Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4(+) and CD8(+) T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation

Depletion of the transcriptional coactivators CREB-binding protein or EP300 downregulates CD20 in diffuse large B-cell lymphoma cells and impairs the cytotoxic effects of anti-CD20 antibodies

Monoclonal antibodies targeting CD20 are central in the treatment of B-cell lymphomas. In diffuse large B-cell lymphoma (DLBCL), inactivating mutations of the histone acetyltransferases CREB-binding protein (CBP) and EP300 are common. Moreover, knockdown of CBP in DLBCL has been shown to result in aberrant transcriptional silencing. Expression of CD20 is sensitive to epigenetic manipulation, and histone deacetylase inhibitors have been found to potentiate treatment with anti-CD20 antibodies. Therefore, we studied the role of CBP and EP300 depletion on CD20 expression and effects of the anti-CD20 antibodies rituximab and obinutuzumab in DLBCL cells. Levels of CBP and EP300 were reduced by shRNA in the germinal centre-derived diffuse large B-cell lymphoma cell line SU-DHL4. The levels of CD20 mRNA and protein were determined by quantitative polymerase chain reaction, Western blot, and flow cytometry. Binding of the transcription factors PU.1 and FOXO1 to the CD20 promoter was determined by chromatin immunoprecipitation coupled with quantitative polymerase chain reaction. Response to the monoclonal anti-CD20 antibodies rituximab and obinutuzumab in CBP- or EP300-depleted cells was assessed by complement-dependent cell death, direct cell death, and antibody-dependent cellular cytotoxicity (ADCC). Our results suggest that depletion of CBP and EP300 levels leads to a strong reduction of CD20 expression, accompanied by reduced binding of PU.1 to the CD20 promoter. In CBP-depleted, but not EP300-depleted cells, increased binding of FOXO1 to the CD20 promoter was observed. Interestingly, CBP or EP300 depletion leads to decreased complement-dependent cell death and direct cell death in response to rituximab and obinutuzumab, which was most pronounced in response to rituximab in CBP-depleted cells. Our data suggest that inactivating mutations of CBP, and to a lesser extent EP300, may impair the response to anti-CD20 antibodies. However, these observations should be analyzed in future clinical trials

The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/ NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies

IBTK Differently Modulates Gene Expression and RNA Splicing in HeLa and K562 Cells

The IBTK gene encodes the major protein isoform IBTKα that was recently characterized as substrate receptor of Cul3-dependent E3 ligase, regulating ubiquitination coupled to proteasomal degradation of Pdcd4, an inhibitor of translation. Due to the presence of Ankyrin-BTB-RCC1 domains that mediate several protein-protein interactions, IBTKα could exert expanded regulatory roles, including interaction with transcription regulators. To verify the effects of IBTKα on gene expression, we analyzed HeLa and K562 cell transcriptomes by RNA-Sequencing before and after IBTK knock-down by shRNA transduction. In HeLa cells, 1285 (2.03%) of 63,128 mapped transcripts were differentially expressed in IBTK-shRNA-transduced cells, as compared to cells treated with control-shRNA, with 587 upregulated (45.7%) and 698 downregulated (54.3%) RNAs. In K562 cells, 1959 (3.1%) of 63128 mapped RNAs were differentially expressed in IBTK-shRNA-transduced cells, including 1053 upregulated (53.7%) and 906 downregulated (46.3%). Only 137 transcripts (0.22%) were commonly deregulated by IBTK silencing in both HeLa and K562 cells, indicating that most IBTKα effects on gene expression are cell type-specific. Based on gene ontology classification, the genes responsive to IBTK are involved in different biological processes, including in particular chromatin and nucleosomal organization, gene expression regulation, and cellular traffic and migration. In addition, IBTK RNA interference affected RNA maturation in both cell lines, as shown by the evidence of alternative 3′- and 5′-splicing, mutually exclusive exons, retained introns, and skipped exons. Altogether, these results indicate that IBTK differently modulates gene expression and RNA splicing in HeLa and K562 cells, demonstrating a novel biological role of this protein

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

<div><p>Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.</p></div

p65-dependent transcriptional activation of eIF4H.

<p>(A) HeLa cells (5×10⁶) were transfected with pRc/CMV-p65 or pRc/CMV-empty vector (5 µg). Forty-eight hours post-transfection, total RNA was extracted and analysed by qRT-PCR to evaluate the expression of the indicated eIF genes. Values (mean ± SD, n = 5) are shown. The asterisk indicates a statistically significant difference between pRc/CMV-p65 and empty vector according to the Student's <i>t</i>-test (<i>p</i>≤0.01). (B) HeLa cells (5×10⁶) were transfected with siRNA control or siRNA p65 (200 pmol). Forty-eight hours post-transfection, total RNA was extracted and analysed by qRT-PCR for the expression of the indicated eIF genes. Values (mean ± SD, n = 5) are shown. The asterisk indicates a statistically significant difference between siRNA p65 and siRNA control according to the Student's <i>t</i>-test (<i>p</i> ≤0.01). (C) Wild type and p65^−/− MEFs (3×10⁵) were lysed, and total RNA was analysed by qRT-PCR for the expression of eIF4H gene. (D) Total cell extracts (20µg) of wild type and p65^−/− MEFs (3×10⁵) were separated by 12% SDS-PAGE and analysed by western blotting using anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the wild type taken as 1. (E) Nuclear extracts of wild type and p65^−/− MEFs (5×10⁶ cells) were analysed for the binding activity of the indicated NF-κB subunits to the NF-κB double-stranded oligonucleotide, as measured by ELISA EMSA using the NF-κB Transcription Factor ELISA assay kit (Cayman). (F) Total RNA from tumour cell lines (MDA-MB-231, MCF-7, SH-SY5Y, U251, D54, MC3, DeFew) (3×10⁵ cells) was analysed by qRT-PCR for the expression of eIF4H gene. (G) Whole protein cell extracts (20µg) of the indicated tumour cell lines were separated by 12% SDS–PAGE and analysed by western blotting using anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the MDA-MB-231 cells taken as 1. (H) Nuclear extracts of the indicated tumor cell lines (5×10⁶ cells) were analysed for the p65 binding to the NF-κB double-stranded oligonucleotide, using the NF-κB Transcription Factor ELISA assay kit (Cayman).</p

TNF-α induces the recruitment of p65 to eIF4H promoter.

<p>(A) HeLa cells (5×10⁶) were transfected with siRNA control or siRNA p65 (200 pmol). Forty-eight hours post-transfection, cells were 45 min-treated with TNF-α (20 ng/mL), or left untreated. Total RNA was extracted and analysed by qRT-PCR to measure the expression of eIF4H and eIF2S3. Values (mean ± SD, n = 3) are shown. Statistically significant differences between the samples are shown according to Student's <i>t</i>-test (p≤0.01). (B) HeLa cells (3×10⁷) were treated with TNF-α (20 ng/mL) for the indicated time, or left untreated. Chromatin was immunoprecipitated with anti-p65, anti-p50, anti-RelB, anti-c-Rel or IgG, and ChIP eluates were analysed by qRT-PCR.</p

p65 increases the protein synthesis rate in eIF4H-dependent manner

<p>. (A) HeLa cells (3×10⁶) were transfected with pRc/CMV empty vector (5µg), or pRc/CMV-3HA-p65, in presence of siRNA control or siRNA p65 (200 pmol). Twenty-four hours post-transfection, cells were incubated in methionine/cysteine-free medium for 30 min before addition of labelling medium containing [³⁵S]-methionine/cysteine (10 µCi/ml). One hour after protein labelling, the protein synthesis rate was evaluated. Values (mean ± SD, n = 3) are shown. Statistically significant differences between the samples are indicated according to Student's <i>t</i>-test (p≤0.01). (B) Protein extracts (20µg) of transfected HeLa cells shown in (A) were separated by 12% SDS–PAGE, and analysed by western blotting using anti-eIF4H, anti-HA, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the control (Rc/CMV plus siRNA control), taken as 1.</p

Bioinformatics-based prediction of NF-κB sites in eIFH promoters.

<p>Jaspar- and TFSEARCH-based computational analysis predicted putative NF-κB enhancers in the promoter regions of core eukaryotic translation initiation factors.</p