NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.
bThe proinflammatory cytokine tumor necrosis factor alpha (TNF-␣) modulates the expression of many genes, primarily through activation of NF-B. Here, we examined the global effects of the elongation factor Spt5 on nascent and mature mRNAs of TNF-␣-induced cells using chromatin and cytosolic subcellular fractions. We identified several classes of TNF-␣-induced genes controlled at the level of transcription, splicing, and chromatin retention. Spt5 was found to facilitate splicing and chromatin release in genes displaying high induction rates. Further analysis revealed striking effects of TNF-␣ on the splicing of 25% of expressed genes; the vast majority were not transcriptionally induced. Splicing enhancement of noninduced genes by TNF-␣ was transient and independent of NF-B. Investigating the underlying basis, we found that Spt5 is required for the splicing facilitation of the noninduced genes. In line with this, Spt5 interacts with Sm core protein splicing factors. Furthermore, following TNF-␣ treatment, levels of RNA polymerase II (Pol II) but not Spt5 are reduced from the splicing-induced genes, suggesting that these genes become enriched with a Pol II-Spt5 form. Our findings revealed the Pol II-Spt5 complex as a highly competent coordinator of cotranscriptional splicing.T he transcription elongation factor DRB sensitivity-inducing factor (DSIF) is a highly conserved complex consisting of a large subunit, Spt5 (p160), and a small subunit, Spt4 (p14). DSIF plays a central role in promoter-proximal pausing by polymerase II (Pol II) (1). In addition, it was shown to facilitate capping (2-5) and to coordinate elongation with mRNA splicing and export of a subset of inflammatory genes (6). Recently, DSIF was also reported to promote 3=-end processing of snRNAs (7) and DNA cleavage during immunoglobulin class switching (8).Tumor necrosis factor alpha (TNF-␣) is a pleiotropic cytokine that modulates many important physiological and pathological processes primarily through inflammation. It induces the production of other proinflammatory cytokines and chemokines and increases its own production (9). TNF-␣, through the TNF receptor (TNFR), triggers a signaling cascade that leads to activation of NF-B, a family of transcription factors that is central to the inflammatory response elicited by TNF-␣ and other extracellular signals. In the resting state NF-B resides in the cytoplasm as a dimer in complex with inhibitory proteins such as IB␣ (10, 11). In response to TNF-␣ or other signaling molecules, an IB kinase complex called IKK is activated and phosphorylates IB␣, targeting it for ubiquitination and degradation by the proteasome (10, 11). Freed of IB␣, the NF-B dimer translocates into the nucleus, where it activates the transcription of genes that control inflammatory responses, cell survival, cell cycle, differentiation, and other functions (12, 13). The mRNAs induced by NF-B are divided into three major groups (I, II, and III) according to the kinetics of their expression, which represent early, slow, and very slow exp...
A subset of inflammatory-response NF-κB target genes is activated immediately following pro-inflammatory signal. Here we followed the kinetics of primary transcript accumulation after NF-κB activation when the elongation factor Spt5 is knocked down. While elongation rate is unchanged, the transcript synthesis at the 5′-end and at the earliest time points is delayed and reduced, suggesting an unexpected role in early transcription. Investigating the underlying mechanism reveals that the induced TFIID–promoter association is practically abolished by Spt5 depletion. This effect is associated with a decrease in promoter-proximal H3K4me3 and H4K5Ac histone modifications that are differentially required for rapid transcriptional induction. In contrast, the displacement of TFIIE and Mediator, which occurs during promoter escape, is attenuated in the absence of Spt5. Our findings are consistent with a central role of Spt5 in maintenance of TFIID–promoter association and promoter escape to support rapid transcriptional induction and re-initiation of inflammatory-response genes.
Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields’ locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)–T–based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag–specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients’ glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.
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