Human neutrophils can mimic myeloid-derived suppressor cells (PMN-MDSC) and suppress microbead or lectin-induced T cell proliferation through artefactual mechanisms.

We report that human conventional CD15+ neutrophils can be isolated in the peripheral blood mononuclear cell (PBMC) layer during Ficoll gradient separation, and that they can impair T cell proliferation in vitro without concomitant neutrophil activation and killing. This effect was observed in a total of 92 patients with organ transplants, lung cancer or anxiety/depression, and in 18 healthy donors. Although such features are typically associated in the literature with the presence of certain myeloid-derived suppressor cell (PMN-MDSC) populations, we found that commercial centrifuge tubes that contained membranes or gels for PBMC isolation led to up to 70% PBMC contamination by CD15+ neutrophils, with subsequent suppressive effects in certain cellular assays. In particular, the suppressive activity of human MDSC should not be evaluated using lectin or microbead stimulation, whereas assays involving soluble or plate-bound antibodies or MLR are unaffected. We conclude that CD15+ neutrophil contamination, and associated effects on suppressor assays, can lead to significant artefacts in studies of human PMN-MDSC.

Human lung tumor FOXP3+ Tregs upregulate four "Treg-locking" transcription factors.

Experimental data indicate that FOXP3+ Tregs can markedly curtail host antitumor immune responses, but the properties of human intratumoral Tregs are still largely unknown, in part due to significant methodologic problems. We studied the phenotypic, functional, epigenetic, and transcriptional features of Tregs in 92 patients with non-small-cell lung cancer, comparing the features of Tregs within tumors versus corresponding blood, lung, and lymph node samples. Intratumoral Treg numbers and suppressive function were significantly increased compared with all other sites but did not display a distinctive phenotype by flow cytometry. However, by undertaking simultaneous evaluation of mRNA and protein expression at the single-cell level, we demonstrated that tumor Tregs have a phenotype characterized by upregulated expression of FOXP3 mRNA and protein as well as significantly increased expression of EOS, IRF4, SATB1, and GATA1 transcription factor mRNAs. Expression of these "Treg-locking" transcription factors was positively correlated with levels of FOXP3 mRNA, with highest correlations for EOS and SATB1. EOS had an additional, FOXP3 mRNA-independent, positive correlation with FOXP3 protein in tumor Tregs. Our study identifies distinctive features of intratumoral Tregs and suggests that targeting Treg-locking transcription factors, especially EOS, may be of clinical importance for antitumor Treg-based therapy.

Sp100A promotes chromatin decondensation at a cytomegalovirus-promoter-regulated transcription site.

Promyelocytic leukemia nuclear bodies (PML-NBs)/nuclear domain 10s (ND10s) are nuclear structures that contain many transcriptional and chromatin regulatory factors. One of these, Sp100, is expressed from a single-copy gene and spliced into four isoforms (A, B, C, and HMG), which differentially regulate transcription. Here we evaluate Sp100 function in single cells using an inducible cytomegalovirus-promoter-regulated transgene, visualized as a chromatinized transcription site. Sp100A is the isoform most strongly recruited to the transgene array, and it significantly increases chromatin decondensation. However, Sp100A cannot overcome Daxx- and α-thalassemia mental retardation, X-linked (ATRX)-mediated transcriptional repression, which indicates that PML-NB/ND10 factors function within a regulatory hierarchy. Sp100A increases and Sp100B, which contains a SAND domain, decreases acetyl-lysine regulatory factor levels at activated sites, suggesting that Sp100 isoforms differentially regulate transcription by modulating lysine acetylation. In contrast to Daxx, ATRX, and PML, Sp100 is recruited to activated arrays in cells expressing the herpes simplex virus type 1 E3 ubiquitin ligase, ICP0, which degrades all Sp100 isoforms except unsumoylated Sp100A. The recruitment Sp100A(K297R), which cannot be sumoylated, further suggests that sumoylation plays an important role in regulating Sp100 isoform levels at transcription sites. This study provides insight into the ways in which viruses may modulate Sp100 to promote their replication cycles.

Single-cell analysis of Daxx and ATRX-dependent transcriptional repression.

Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function.

Sp100 as a potent tumor suppressor: accelerated senescence and rapid malignant transformation of human fibroblasts through modulation of an embryonic stem cell program.

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.

LIM protein Ajuba functions as a nuclear receptor corepressor and negatively regulates retinoic acid signaling.

Corepressors play an essential role in nuclear receptor-mediated transcriptional repression. In general, corepressors directly bind to nuclear receptors via CoRNR boxes (L/I-X-X-I/V-I) in the absence of ligand and appear to act as scaffolds to further recruit chromatin remodeling complexes to specific target genes. Here, we describe the identification of the multiple LIM domain protein Ajuba as a unique corepressor for a subset of nuclear hormone receptors. Ajuba contains functional nuclear-receptor interacting motifs and selectively interacts with retinoic acid receptors (RARs) and rexinoid receptor (RXRs) subtypes in a ligand-dependent manner. Simultaneous mutation of these motifs abolishes RAR binding and concomitantly leads to loss of repression on RARE reporter genes. P19 cells depleted of Ajuba are highly sensitized to all-trans retinoic acid (atRA)-induced transcription and differentiation. In the absence of atRA, Ajuba can be readily found at the RARE control elements of RAR endogenous target genes. Stimulation of cells with atRA results in the dissociation of Ajuba from these regions. Moreover, we observed that coexpression of the known Ajuba binding partner Prmt5 (protein arginine methyltransferase-5) inhibited the Ajuba/RAR interaction. The high-affinity Ajuba-RAR/RXR interaction site overlaps the region responsible for Ajuba/Prmt5 binding, and thus binding appears to be mutually exclusive, providing a potential mechanism for these observations. Identification of Ajuba as a unique corepressor for nuclear receptors sheds new light on mechanisms for nuclear receptor-mediated repression and provides a unique target for developing more effective therapeutics to modulate this important pathway.

Differential functions of interferon-upregulated Sp100 isoforms: herpes simplex virus type 1 promoter-based immediate-early gene suppression and PML protection from ICP0-mediated degradation.

Cells have intrinsic defenses against virus infection, acting before the innate or the adaptive immune response. Preexisting antiviral proteins such as PML, Daxx, and Sp100 are stored in specific nuclear domains (ND10). In herpes simplex virus type 1 (HSV-1), the immediate-early protein ICP0 serves as a counterdefense through degradation of the detrimental protein PML. We asked whether interferon (IFN)-upregulated Sp100 is similarly antagonized by ICP0 in normal human fibroblasts by using a selective-knockdown approach. We find that of the four Sp100 isoforms, the three containing a SAND domain block the transcription of HSV-1 proteins ICP0 and ICP4 at the promoter level and that IFN changes the differential splicing of the Sp100 transcript in favor of the inhibitor Sp100C. At the protein level, ICP0 activity does not lead to the hydrolysis of any of the Sp100 isoforms. The SAND domain-containing isoforms are not general inhibitors of viral promoters, as the activity of the major immediate-early cytomegalovirus promoter is not diminished, whereas the long terminal repeat of a retrovirus, like the ICP0 promoter, is strongly inhibited. Since we could not find a specific promoter region in the ICP0 gene that responds to the SAND domain-containing isoforms, we questioned whether Sp100 could act through other antiviral proteins such as PML. We find that all four Sp100 isoforms stabilize ND10 and protect PML from ICP0-based hydrolysis. Loss of either all PML isoforms or all Sp100 isoforms reduces the opposite constituent ND10 protein, suggesting that various interdependent mechanisms of ND10-based proteins inhibit virus infection at the immediate-early level.

Dynamics of component exchange at PML nuclear bodies.

PML nuclear bodies (NBs) are involved in the regulation of key nuclear pathways but their biochemical function in nuclear metabolism is unknown. In this study PML NB assembly dynamics were assessed by live cell imaging and mathematic modeling of its major component parts. We show that all six nuclear PML isoforms exhibit individual exchange rates at NBs and identify PML V as a scaffold subunit. SP100 exchanges at least five times faster at NBs than PML proteins. Turnover dynamics of PML and SP100 at NBs is modulated by SUMOylation. Exchange is not temperature-dependent but depletion of cellular ATP levels induces protein immobilization at NBs. The PML-RARalpha oncogene exhibits a strong NB retention effect on wild-type PML proteins. HIPK2 requires an active kinase for PML NB targeting and elevated levels of PML IV increase its residence time. DAXX and BLM turn over rapidly and completely at PML NBs within seconds. These findings provide a kinetics model for factor exchange at PML NBs and highlight potential mechanisms to regulate intranuclear trafficking of specific factors at these domains.

Differences between mouse and human cytomegalovirus interactions with their respective hosts at immediate early times of the replication cycle.

The promise of the mouse model of cytomegalovirus (CMV) research lies in a cost effective way to obtain significant data in in vivo settings. Keeping that promise requires a high degree of equivalency in the human and mouse virus. While genomic structure and many common proteins suggest that this system is appropriate to develop and test concepts in an organismal context, areas of difference have not been evaluated. Here we show that the major immediate early protein 1 (IE1) in MCMV binds the repressor Daxx suggesting that it serves a function performed by pp71 in HCMV. A Daxx binding pp71 equivalent at M82 could not be identified for MCMV. Differences in the mouse and human interferon upregulation of Daxx may have driven the need to have a Daxx-defeating function during reactivation, when pp71 is not present. The major immediate early protein 1 also differs in its chromatin binding properties between the two viruses. MCMV IE1 does not bind to chromatin, but HCMV IE1 does. It remains unclear whether this difference is functionally significant. The HCMV major immediate early protein 2 and its MCMV equivalent IE3 differ in their effect on the cell cycle; HCMV IE2 blocks the cell cycle, but MCMV IE3 does not, allowing MCMV to spread in infected mouse cells by cell division with continued expression of the major transactivating viral proteins. Actively transcribing genomes inducing immediate transcript environments are usually silenced and diminish during cell cycle progression. However, a recognizable desilencing and increase in immediate transcript environments takes place immediately after mitosis in MCMV infected cells. This raises the possibility that desilencing happens during tissue transplantation, wound healing, or other injury where cells are induced to proliferate.

PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing.

Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO-interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a function of the PHD domain as an intramolecular E3 SUMO ligase.

KAP1, a novel substrate for PIKK family members, colocalizes with numerous damage response factors at DNA lesions.

The DNA damage response requires a coordinated nucleo-cytoplasmic cascade of events, which ultimately converge on damaged DNA packaged in chromatin. Few connections between the proteins that remodel chromatin and the proteins that mediate this damage response have been shown. We have investigated the DNA damage-induced phosphorylation of the KRAB-ZFP-associated protein 1 (KAP1), the dedicated corepressor for Krüppel-associated box (KRAB) zinc finger protein (ZFP) proteins. We show that KAP1 is rapidly phosphorylated following DNA damage by members of the phosphatidylinositol-3 kinase-like family of kinases. This phosphorylation occurs at a single amino acid residue that is conserved from mice to humans and is located adjacent to the bromodomain, suggesting that it may regulate chromatin recognition by that module. Phosphorylated KAP1 rapidly localizes to sites of DNA strand breaks in the nucleus in response to ionizing radiation. This discovery provides a novel link between chromatin-mediated transcriptional repression and the recognition/repair of DNA, which must be accomplished by the cellular DNA damage response.

Differential role of Sp100 isoforms in interferon-mediated repression of herpes simplex virus type 1 immediate-early protein expression.

Nuclear domains called ND10 or PML nuclear bodies contain interferon (IFN)-upregulated proteins like PML and Sp100. Paradoxically, herpes simplex virus 1 (HSV-1) begins its transcriptional cascade at aggregates of ND10-associated proteins, which in turn are destroyed by the HSV-1 immediate-early protein ICP0. While PML is essential in the formation of ND10, the function of Sp100 in the cells' defense against viral infection is unknown. In this study we investigated the potential antiviral effect of IFN-beta-induced Sp100. We found that IFN-beta treatment leads to a differential accumulation of four Sp100 isoforms in different cell lines. Using an HEK293 cell line derivative, 293-S, producing no detectable amounts of Sp100 even after IFN exposure, we analyzed individual Sp100 isoforms for their effect on HSV-1 infection. Sp100 isoforms B, C, and HMG, but not Sp100A, suppressed ICP0 and ICP4 early after infection. Isoforms B, C, and HMG suppressed expression from the ICP0 promoter in transient transfection, whereas Sp100A enhanced expression. Moreover, Sp100A localized in ND10, whereas the repressive isoforms were either dispersed within the nucleus or, at unphysiologically higher expression levels, formed new aggregates. The repressive activity was dependent on an intact SAND domain, since Sp100B bearing a W655Q mutation in the SAND domain lost this repressive activity and accumulated in ND10. Using RNA interference to knock down the repressive Sp100 isoforms B, C, and HMG, we find that they are an essential part of the IFN-beta-mediated suppression of ICP0 expression. These data suggest that repression by the Sp100 isoforms B, C, and HMG takes place outside of ND10 and raise the possibility that viral genomes at Sp100A accumulations are more likely to start their transcription program because of a more permissive local environment.

The mammalian heterochromatin protein 1 binds diverse nuclear proteins through a common motif that targets the chromoshadow domain.

The HP1 proteins regulate epigenetic gene silencing by promoting and maintaining chromatin condensation. The HP1 chromodomain binds to methylated histone H3. More enigmatic is the chromoshadow domain (CSD), which mediates dimerization, transcription repression, and interaction with multiple nuclear proteins. Here we show that KAP-1, CAF-1 p150, and NIPBL carry a canonical amino acid motif, PxVxL, which binds directly to the CSD with high affinity. We also define a new class of variant PxVxL CSD-binding motifs in Sp100A, LBR, and ATRX. Both canonical and variant motifs recognize a similar surface of the CSD dimer as demonstrated by a panel of CSD mutants. These in vitro binding results were confirmed by the analysis of polypeptides found associated with nuclear HP1 complexes and we provide the first evidence of the NIPBL/delangin protein in human cells, a protein recently implicated in the developmental disorder, Cornelia de Lange syndrome. NIPBL is related to Nipped-B, a factor participating in gene activation by remote enhancers in Drosophila melanogaster. Thus, this spectrum of direct binding partners suggests an expanded role for HP1 as factor participating in promoter-enhancer communication, chromatin remodeling/assembly, and sub-nuclear compartmentalization.

Heat shock and Cd2+ exposure regulate PML and Daxx release from ND10 by independent mechanisms that modify the induction of heat-shock proteins 70 and 25 differently.

Nuclear domains called ND10 or PML bodies might function as nuclear depots by recruiting or releasing certain proteins. Although recruitment of proteins through interferon-induced upregulation and SUMO-1 modification level of PML had been defined, it is not known whether release of proteins is regulated and has physiological consequences. Exposure to sublethal environmental stress revealed a sequential release of ND10-associated proteins. Upon heat shock Daxx and Sp100 were released but PML remained, whereas exposure to subtoxic concentrations of CdCl(2) induced the release of ND10-associated proteins, including PML, with Sp100 remaining in a few sites. In both cases, recovery times were similar and were followed by a burst of mitotic activity. Cadmium-induced release of proteins from ND10 could be blocked by inhibiting activation of p38 MAPK or ERK1/2. By contrast, heat-shock-induced desumolation of PML and release of proteins from ND10 are unaffected by these inhibitors but can be recapitulated by overexpression of the SUMO isopeptidase SENP-1. Therefore, activation of SENP-1-like SUMO isopeptidase(s) during heat shock is not affected by these kinases. Thus, the release of ND10-associated proteins is not due to a general dispersal of nuclear domains but seems to be regulated by rapid desumolation during thermal stress and through the phosphorylation cascade of stress and mitogenic signaling pathways in the case of CdCl(2). Whether the release of certain proteins had consequences was tested for heat-shock-protein transcription and synthesis. Release of Daxx correlated with Hsp25 suppression, suggesting that Daxx normally inhibits immediate Hsp25 production. Release of PML correlated with lower production of Hsp70. These results suggest that segregation or release of PML or Daxx have differential physiological relevance during the stress response. The fact that enzymatic activation of protein release or segregation after stress modifies the heat-shock response strengthens the concept of ND10 as a regulated depot of effector proteins.

Increased chromosomal alterations and micronuclei formation in human hepatoma HepG2 cells transfected with the hepatitis B virus HBX gene.

The protein encoded by the hepatitis B virus (HBV)-X gene, HBX, has been implicated to be involved in the development of HBV-associated liver cancer. HBX is a multifunctional regulatory protein that has been identified as a potential oncogene but its exact function remains unclear. HBX was documented to interact with several factors involved in cellular DNA repair as well as compromise the cell's ability to repair damaged DNA. We previously documented an accumulation of genetic alterations in two HepG2 cell lines independently transfected with HBV. In this report, we investigate the effect of the HBV-X gene (HBX) on the stability of the host genome using HepG2 stable transfectants (HepG2-HBX) and vector controls (HepG2-neo). We document that all HepG2-HBX clones analyzed contain HBX gene integrated and HBX transcript. Our data demonstrate that HepG2-HBX cells have an increased number of chromosome alterations and micronuclei formation compared to vector controls. A total of 10 de novo chromosomal rearrangements involving nine different chromosomes were detected in the HepG2-HBX clones, while no new rearrangements were found in vector controls. Each HepG2-HBX clone contained independently occurring de novo alterations not found in other HBX or vector clones. A three-fold increase of micronuclei formation was detected in HepG2-HBX cells compared to vector controls. Micronuclei originated from all chromosomes, however, preliminary data indicated that micronuclei originating from chromosomes 2, 3, 7, 18 and 20 were found in a greater amount in cells expressing the HBX gene. Interestingly, chromosomes 2, 18 and 20 were three of the chromosomes found rearranged in HepG2-HBX clones. These data provide evidence that genomic integrity was affected in cells expressing the HBX gene. De novo cytogenetic alterations identified in HepG2-HBX clones implicate the involvement of HBX in the process and support the hypothesis that HBX may interfere with normal cellular processes responsible for genomic integrity, increasing the risk for acquiring genetic mutations in infected hepatocytes.

SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins.

Posttranslational modification of histones has emerged as a key regulatory signal in eukaryotic gene expression. Recent genetic and biochemical studies link H3-lysine 9 (H3-K9) methylation to HP1-mediated heterochromatin formation and gene silencing. However, the mechanisms that target and coordinate these activities to specific genes is poorly understood. Here we report that the KAP-1 corepressor for the KRAB-ZFP superfamily of transcriptional silencers binds to SETDB1, a novel SET domain protein with histone H3-K9-specific methyltransferase activity. Although acetylation and phosphorylation of the H3 N-terminal tail profoundly affect the efficiency of H3-K9 methylation by SETDB1, we found that methylation of H3-K4 does not affect SETDB1-mediated methylation of H3-K9. In vitro methylation of the N-terminal tail of histone H3 by SETDB1 is sufficient to enhance the binding of HP1 proteins, which requires both an intact chromodomain and chromoshadow domain. Indirect immunofluoresence staining of interphase nuclei localized SETDB1 predominantly in euchromatic regions that overlap with HP1 staining in nonpericentromeric regions of chromatin. Moreover, KAP-1, SETDB1, H3-MeK9, and HP1 are enriched at promoter sequences of a euchromatic gene silenced by the KRAB-KAP-1 repression system. Thus, KAP-1 is a molecular scaffold that is targeted by KRAB-ZFPs to specific loci and coordinates both histone methylation and the deposition of HP1 proteins to silence gene expression.