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

AbstractWe 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.

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Diversification of Transcription Factor NF-κB in Protists

10.1101/2021.03.15.435342 ◽

2021 ◽

Author(s):

Leah M. Williams ◽

Sainetra Sridhar ◽

Jason Samaroo ◽

Ebubechi K. Adindu ◽

Anvitha Addanki ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activation ◽

Functional Characterization ◽

Binding Activity ◽

Life Stages ◽

Rna Seq ◽

Dna Binding Activity ◽

Inhibitory Domain

In this report, we investigate the evolution of transcription factor NF-κB by examining its structure, activity, and regulation in two protists using phylogenetic, cellular, and biochemical techniques. In Capsaspora owczarzaki (Co), we find that full-length NF-κB has an N-terminal DNA-binding domain and a C-terminal Ankyrin (ANK) repeat inhibitory domain, and its DNA-binding activity is more similar to metazoan NF-κB rather than Rel proteins. As with mammalian NF-κB proteins, removal of the ANK repeats is required for Co-NF-κB to enter the nucleus, bind DNA, and activate transcription. However, C-terminal processing of Co-NF-κB is not induced by co-expression of IKK in human cells. Exogenously expressed Co-NF-κB localizes to the nucleus in Co cells. NF-κB mRNA and DNA-binding levels differ across three life stages of Capsaspora, suggesting distinct roles for NF-κB in these life stages. RNA-seq and GO analyses identify possible gene targets and biological functions of Co-NF-κB. We also show that three NF-κB-like proteins from the choanoflagellate Acanthoeca spectabilis (As) all consist of primarily the N-terminal conserved Rel Homology domain sequences of NF-κB, and lack C-terminal ANK repeats. All three As-NF-κB proteins constitutively enter the nucleus of human and Co cells, but differ in their DNA-binding and transcriptional activation activities. Furthermore, all three As-NF-κB proteins can form heterodimers, indicating that NF-κB diversified into multi-subunit families at least two times during evolution. Overall, these results present the first functional characterization of NF-κB in a taxonomic kingdom other than Animalia and provide information about the evolution and diversification of this biologically important transcription factor.

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Functional characterization of GATA3 mutations causing the hypoparathyroidism-deafness-renal (HDR) dysplasia syndrome: insight into mechanisms of DNA binding by the GATA3 transcription factor

Human Molecular Genetics ◽

10.1093/hmg/ddl454 ◽

2006 ◽

Vol 16 (3) ◽

pp. 265-275 ◽

Cited By ~ 103

Author(s):

A. Ali ◽

P. T. Christie ◽

I. V. Grigorieva ◽

B. Harding ◽

H. Van Esch ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Functional Characterization ◽

Insight Into

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Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II (NF-kappa B) element

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5217-5227.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5217-5227

Author(s):

A L Lattion ◽

E Espel ◽

P Reichenbach ◽

C Fromental ◽

P Bucher ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

B Lymphocytes ◽

Binding Proteins ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Binding Proteins ◽

Nf Kappa B ◽

Dna Binding Specificity

We have biochemically and functionally characterized a new transcription factor, NP-TCII, which is present in nuclei from unstimulated T and B lymphocytes but is not found in nonhematopoietic cells. This factor has a DNA-binding specificity similar to that of NF-kappa B but is unrelated to this or other Rel proteins by functional and biochemical criteria. It can also be distinguished from other previously described lymphocyte-specific DNA-binding proteins.

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Characterization of a new tissue-specific transcription factor binding to the simian virus 40 enhancer TC-II (NF-kappa B) element.

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.5217 ◽

1992 ◽

Vol 12 (11) ◽

pp. 5217-5227 ◽

Cited By ~ 9

Author(s):

A L Lattion ◽

E Espel ◽

P Reichenbach ◽

C Fromental ◽

P Bucher ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

B Lymphocytes ◽

Binding Proteins ◽

Simian Virus 40 ◽

Simian Virus ◽

Dna Binding Proteins ◽

Nf Kappa B ◽

Dna Binding Specificity

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Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation

10.1101/691097 ◽

2019 ◽

Author(s):

Leah M. Williams ◽

Melissa M. Inge ◽

Katelyn M. Mansfield ◽

Anna Rasmussen ◽

Jamie Afghani ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Transcriptional Activation ◽

Functional Characterization ◽

Binding Activity ◽

Sponge Tissue ◽

Dimerization Domain ◽

Dna Binding Activity ◽

Iκb Kinase

ABSTRACTBiological and biochemical functions of immunity transcription factor NF-κB in basal metazoans are largely unknown. Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq), in the phylum Porifera. Structurally and phylogenetically, the Aq-NF-κB protein is most similar to NF-κB p100 and p105 among vertebrate proteins, with an N-terminal DNA-binding/dimerization domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile more similar to human NF-κB proteins than Rel proteins. Aq-NF-κB also resembles the mammalian NF-κB protein p100 in that C-terminal truncation results in translocation of Aq-NF-κB to the nucleus and increases its transcriptional activation activity. Overexpression of a human or sea anemone IκB kinase (IKK) can induce C-terminal processing of Aq-NF-κB in vivo, and this processing requires C-terminal serine residues in Aq-NF-κB. Unlike human NF-κB p100, however, the C-terminal sequences of Aq-NF-κB do not effectively inhibit its DNA-binding activity when expressed in human cells. Tissue of another demosponge, a black encrusting sponge, contains NF-κB site DNA-binding activity and an NF-κB protein that appears mostly processed and in the nucleus of cells. NF-κB DNA-binding activity and processing is increased by treatment of sponge tissue with LPS. By transcriptomic analysis of A. queenslandica we identified likely homologs to many upstream NF-κB pathway components. These results present a functional characterization of the most ancient metazoan NF-κB protein to date, and show that many characteristics of mammalian NF-κB are conserved in sponge NF-κB, but the mechanism by which NF-κB functions and is regulated in the sponge may be somewhat different.

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