Comparison of NF-κB from the protists Capsaspora owczarzaki and Acanthoeca spectabilis reveals extensive evolutionary diversification of this transcription factor

We provide a functional characterization of transcription factor NF-κB in protists and provide information about the evolution and diversification of this biologically important protein. We characterized NF-κB in two protists using phylogenetic, cellular, and biochemical techniques. NF-κB of the holozoan Capsaspora owczarzaki (Co) has an N-terminal DNA-binding domain and a C-terminal Ankyrin repeat (ANK) domain, and its DNA-binding specificity is more similar to metazoan NF-κB proteins than to Rel proteins. Removal of the ANK domain allows Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by IκB kinases in human cells. Overexpressed Co-NF-κB localizes to the cytoplasm in Co cells. Co-NF-κB mRNA and DNA-binding levels differ across three Capsaspora life stages. RNA-sequencing and GO analyses identify possible gene targets of Co-NF-κB. Three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) contain conserved Rel Homology domain sequences, but lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of cells, but differ in their DNA-binding abilities, transcriptional activation activities, and dimerization properties. These results provide a basis for understanding the evolutionary origins of this key transcription factor and could have implications for the origins of regulated immunity in higher taxa.

Phenotypic characterization with somatic genome editing and gene transfer reveals the diverse oncogenicity of ependymoma fusion genes

Recurrent RELA and YAP1 fusions are intimately associated with tumorigenesis in supratentorial ependymomas. Chromothripsis and focal copy number alterations involving 11q are hallmarks of these tumors. However, it is unknown whether the chromosomal alterations are a direct causal event resulting in fusion transcripts. In addition, the biological significance of the RELA fusion variants and YAP1 fusions is not yet fully characterized. In this study, we generated gene rearrangements on 11q with the CRISPR/Cas9 system and investigated the formation of oncogenic ependymoma fusion genes. Further, we examined the oncogenic potential of RELA fusion variants and YAP1 fusions in a lentiviral gene transfer model. We observed that endogenous RELA fusion events were successfully induced by CRISPR/Cas9-mediated genome rearrangement in cultured cells. In vivo genome editing in mouse brain resulted in the development of ependymoma-like brain tumors that harbored the Rela fusion gene. All RELA fusion variants tested, except a variant lacking the Rel homology domain, were able to induce tumor formation, albeit with different efficacy. Furthermore, expression of YAP1-FAM118B and YAP1-MAMLD1 fusions induced the formation of spindle-cell-like tumors at varying efficacy. Our results indicate that chromosomal rearrangements involving the Rela locus are the causal event for the formation of Rela fusion-driven ependymomas in mice. Furthermore, the type of RELA. fusion might affect the aggressiveness of tumors and that the Rel homology domain is essential for the oncogenic functions of RELA. fusions. The YAP1 fusion genes are also oncogenic when expressed in mice.

IκB Kinase α-Mediated Derepression of SMRT Potentiates Acetylation of RelA/p65 by p300

ABSTRACT Over the last several years, significant progress has been made in identifying chromatin-regulated events that govern NF-κB transcription. Using either laminin attachment or tumor necrosis factor alpha as a physiological stimulus of NF-κB activation, we demonstrate that IκB kinase α (IKKα) is recruited to chromatin in distinct phases. In the initial phase, IKKα is responsible for derepressing the silencing mediator for retinoic acid and thyroid hormone receptor (SMRT)-histone deacetylase 3 (HDAC3) corepressor complex from the p50 homodimer. However, in the latter phase, chromatin-bound IKKα coordinates the simultaneous phosphorylation of RelA/p65(S536) and SMRT(S2410) as detected by chromatin immunoprecipitation (ChIP) assays. Although phosphorylated SMRT remains bound to the active p50-RelA/p65 heterodimer of NF-κB, derepression of SMRT is evidenced by the loss of chromatin-associated HDAC3 activity. ChIP and re-ChIP analysis demonstrates that phosphorylation of RelA/p65(S536) and SMRT(S2410) occurs prior to acetylation of RelA/p65 at K310. Moreover, IKKα-induced phosphorylation of RelA/p65(S536) displaces corepressor activity, allowing p300-mediated acetylation of RelA/p65. Introduction of nonphosphorylatable mutants of RelA/p65 and SMRT proteins or the inhibition of IKK activity results in active repression of NF-κB promoters by tethering the SMRT-HDAC3 complex. Similar to phosphorylation within the Rel homology domain of RelA/p65, which governs an exchange of HDAC1 for CBP/p300 acetyltransferases, we demonstrate that phosphorylation within the transactivation domain of RelA/p65(S536) displaces SMRT-HDAC3 repressor activity, allowing p300 to acetylate RelA/p65.