Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly.

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eµ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans.

BACKGROUND: Non-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer's disease (AD) shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28-31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. RESULTS: Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at -31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at -31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors. CONCLUSION: The results suggest that the clock-regulated and immune system modulator transcription factor E4BP4/ NFIL3 likely regulates the expression of both appb in zebrafish and APP in humans. It suggests potential human APP gene regulatory pathways, not on the basis of comparing DNA primary sequences with zebrafish appb but on the model of conservation of transcription factors.

IL-10 transcription is negatively regulated by BAF180, a component of the SWI/SNF chromatin remodeling enzyme.

BACKGROUND: SWI/SNF chromatin remodeling enzymes play a critical role in the development of T helper lymphocytes, including Th2 cells, and directly program chromatin structure at Th2 cytokine genes. Different versions of SWI/SNF complexes, including BAF and PBAF, have been described based on unique subunit composition. However, the relative role of BAF and PBAF in Th cell function and cytokine expression has not been reported. RESULTS: Here we examine the role of the PBAF SWI/SNF complex in Th cell development and gene expression using mice deficient for a PBAF-specific component, BAF180. We find that T cell development in the thymus and lymphoid periphery is largely normal when the BAF180 gene is deleted late in thymic development. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. CONCLUSIONS: These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription.

CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes.

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease.

Nontelomeric splice variant of telomere repeat-binding factor 2 maintains neuronal traits by sequestering repressor element 1-silencing transcription factor.

Telomere repeat-binding factor 2 (TRF2) is critical for telomere integrity in dividing stem and somatic cells, but its role in postmitotic neurons is unknown. Apart from protecting telomeres, nuclear TRF2 interacts with the master neuronal gene-silencer repressor element 1-silencing transcription factor (REST), and disruption of this interaction induces neuronal differentiation. Here we report a developmental switch from the expression of TRF2 in proliferating neural progenitor cells to expression of a unique short nontelomeric isoform of TRF2 (TRF2-S) as neurons establish a fully differentiated state. Unlike nuclear TRF2, which enhances REST-mediated gene repression, TRF2-S is located in the cytoplasm where it sequesters REST, thereby maintaining the expression of neuronal genes, including those encoding glutamate receptors, cell adhesion, and neurofilament proteins. In neurons, TRF2-S-mediated antagonism of REST nuclear activity is greatly attenuated by either overexpression of TRF2 or administration of the excitatory amino acid kainic acid. Overexpression of TRF2-S rescues kainic acid-induced REST nuclear accumulation and its gene-silencing effects. Thus, TRF2-S acts as part of a unique developmentally regulated molecular switch that plays critical roles in the maintenance and plasticity of neurons.

NF-κB and BRG1 bind a distal regulatory element in the IL-3/GM-CSF locus.

We investigated gene regulation at the IL-3/GM-CSF gene cluster. We found BRG1, a SWI/SNF remodeling ATPase, bound a distal element, CNSa. BRG1 binding was strongest in differentiated, stimulated T helper cells, paralleling IL-3 and GM-CSF expression. Depletion of BRG1 reduced IL-3 and GM-CSF transcription. BAF-specific SWI/SNF subunits bound to this locus and regulated IL-3 expression. CNSa was in closed chromatin in fibroblasts, open chromatin in differentiated T helper cells, and moderately open chromatin in naïve (undifferentiated) T helper cells; BRG1 was required for the most open state. CNSa increased transcription of a reporter in an episomal expression system, in a BRG1-dependent manner. The NF-κB subunit RelA/p65 bound CNSa in activated T helper cells. Inhibition of NF-κB blocked BRG1 binding to CNSa, chromatin opening at CNSa, and activation of IL-3 and GM-CSF. Together, these findings suggest CNSa is a distal enhancer that binds BRG1 and NF-κB.

Dynamic BRG1 recruitment during T helper differentiation and activation reveals distal regulatory elements.

T helper cell differentiation and activation require specific transcriptional programs accompanied by changes in chromatin structure. However, little is known about the chromatin remodeling enzymes responsible. We performed genome-wide analysis to determine the general principles of BRG1 binding, followed by analysis of specific genes to determine whether these general rules were typical of key T cell genes. We found that binding of the remodeling protein BRG1 was programmed by both lineage and activation signals. BRG1 binding positively correlated with gene activity at protein-coding and microRNA (miRNA) genes. BRG1 binding was found at promoters and distal regions, including both novel and previously validated distal regulatory elements. Distal BRG1 binding correlated with expression, and novel distal sites in the Gata3 locus possessed enhancer-like activity, suggesting a general role for BRG1 in long-distance gene regulation. BRG1 recruitment to distal sites in Gata3 was impaired in cells lacking STAT6, a transcription factor that regulates lineage-specific genes. Together, these findings suggest that BRG1 interprets both differentiation and activation signals and plays a causal role in gene regulation, chromatin structure, and cell fate. Our findings suggest that BRG1 binding is a useful marker for identifying active cis-regulatory regions in protein-coding and miRNA genes.

Activation of heat shock factor 1 plays a role in pyrrolidine dithiocarbamate-mediated expression of the co-chaperone BAG3.

Adaptive responses to physical and inflammatory stressors are mediated by transcription factors and molecular chaperones. The transcription factor heat shock factor 1 (HSF1) has been implicated in extending lifespan in part by increasing expression of heat shock response genes. Pyrrolidine dithiocarbamate (PDTC) is a small thiol compound that exerts in vivo and in vitro anti-inflammatory properties through mechanisms that remain unclear. Here we report that PDTC induced the release of monomeric HSF1 from the molecular chaperone heat shock protein 90 (Hsp90), with concomitant increase in HSF1 trimer formation, translocation to the nucleus, and binding to promoter of target genes in human HepG2 cells. siRNA-mediated silencing of HSF1 blocked BAG3 gene expression by PDTC. The protein levels of the co-chaperone BAG3 and its interaction partner Hsp72 were stimulated by PDTC in a dose-dependent fashion, peaking at 6h. Inhibition of Hsp90 function by geldanamycin derivatives and novobiocin elicited a pattern of HSF1 activation and BAG3 expression that was similar to PDTC. Chromatin immunoprecipitation studies showed that PDTC and the inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin enhanced the binding of HSF1 to the promoter of several target genes, including BAG3, HSPA1A, HSPA1B, FKBP4, STIP1 and UBB. Cell treatment with PDTC increased significantly the level of Hsp90α thiol oxidation, a posttranslational modification known to inhibit its chaperone function. These results unravel a previously unrecognized mechanism by which PDTC and related compounds could confer cellular protection against inflammation through HSF1-induced expression of heat shock response genes.

The SNF2H chromatin remodeling enzyme has opposing effects on cytokine gene expression.

Cytokine gene expression is a key control point in the function of the immune system. Cytokine gene regulation is linked to changes in chromatin structure; however, little is known about the remodeling enzymes mediating these changes. Here we investigated the role of the ATP-dependent chromatin remodeling enzyme SNF2H in mouse T cells; to date, SNF2H has not been investigated in T cells. We found that SNF2H repressed expression of IL-2 and other cytokines in activated cells. By contrast, SNF2H activated expression of IL-3. The ISWI components SNF2H and ACF1 bound to the tested loci, suggesting the regulation was direct. SNF2H decreased accessibility at some binding sites within the IL2 locus, and increased accessibility within some IL3 binding sites. The changes in gene expression positively correlated with accessibility changes, suggesting a simple model that accessibility enables transcription. We also found that loss of the ISWI ATPase SNF2H reduced binding to target genes and protein expression of ACF1, a binding partner for SNF2H, suggesting complex formation stabilized ACF1. Together, these findings reveal a direct role for SNF2H in both repression and activation of cytokine genes.

S-glutathionylation impairs signal transducer and activator of transcription 3 activation and signaling.

S-glutathionylation is a physiological, reversible protein modification of cysteine residues with glutathione in response to mild oxidative stress. Because the key cell growth regulator signal transducer and activator of transcription (STAT) 3 is particularly susceptible to redox regulation, we hypothesized that oxidative modification of cysteine residues of STAT3 by S-glutathionylation may occur. Herein, we show that the cysteine residues of STAT3 are modified by a thiol-alkylating agent and are the targets of S-glutathionylation. STAT3 protein thiol reactivity was reversibly attenuated with concomitant increase in the S-glutathionylation of STAT3 upon treatment of human HepG2 hepatoma cells with pyrrolidine dithiocarbamate, glutathione disulfide, or diamide. Under these conditions there was a marked reduction in IL-6-dependent STAT3 signaling, including decreased STAT3 tyrosine phosphorylation, loss in nuclear accumulation of STAT3, and impaired expression of target genes, such as fibrinogen-gamma. In a cell-free system, diamide induced glutathionylation of STAT3, which was decreased upon addition of glutaredoxin (GRX)-1, a deglutathionylation enzyme, or the reducing agent, dithiothreitol. Glutathionylated STAT3 was a poor Janus protein tyrosine kinase 2 substrate in vitro, and it exhibited low DNA-binding activity. Cellular GRX-1 activity was inhibited by diamide and pyrrolidine dithiocarbamate treatment; however, ectopic expression of GRX-1 was accompanied by a modest increase in phosphorylation, nuclear translocation, and DNA-binding ability of STAT3 in response to IL-6. These results are the first to show S-glutathionylation of STAT3, a modification that may exert regulatory function in STAT3 signaling.

Deficiency of ADAP/Fyb/SLAP-130 destabilizes SKAP55 in Jurkat T cells.

ADAP (adhesion and degranulation-promoting adaptor protein) and SKAP55 (Src kinase-associated phosphoprotein of 55 kDa) are T cell adaptors that mediate inside-out signaling from the T cell antigen receptor to integrins, giving rise to increased integrin affinity/avidity and formation of the immunological synapse between the T cell and the antigen-presenting cell. These two proteins are tightly and constitutively associated with one another, and their ability to interact is required for inside-out signaling. Here we show in an ADAP-deficient Jurkat T cell line that the co-dependence of ADAP and SKAP55 extends beyond their functional and physical interactions and show that SKAP55 protein is unstable in the absence of ADAP. Restoration of ADAP to the ADAP-deficient Jurkat T cell line restores SKAP55 expression by causing a 5-fold decrease in the rate of SKAP55 proteolysis. Inactivation of the Src homology 3 domain of SKAP55, which mediates the association between SKAP55 with ADAP, blocks the protective effect of ADAP. The half-life of SKAP55, in the absence of ADAP, is approximately 15-20 min, increasing to 90 min in the presence of ADAP. This is a remarkably rapid rate of turnover for a signaling protein and suggests the possibility that stimuli that signal for the stabilization of SKAP55 may play an important role in T cell adhesion and conjugate formation.

Modulation of specific protein expression levels by PTEN: identification of AKAP121, DHFR, G3BP, Rap1, and RCC1 as potential targets of PTEN.

The tumor suppressor PTEN is mutated in a high percentage of human cancers, and is implicated in pathways regulating cell growth, proliferation, survival, and migration. Despite significant advances, our understanding of its mechanisms of action remains incomplete. We have used a high-throughput proteomic immunoblotting approach to identify proteins whose expression levels are modulated by PTEN. Out of over 800 proteins screened, 22 proteins showed significant changes in expression. Five proteins that exhibited two-fold or greater changes in expression level were further characterized. AKAP121 and G3BP expression was reduced, while dihydrofolate reductase (DHFR), Rap1 and RCC1 expression was elevated in response to PTEN expression in a PTEN-null T-cell leukemia line. The phosphatase activity of PTEN was required for these effects. However, direct inhibition of PI-3 Kinase could mimic PTEN in modulating expression of DHFR, G3BP, Rap1 and RCC1, but not AKAP121. Real-time PCR showed that the effects of PTEN were primarily post-transcriptional, and would not have been revealed by mRNA-based screens. We conclude from these data that PTEN can modulate the expression level of a number of different proteins. The identified proteins have the potential to serve as previously unrecognized effectors of PTEN, and suggest the existence of additional complexity in the modes by which PTEN can regulate cellular biology.

Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.

The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

PTEN permits acute increases in D3-phosphoinositide levels following TCR stimulation but inhibits distal signaling events by reducing the basal activity of Akt.

Phosphoinositide 3-kinase (PI3K) is important in TCR signaling. PI3K generates phosphatidylinositol 3, 4, 5-trisphosphate (PI-3,4,5-P3), which regulates membrane localization and/or activity of multiple signaling proteins. PTEN (phosphatase and tensin homologue deleted on chromosome 10) opposes PI3K, reversing this reaction. Maintaining the balance between these two enzymes is important for normal T cell function. Here we use the PTEN-null Jurkat T cell line to address the role of PTEN in modulating proximal and distal TCR-signaling events. PTEN expression at levels that restored low basal Akt phosphorylation (an indicator of PI-3,4,5-P3 levels), but which were not themselves cytotoxic, had minimal effect on TCR-stimulated activation of phospholipase Cgamma1 and Ca2+ flux, but reduced the duration of extracellular signal-regulated kinase (Erk) activation. Distal signaling events, including nuclear factor of activated T cells (NFAT) activation, CD69 expression and IL-2 production, were all inhibited by PTEN expression. Notably, PTEN did not block TCR-stimulated PI-3,4,5-P3 accumulation. The effect of PTEN on distal TCR signaling events was strongly correlated with the loss of the constitutive Akt activation and glycogen synthase kinase-3 (GSK3) inhibition that is typical of Jurkat cells, and could be reversed by expression of activated Akt or pharmacologic inhibition of GSK3. These results suggest that PTEN acts in T cells primarily to control basal PI-3,4,5-P3 levels, rather than opposing PI3K acutely during TCR stimulation.

PTEN expression in PTEN-null leukaemic T cell lines leads to reduced proliferation via slowed cell cycle progression.

The balance of activities between the proto-oncogene phosphoinositide 3-kinase (PI3K) and the tumour suppressor gene PTEN has been shown to affect cellular growth and proliferation, as well as tumorigenesis. Previously, PTEN expression in the PTEN-null Jurkat T cell leukaemia line was shown to cause reduced proliferation without cell cycle arrest. Here, we further these investigations by determining the basis for this phenomenon. By BrdU pulse-chase and cell cycle arrest and release assays, we find that PTEN expression reduced proliferation by slowing progression through all phases of the cell cycle. This was associated with reduced levels of cyclins A, B1 and B2, cdk4, and cdc25A and increased p27KIP1 expression. Apoptosis played no role in the antiproliferative effect of PTEN, since only marginal increases in the rate of apoptosis were detected upon PTEN expression, and inhibitors of effector caspases did not restore proliferative capacity. Active Akt blocked the antiproliferative effects of PTEN, indicating that PTEN mediates its effects through conventional PI3K-linked signalling pathways. Similar results were obtained from a different PTEN-null leukaemia T cell line, CEM. Together, these results show that PTEN expression in leukaemic T cells leads to reduced proliferation via an apoptosis-independent mechanism involving slower passage through the cell cycle.