Of these, 46 make physical contact with INS We analyze a subset of the contacted genes and show that all are associated with acetylation of histone H3 lysine 27, a marker of actively expressed genes. To demonstrate the usefulness of this approach in revealing regulatory pathways, we identify from among the contacted sites the previously uncharacterized gene SSTR5-AS1 and show that it plays an important role in controlling the effect of somatostatin-28 on insulin secretion. These results are consistent with models in which clustering of genes supports transcriptional activity. This may be a particularly important mechanism in pancreatic ß cells and in other cells where a small subset of genes is expressed at high levels.

Interactome mapping defines BRG1, a component of the SWI/SNF chromatin remodeling complex, as a new partner of the transcriptional regulator CTCF.

The highly conserved zinc finger CCCTC-binding factor (CTCF) regulates genomic imprinting and gene expression by acting as a transcriptional activator or repressor of promoters and insulator of enhancers. The multiple functions of CTCF are accomplished by co-association with other protein partners and are dependent on genomic context and tissue specificity. Despite the critical role of CTCF in the organization of genome structure, to date, only a subset of CTCF interaction partners have been identified. Here we present a large-scale identification of CTCF-binding partners using affinity purification and high-resolution LC-MS/MS analysis. In addition to functional enrichment of specific protein families such as the ribosomal proteins and the DEAD box helicases, we identified novel high-confidence CTCF interactors that provide a still unexplored biochemical context for CTCF's multiple functions. One of the newly validated CTCF interactors is BRG1, the major ATPase subunit of the chromatin remodeling complex SWI/SNF, establishing a relationship between two master regulators of genome organization. This work significantly expands the current knowledge of the human CTCF interactome and represents an important resource to direct future studies aimed at uncovering molecular mechanisms modulating CTCF pleiotropic functions throughout the genome.

Sodium valproate rescues expression of TRANK1 in iPSC-derived neural cells that carry a genetic variant associated with serious mental illness.

Biological characterization of genetic variants identified in genome-wide association studies (GWAS) remains a substantial challenge. Here we used human-induced pluripotent stem cells (iPSC) and their neural derivatives to characterize common variants on chromosome 3p22 that have been associated by GWAS with major mental illnesses. IPSC-derived neural progenitor cells carrying the risk allele of the single nucleotide polymorphism (SNP), rs9834970, displayed lower baseline TRANK1 expression that was rescued by chronic treatment with therapeutic dosages of valproic acid (VPA). VPA had the greatest effects on TRANK1 expression in iPSC, NPC, and astrocytes. Although rs9834970 has no known function, we demonstrated that a nearby SNP, rs906482, strongly affects binding by the transcription factor, CTCF, and that the high-affinity allele usually occurs on haplotypes carrying the rs9834970 risk allele. Decreased expression of TRANK1 perturbed expression of many genes involved in neural development and differentiation. These findings have important implications for the pathophysiology of major mental illnesses and the development of novel therapeutics.

Order from chaos in the nucleus.

Although it might appear that chromatin is randomly packed within the nucleus, recent data (Hou et al., 2012, in this issue of Molecular Cell) show that it is organized into defined and functionally important domains marked by preferred intradomain physical contacts, and with boundaries associated with insulator protein occupancy.

Association of the Long Non-coding RNA Steroid Receptor RNA Activator (SRA) with TrxG and PRC2 Complexes.

Long non-coding RNAs (lncRNAs) have been recognized as key players in transcriptional regulation. We show that the lncRNA steroid receptor RNA activator (SRA) participates in regulation through complex formation with trithorax group (TrxG) and polycomb repressive complex 2 (PRC2) complexes. Binding of the SRA-associated RNA helicase p68 preferentially stabilizes complex formation between SRA and a TrxG complex but not PRC2. In human pluripotent stem cells NTERA2, SRA binding sites that are also occupied by p68 are significantly enriched for H3K4 trimethylation. Consistent with its ability to interact with TrxG and PRC2 complexes, some SRA binding sites in human pluripotent stem cells overlap with bivalent domains. CTCF sites associated with SRA appear also to be enriched for bivalent modifications. We identify NANOG as a transcription factor directly interacting with SRA and co-localizing with it genome-wide in NTERA2. Further, we show that SRA is important for maintaining the stem cell state and for reprogramming of human fibroblasts to achieve the pluripotent state. Our results suggest a mechanism whereby the lncRNA SRA interacts with either TrxG or PRC2. These complexes may then be recruited by various DNA binding factors to deliver either activating or silencing signals, or both, to establish bivalent domains.

Mediation of CTCF transcriptional insulation by DEAD-box RNA-binding protein p68 and steroid receptor RNA activator SRA.

CCCTC-binding factor (CTCF) is a DNA-binding protein that plays important roles in chromatin organization, although the mechanism by which CTCF carries out these functions is not fully understood. Recent studies show that CTCF recruits the cohesin complex to insulator sites and that cohesin is required for insulator activity. Here we showed that the DEAD-box RNA helicase p68 (DDX5) and its associated noncoding RNA, steroid receptor RNA activator (SRA), form a complex with CTCF that is essential for insulator function. p68 was detected at CTCF sites in the IGF2/H19 imprinted control region (ICR) as well as other genomic CTCF sites. In vivo depletion of SRA or p68 reduced CTCF-mediated insulator activity at the IGF2/H19 ICR, increased levels of IGF2 expression, and increased interactions between the endodermal enhancer and IGF2 promoter. p68/SRA also interacts with members of the cohesin complex. Depletion of either p68 or SRA does not affect CTCF binding to its genomic sites, but does reduce cohesin binding. The results suggest that p68/SRA stabilizes the interaction of cohesin with CTCF by binding to both, and is required for proper insulator function.

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions.

The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA cross-linking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.

Mapping of long-range INS promoter interactions reveals a role for calcium-activated chloride channel ANO1 in insulin secretion.

We used circular chromatin conformation capture (4C) to identify a physical contact in human pancreatic islets between the region near the insulin (INS) promoter and the ANO1 gene, lying 68 Mb away on human chromosome 11, which encodes a Ca(2+)-dependent chloride ion channel. In response to glucose, this contact was strengthened and ANO1 expression increased, whereas inhibition of INS gene transcription by INS promoter targeting siRNA decreased ANO1 expression, revealing a regulatory effect of INS promoter on ANO1 expression. Knockdown of ANO1 expression caused decreased insulin secretion in human islets, establishing a physical proximity-dependent feedback loop involving INS transcription, ANO1 expression, and insulin secretion. To explore a possible role of ANO1 in insulin metabolism, we carried out experiments in Ano1(+/-) mice. We observed reduced serum insulin levels and insulin-to-glucose ratios in high-fat diet-fed Ano1(+/-) mice relative to Ano1(+/+) mice fed the same diet. Our results show that determination of long-range contacts within the nucleus can be used to detect novel and physiologically relevant mechanisms. They also show that networks of long-range physical contacts are important to the regulation of insulin metabolism.

Induced pluripotency enables differentiation of human nullipotent embryonal carcinoma cells N2102Ep.

Embryonal carcinoma (EC) cells, which are considered to be malignant counterparts of embryonic stem cells, comprise the pluripotent stem cell component of teratocarcinomas, a form of testicular germ cell tumors (GCTs). Nevertheless, many established human EC cell lines are nullipotent with limited or no capacity to differentiate under normal circumstances. In this study, we tested whether an over-expression of Yamanaka's reprogramming factors OCT4, SOX2, c-MYC and KLF4 might enable differentiation of the human nullipotent EC cells N2102Ep. Using OCT4 knockdown differentiated N2102Ep cells, we are able to derive reprogrammed N2102Ep cell lines. The induced pluripotency of N2102Ep allows the cells to differentiate toward neural lineage by retinoic acid; the expression of SSEA3 and SSEA4 is down-regulated, whereas that of neural surface markers is up-regulated. Consistent with the up-regulation of neural surface markers, the expression of the master neuroectodermal transcription factor PAX6 is also induced in reprogrammed N2102Ep. We next investigated whether PAX6 might induce spontaneous differentiation of nullipotent stem cells N2102Ep. However, while an ectopic expression of PAX6 promotes differentiation of NTERA2, it induces cell death in N2102Ep. We nevertheless find that upon induction of retinoic acid, the reprogrammed N2102Ep cells form mature neuronal morphology similar to differentiated pluripotent stem cells NTERA2 as determined by TUJ1 expression, which is absent in N2102Ep parental cells. Altogether, we conclude that the nullipotent state of human EC cells can be reprogrammed to acquire a more relaxed state of differentiation potential by Yamanaka's factors.

Vezf1 protein binding sites genome-wide are associated with pausing of elongating RNA polymerase II.

The protein Vezf1 plays multiple roles important for embryonic development. In Vezf1(-/-) mouse embryonic stem (mES) cells, our earlier data showed widespread changes in gene-expression profiles, including decreased expression of the full-length active isoform of Dnmt3b methyltransferase and concomitant genome-wide reduction in DNA methylation. Here we show that in HeLaS3 cells there is a strong genome-wide correlation between Vezf1 binding and peaks of elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of elongation. In WT mES cells, the elongating form of RNA pol II accumulates near Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and this accumulation is significantly reduced at these sites in Vezf1(-/-) mES cells. Depending upon genomic location, Vezf1-mediated Pol II pausing can have different regulatory roles in transcription and splicing. We find examples of genes in which Vezf1 binding sites are located near cassette exons, and in which loss of Vezf1 leads to a change in the relative abundance of alternatively spliced messages. We further show that Vezf1 interacts with Mrg15/Mrgbp, a protein that recognizes H3K36 trimethylation, consistent with the role of histone modifications at alternatively spliced sites.

The evolution of epigenetics.

Early studies of the developing embryo raised the question of how a fertilized egg could give rise to a complex multicellular organism containing many different kinds of cells. The term epigenetics originally referred to the study of these processes. With the advent of detailed knowledge of mechanisms of gene expression, this definition was superseded by another: epigenetics concerned the transmission of phenotype through mitosis or the germ line by mechanisms that did not involve changes in the DNA sequence. Much effort has been spent in attempting to identify and characterize these events. Work initially focused on DNA methylation as an epigenetic mark, but more recently there has been an emphasis on histone modifications as possible carriers of epigenetic information. However, there is confusion between situations in which the modifications may be propagated through cell division, thus helping to maintain a pattern of gene expression, and situations in which the modifications are simply part of the transcriptional apparatus. Arguments about the role of the histones have led to a reexamination of the definition of epigenetics and the primary events in development leading to cell type specific gene expression patterns.

Chromatin domains, insulators, and the regulation of gene expression.

The DNA sequence elements called insulators have two basic kinds of properties. Barrier elements block the propagation of heterochromatic structures into adjacent euchromatin. Enhancer blocking elements interfere with interaction between an enhancer and promoter when placed between them. We have dissected a compound insulator element found at the 5' end of the chicken ß-globin locus, which possesses both activities. Barrier insulation is mediated by two kinds of DNA binding proteins: USF1/USF2, a heterodimer which recruits multiple enzyme complexes capable of marking histone on adjacent nucleosomes with 'activating' marks, and Vezf1, which protects against DNA methylation. We have found that the heterochromatic region upstream of the insulator element is maintained in its silent state by a dicer-dependent mechanism, suggesting a mechanism for Vezf1 function in the insulator. Enhancer blocking function in the ß-globin insulator element is conferred by a binding site for CTCF. Consistent with this property, CTCF binding was found some years ago to be essential for imprinted expression at the Igf2/H19 locus. Work in many laboratories has since demonstrated that CTCF helps stabilize long-range interactions in the nucleus. We have recently shown that in the case of the human insulin locus such an interaction, over a distance of ~300kb, can result in stimulation of a target gene which itself is important for insulin secretion. This article is part of a Special Issue entitled: Chromatin in time and space.

Chromatin boundaries require functional collaboration between the hSET1 and NURF complexes.

Chromatin insulators protect erythroid genes from being silenced during erythropoiesis, and the disruption of barrier insulator function in erythroid membrane gene loci results in mild or severe anemia. We showed previously that the USF1/2-bound 5'HS4 insulator mediates chromatin barrier activity in the erythroid-specific chicken ß-globin locus. It is currently not known how insulators establish such a barrier. To understand the function of USF1, we purified USF1-associated protein complexes and found that USF1 forms a multiprotein complex with hSET1 and NURF, thus exhibiting histone H3K4 methyltransferase- and ATP-dependent nucleosome remodeling activities, respectively. Both SET1 and NURF are recruited to the 5'HS4 insulator by USF1 to retain the active chromatin structure in erythrocytes. Knock-down of NURF resulted in a rapid loss of barrier activity accompanied by an alteration of nucleosome positioning, increased occupancy of the nucleosome-free linker region at the insulator site, and increased repressive H3K27me3 levels in the vicinity of the HS4 insulator. Furthermore, suppression of SET1 reduced barrier activity, decreased H3K4me2 and acH3K9/K14, and diminished the recruitment of BPTF at several erythroid-specific barrier insulator sites. Therefore, our data reveal a synergistic role of hSET1 and NURF in regulating the USF-bound barrier insulator to prevent erythroid genes from encroachment of heterochromatin.

Chromatin structure outside and inside the nucleus.

The structure of the 30-nm chromatin fiber has provided, over the years, an important reference in chromatin studies. Originally derived from electron microscopic studies of soluble chromatin fibers released by restriction digestion, the gross structural features of such fragments have been supported by biophysical methods such as low angle X-ray and neutron scattering, sedimentation, light scattering, and electric dichroism. Electron microscopy and sedimentation velocity measurements demonstrated that reconstituted chromatin fibers, prepared from repeating arrays of high affinity nucleosome positioning sequences, retain the same overall features as observed for native chromatin fibers. It had been suggested that the 30 nm fiber might be the form assumed in vivo by transcriptionally silent chromatin, but individual gene or genome-wide studies of chromatin released from nuclei do not reveal any such simple correlation. Furthermore, even though the 30 nm fiber has been thought to represent an intermediate in the hierarchical folding of DNA into chromosomes, most analyses of chromatin folding within the nucleus do not detect any regular extended compact structures. However, there are important exceptions in chicken erythroid cell nuclei as well as in transcribed regions that form extended loops. Localized domains within the nucleus, either at the surface of chromosome domains or constrained as a specialized kind of constitutive heterochromatin by specific DNA binding proteins, may adopt 30 nm fiber-like structures.

Physical chemistry of nucleic acids and their complexes.

Studies of the physical properties of nucleic acids began almost immediately following the discovery of the DNA structure. Early investigations focused on the stability and specificity of multi-strand polynucleotide complexes, then gradually on their interaction with other molecules, particularly proteins. As molecular and structural biology expanded to provide detailed information about biochemical mechanisms, physical studies eventually acquired the additional constraint that they should be relevant to functioning biological systems. We describe work in our laboratory that began with investigations of relatively simple questions about the role of electrostatic interactions in the stabilization of multi-strand nucleic acid structures, and evolved to studies of chromatin structure in vitro and within the nucleus.

VEZF1 elements mediate protection from DNA methylation.

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.