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STAT3 is a pleiotropic transcription factor involved in homeostatic and host defense processes in the human body. It is activated by numerous cytokines and growth factors and generates a series of cellular effects. Of the STAT-mediated signal transduction pathways, STAT3 transcriptional control is best understood. Jak kinase dependent activation of STAT3 relies on Y705 phosphorylation triggering a conformational switch that is stabilized by intermolecular interactions between SH2 domains and the pY705 motif. We here show that a second tyrosine phosphorylation within the SH2 domain at position Y640, induced by Tyk2, negatively controls STAT3 activity. The Y640F mutation leads to stabilization of activated STAT3 homodimers, accelerated nuclear translocation and superior transcriptional activity following IL-6 and LIF stimulation. Moreover, it unlocks type I IFN-dependent STAT3 signalling in cells that are normally refractory to STAT3 transcriptional activation.

Actin and nuclear myosin 1c (NM1) cooperate in RNA polymerase I (pol I) transcription. NM1 is also part of a multiprotein assembly, B-WICH, which is involved in transcription. This assembly contains the chromatin remodeling complex WICH with its subunits WSTF and SNF2h. We report here that NM1 binds SNF2h with enhanced affinity upon impairment of the actin-binding function. ChIP analysis revealed that NM1, SNF2h, and actin gene occupancies are cell cycle-dependent and require intact motor function. At the onset of cell division, when transcription is temporarily blocked, B-WICH is disassembled due to WSTF phosphorylation, to be reassembled on the active gene at exit from mitosis. NM1 gene knockdown and motor function inhibition, or stable expression of NM1 mutants that do not interact with actin or chromatin, overall repressed rRNA synthesis by stalling pol I at the gene promoter, led to chromatin alterations by changing the state of H3K9 acetylation at gene promoter, and delayed cell cycle progression. These results suggest a unique structural role for NM1 in which the interaction with SNF2h stabilizes B-WICH at the gene promoter and facilitates recruitment of the HAT PCAF. This leads to a permissive chromatin structure required for transcription activation., Author Summary Actin and myosin are key regulators of several processes that occur in the cell nucleus. In rRNA biogenesis, actin in complex with nuclear myosin 1c (NM1) is involved in several phases of rDNA transcription. Further, NM1 interacts with the chromatin remodelling complex WICH, with the subunits WSTF and SNF2h. The multiprotein assembly thus formed, termed B-WICH, is engaged in the post-initiation phase of pol I transcription. These observations have led to the proposal that the actin-NM1 interaction mediates the recruitment of the WICH complex to activate transcription. Recent evidence indicates that the WSTF component of the B-WICH complex facilitates SNF2h-dependent nucleosomes repositioning and remodels in this way the chromatin at the rRNA gene promoter. We show here that NM1 interacts with the WICH complex and that this interaction is required to establish permissive chromatin by promoting H3K9 acetylation. This mechanism leads to transcription activation and facilitates cell cycle progression. NM1 performs these actions by interacting with SNF2h, presumably stabilizing B-WICH at the gene promoter. We show also that NM1 is needed for association of the polymerase-associated actin with the rRNA gene. Actin and SNF2h compete for NM1 binding. Therefore we propose a two-step mechanism of gene activation where NM1 functions as a structural switch that connects pol I with chromatin for transcription activation and cell cycle progression.

Tyrosine phosphorylation is a hallmark for activation of STAT proteins, but their transcriptional activity also depends on other secondary modifications. Type I IFNs can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as an important mediator of this STAT3-targeted transcriptional repression. Sin3a directly interacts with STAT3 and promotes its deacetylation. SIN3A silencing results in a prolonged nuclear retention of activated STAT3 and enhances its recruitment to the SOCS3 promoter, concomitant with histone hyperacetylation and enhanced STAT3-dependent transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.

The antiviral and antiproliferative responses mediated by type I interferons (IFNs) depend on JAK/STAT signaling and ISGF3 (STAT1:STAT2:IRF9)-dependent transcription. In addition, type I IFNs stimulate STAT3 activation in many cell types, an event generally associated with cell cycle progression, survival, and proliferation. To gather more insight into this functionally contradictive phenomenon, we studied the regulation of STAT3 transcriptional activity upon type I IFN treatment. We show that IFN alpha 2 stimulation strongly induces STAT3 phosphorylation, nuclear translocation, and promoter binding, yet the activation of transcription of a STAT3-dependent reporter and endogenous genes, such as SOCS3 and c-FOS, is impaired. Simultaneous treatment with IFN alpha 2 and trichostatin A, as well as combined HDAC1/HDAC2 silencing, restores STAT3-dependent reporter gene and endogenous gene expression, strongly suggesting that HDAC1 and HDAC2 are directly involved in repressing IFN alpha 2-activated STAT3. Of note, single silencing of only one of the two HDACs does not lead to enhanced STAT3 activity, supporting a functional redundancy between these two enzymes. In sharp contrast, HDAC1 and HDAC2 activities are required for ISGF3-dependent gene expression. We conclude that HDAC1 and HDAC2 differentially modulate STAT activity in response to IFN alpha 2: while they are required for the induction of ISGF3-responsive genes, they impair the transcription of STAT3-dependent genes.

The activity of Signal Transducer and Activator of Transcription (STAT) proteins is regulated by a multitude of posttranslational modifications, which control different aspects of signal transduction. In particular, lysine acetylation and deacetylation has emerged as a critical modification, regulating the activity of all seven STAT members. Unlike tyrosine phosphorylation/dephosphorylation, which mainly acts as an on/off switch of STAT activity, the control exerted by acetylation appears multifaceted and by far more complex, exerting distinct and even opposed regulation to different STAT members. We observed that type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide siRNA screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as a novel regulator of STAT activity. Sin3a directly interacts with STAT3 and controls its acetylation status, its nuclear accumulation and DNA binding, strongly influencing the transcription of a subset of STAT3-responsive genes. Interfering with Sin3a expression restores, at least partially, the STAT3 transcriptional blockade observed upon IFN treatment. Strikingly, Sin3a exerts opposed regulation on ISGF3 activity, as it is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. We conclude that Sin3a contributes to the regulation of the balance between the activities of different STAT members, and interfering with its activity or expression may re-direct cytokine signals, favoring the onset of pathological conditions, such as autoimmune syndromes, inflammation and cancer.

Introduction Tyrosine phosphorylation is a hallmark for activation of Signal Transducer and Activator of Transcription (STAT) proteins, but their transcriptional activity also depends on other secondary modifications. Type I interferons (IFNs) can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Methods Simultaneous treatment with IFNa2 and Trichostatin A, as well as combined HDAC1/HDAC2 silencing, restores STAT3-dependent reporter gene and endogenous genes expression, strongly suggesting that HDAC1 and HDAC2 are directly involved in repressing IFNa-activated STAT3. We used the IFN-dependent STAT3 transcriptional blockade as read-out for a genome-wide RNAi repressor screen. Results Quite significantly, we identified multiple partners of the Sin3a/HDAC transcriptional repressor complex as negative regulators of STAT3 transcriptional activity, regardless to the STAT3 activating stimulus. Sin3a directly interacts with STAT3 and controls its acetylation status, its nuclear accumulation and DNA binding, strongly influencing the transcription of a subset of STAT3-responsive genes. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. Conclusion The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.