Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies.

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (-7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of -7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

G1P3, an interferon- and estrogen-induced survival protein contributes to hyperplasia, tamoxifen resistance and poor outcomes in breast cancer.

Hormonally regulated survival factors can have an important role in breast cancer. Here we elucidate G1P3, a survival protein induced by interferons (IFNs), as a target of estrogen signaling and a contributor to poor outcomes in estrogen receptor-positive (ER(+)) breast cancer. Compared with normal breast tissue, G1P3 was upregulated in the malignant epithelium (50 × higher) and was induced by estrogen ex vivo. In accord with its overexpression in early stages of breast cancer (hyperplasia and ductal carcinoma in situ), in morphogenesis assays G1P3 enhanced the survival of MCF10A acinar luminal cells causing hyperplasia by suppressing detachment-induced loss of mitochondrial potential and apoptosis (anoikis). In cells undergoing anoikis, G1P3 attenuated the induction of Bim protein, a proapoptotic member of the Bcl-2 family and reversed the downmodulation of Bcl-2 protein. Downregulation of G1P3 induced spontaneous apoptosis in BT-549 breast cancer cells and significantly reduced the growth of ER(+) breast cancer cell MCF7 (P≤0.01), further suggesting its prosurvival activity. In agreement with its induction by estrogen, G1P3 antagonized tamoxifen, an inhibitor of ER in MCF7 cells. More importantly, elevated expression of G1P3 was significantly associated with decreased relapse-free and overall survival in ER(+) breast cancer patients (P≤0.01). Our studies suggest that elevated expression of G1P3 may perturb canonical tumor-suppressing activity of IFNs partly by affecting the balance of pro- and antiapoptotic members of Bcl-2 family proteins, leading to breast cancer development and resistance to therapies.

Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma.

BACKGROUND: Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas. RESULTS: At IC(50) concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3-7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (P<0.0001) reduced the survival of primary myeloma cells. CONCLUSIONS: Cathepsin B has a prominent function in mediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL.

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-alpha2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a >3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-alpha2b and IFN-beta; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-beta or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-beta and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.

Identification of TFII-I as the endoplasmic reticulum stress response element binding factor ERSF: its autoregulation by stress and interaction with ATF6.

When mammalian cells are subjected to stress targeted to the endoplasmic reticulum (ER), such as depletion of the ER Ca(2+) store, the transcription of a family of glucose-regulated protein (GRP) genes encoding ER chaperones is induced. The GRP promoters contain multiple copies of the ER stress response element (ERSE), consisting of a unique tripartite structure, CCAAT(N(9))CCACG. Within a subset of mammalian ERSEs, N(9) represents a GC-rich sequence of 9 bp that is conserved across species. A novel complex (termed ERSF) exhibits enhanced binding to the ERSE of the grp78 and ERp72 promoters using HeLa nuclear extracts prepared from ER-stressed cells. Optimal binding of ERSF to ERSE and maximal ERSE-mediated stress inducibility require the conserved GGC motif within the 9-bp region. Through chromatographic purification and subsequent microsequencing, we have identified ERSF as TFII-I. Whereas TFII-I remains predominantly nuclear in both nontreated NIH 3T3 cells and cells treated with thapsigargin (Tg), a potent inducer of the GRP stress response through depletion of the ER Ca(2+) store, the level of TFII-I transcript was elevated in Tg-stressed cells, correlating with an increase in TFII-I protein level in the nuclei of Tg-stressed cells. Purified recombinant TFII-I isoforms bind directly to the ERSEs of grp78 and ERp72 promoters. The stimulation of ERSE-mediated transcription by TFII-I requires the consensus tyrosine phosphorylation site of TFII-I and the GGC sequence motif of the ERSE. We further discovered that TFII-I is an interactive protein partner of ATF6 and that optimal stimulation of ERSE by ATF6 requires TFII-I.

Structure-function analysis of TFII-I. Roles of the N-terminal end, basic region, and I-repeats.

The transcription factor TFII-I can bind specifically to several DNA sequence elements and is implicated in both basal and activated transcription. There are four alternatively spliced isoforms of TFII-I, all characterized by the presence of six I-repeats, R1-R6, each containing a potential helix-loop-helix motif implicated in protein-protein interactions. These isoforms exhibit both homomeric and heteromeric interactions that lead to nuclear localization. In this study we mapped two distinct regions in TFII-I that affect its DNA binding. Deletion of either of these regions led to abrogation of DNA binding and transcriptional activation from both the Vbeta and c-fos promoters. The I-repeats, as expected, were capable of mediating homomeric interactions either individually or in combination. Unexpectedly, an additional homomeric interaction domain was found within the N-terminal end of TFII-I that includes a putative leucine zipper motif. These data suggest a model in which TFII-I undergoes regulated homomeric interaction mediated by both the N-terminal end and the I-repeats.

Purification and characterization of a 29 kDa poly(A)-binding protein from chickpea (Cicer arietinum) epicotyl.

A poly(A)-binding protein (PABP) with mol wt 29,000 has been purified from chickpea (Cicer arietinum) epicotyl by ammonium sulfate fractionation and Cibacron blue F3-GA chromatography, making a complex with poly(A) and elution of PABP-poly(A) complex at 45 degrees C from oligo d(T)-cellulose. The elution pattern and binding properties show that the purified protein is different from the PABP (mol. wt 72,000) reported earlier from our laboratory.

Purification and characterization of a poly(A)-binding protein from chickpea (Cicer arietinum) epicotyl.

A poly(A)-binding protein (PABP) with mol wt 72,000 has been purified from chickpea (Cicer arietinum) epicotyls by ammonium sulfate fractionation, Cibacron blue F3-GA and poly(A) agarose chromatography. The binding properties and the specificity of binding show that the purified protein is an analogue of PABPs in other eukaryotes. This PABP is highly susceptible to proteolysis and upon degradation forms a polypeptide fragment of mol wt 21,000 which has an independent poly(A) binding activity.

Alternatively spliced isoforms of TFII-I. Complex formation, nuclear translocation, and differential gene regulation.

TFII-I is a multifunctional phosphoprotein with roles in transcription and signal transduction. Here we report characterization of three additional alternatively spliced isoforms of TFII-I. Employing isoform-specific antibodies, we show that the isoforms form a stable complex in vivo preferentially in the nucleus compared with the cytoplasm. We further show that both homomeric and heteromeric interactions are possible and that the heteromeric interactions between a wild type and a nuclear localization-deficient mutant result in nuclear translocation of the complex, leading us to postulate that complex formation might aid in nuclear translocation. In functional assays all four isoforms individually bind to DNA and transactivate reporter genes to a similar extent. However, although co-expression of different TFII-I isoforms leads to enhanced basal activity, it results in attenuated signal responsive activity. Thus, TFII-I might differentially regulate its target genes via complex or subcomplex formation.

Regulation of nuclear localization and transcriptional activity of TFII-I by Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is required for normal B-cell development, as defects in Btk lead to X-linked immunodeficiency (xid) in mice and X-linked agammaglobulinemia (XLA) in humans. Here we demonstrate a functional interaction between the multifunctional transcription factor TFII-I and Btk. Ectopic expression of wild-type Btk enhances TFII-I-mediated transcriptional activation and its tyrosine phosphorylation in transient-transfection assays. Mutation of Btk in either the PH domain (R28C, as in the murine xid mutation) or the kinase domain (K430E) compromises its ability to enhance both the tyrosine phosphorylation and the transcriptional activity of TFII-I. TFII-I associates constitutively in vivo with wild-type Btk and kinase-inactive Btk but not xid Btk. However, membrane immunoglobulin M cross-linking in B cells leads to dissociation of TFII-I from Btk. We further show that while TFII-I is found in both the nucleus and cytoplasm of wild-type and xid primary resting B cells, nuclear TFII-I is greater in xid B cells. Most strikingly, receptor cross-linking of wild-type (but not xid) B cells results in increased nuclear import of TFII-I. Taken together, these data suggest that although the PH domain of Btk is primarily responsible for its physical interaction with TFII-I, an intact kinase domain of Btk is required to enhance transcriptional activity of TFII-I in the nucleus. Thus, mutations impairing the physical and/or functional association between TFII-I and Btk may result in diminished TFII-I-dependent transcription and contribute to defective B-cell development and/or function.

Regulation of TFII-I activity by phosphorylation.

The transcription factor TFII-I binds to distinct promoter sequences including an initiator element in several eukaryotic genes. Here we demonstrate that TFII-I is phosphorylated in vivo at serine/threonine and tyrosine residues in the absence of any apparent extracellular signals. This "basal" phosphorylation of TFII-I is not required and does not affect its specific DNA binding, but is critical for its in vitro transcriptional properties via the Vbeta promoter. To better assess the functional role of phosphorylation in regulating TFII-I activity, we focused on tyrosine phosphorylation of TFII-I. Ectopically expressed recombinant TFII-I, like its native counterpart, exhibits tyrosine phosphorylation in the absence of distinct extracellular signals. More important, mutation of a potential consensus tyrosine phosphorylation site in TFII-I leads to severe reduction in its basal transcriptional activation of the Vbeta promoter in vivo. Taken together, these data suggest that tyrosine phosphorylation of TFII-I is important for its initiator-dependent transcriptional activity.

TFII-I regulates Vbeta promoter activity through an initiator element.

In our effort to understand the transcriptional regulation of naturally occurring TATA-less but initiator (Inr)-containing genes, we have employed the murine T-cell receptor Vbeta 5.2 promoter as a model. Here we show by transient-transfection assays that the Inr binding transcription factor TFII-I is required for efficient expression of the Vbeta promoter in vivo. Mutations in the Inr element that reduced binding of TFII-I also abolished the Vbeta promoter activity by ectopic TFII-I. We further biochemically identified a protease-resistant N-terminal DNA binding fragment of TFII-I, p70. When ectopically expressed, recombinant p70 bound to the Vbeta Inr element with a specificity similar to that of wild-type TFII-I. More importantly, p70, which lacks independent activation functions, behaved as a dominant negative mutant that inhibited Inr-specific function of wild-type TFII-I. However, the activation functions of p70 were restored when fused to the heterologous activation domain of the yeast activator protein GAL4. Taken together, these data suggest that TFII-I functions in vivo require an intact Inr element and that the Inr-specific transcriptional functions of TFII-I are solely dictated by its N-terminal DNA binding domain and do not require its own C-terminal activation domain.

TFII-I enhances activation of the c-fos promoter through interactions with upstream elements.

The transcription factor TFII-I was initially isolated as a factor that can bind to initiator elements in core promoters. Recent evidence suggests that TFII-I may also have a role in signal transduction. We have found that overexpression of TFII-I can enhance the response of the wild-type c-fos promoter to a variety of stimuli. This effect depends on the c-fos c-sis-platelet-derived growth factor-inducible factor binding element (SIE) and serum response element (SRE). There is no effect of cotransfected TFII-I on the TATA box containing the c-fos basal promoter. Three TFII-I binding sites can be found in c-fos promoter. Two of these overlap the c-fos SIE and SRE, and another is located just upstream of the TATA box. Mutations that distinguish between serum response factor (SRF), STAT, and TFII-I binding to the c-fos SIE and SRE suggest that the binding of TFII-I to these elements is important for c-fos induction in conjunction with the SRF and STAT transcription factors. Moreover, TFII-I can form in vivo protein-protein complexes with the c-fos upstream activators SRF, STAT1, and STAT3. These results suggest that TFII-I may mediate the functional interdependence of the c-fos SIE and SRE elements. In addition, the ras pathway is required for TFII-I to exert its effects on the c-fos promoter, and growth factor stimulation enhances tyrosine phosphorylation of TFII-I. These results indicate that TFII-I is involved in signal transduction as well as transcriptional activation of the c-fos promoter.

A multifunctional DNA-binding protein that promotes the formation of serum response factor/homeodomain complexes: identity to TFII-I.

The human homeodomain protein Phox1 interacts functionally with serum response factor (SRF) to impart serum responsive transcriptional activity to SRF-binding sites in a HeLa cell cotransfection assay. However, stable ternary complexes composed of SRF, Phox1, and DNA, which presumably mediate the transcriptional effects of Phox1 in vivo, have not been observed in vitro. Here, we report the identification, purification, and molecular cloning of a human protein that promotes the formation of stable higher-order complexes of SRF and Phox1. We show that this protein, termed SPIN, interacts with SRF and Phox1 in vitro and in vivo. Furthermore, SPIN binds specifically to multiple sequences in the c-fos promoter and interacts cooperatively with Phox1 to promote serum-inducible transcription of a reporter gene driven by the c-fos serum response element (SRE). SPIN is identical to the initiator-binding protein TFII-I. Consistent with this hypothesis, SPIN exhibits modest affinity for a characterized initiator sequence in vitro. We propose that this multifunctional protein coordinates the formation of an active promoter complex at the c-fos gene, including the linkage of specific signal responsive activator complexes to the general transcription machinery.

Methods for studying the biochemical properties of an Inr element binding protein: TFII-I.

Transcription initiation in eukaryotic mRNA coding genes is brought about by a host of general transcription factors, which assemble into a functional preinitiation complex (PIC) at the core promoter region, and gene-specific factors, which exert their effects on the rate and/or stability of the PIC. The core promoter region consists of a well-characterized TATA box and/or a less well-characterized pyrimidine-rich initiator element (Inr). While the biochemical mechanisms of TATA-mediated transcription initiation are extensively studied and known to be directed by the TATA binding protein, the mechanisms via the Inr element are poorly understood, as several factors have been shown to bind to an Inr. Here, we describe the biochemical properties of an Inr binding protein, TFII-I, employing the naturally occurring TATA-less but Inr-containing promoter derived from the T-cell receptor beta chain gene (V beta).