Identification and evolution of transcription factors RHR gene family (NFAT and RBPJ) involving lamprey (Lethenteron reissneri) innate immunity

Highlights

• Four RHR genes identified and cloned from Lethenteron reissneri.

• The evolutionary relationship of NFAT and RBPJ genes among different species is indicated.

• RHR gene family plays a significant role in the immune defense of lamprey.

• The study provides a new understanding for origin and evolution of the RHR gene family.

Abstract

Nuclear factor of activated T cells (NFAT) and recombination signal binding protein (RBP) belong to the family of Rel homology region (RHR) transcription factors which regulate the expression of genes involved in different aspects of the immune response. To gain insights into the evolution and characterisation of RHR genes in lampreys, a jawless vertebrate, four RHR genes, including nuclear factor of activated T cells (NFAT) and recombination signal binding protein for immunoglobulin kappa J region (RBPJ), have been identified and cloned from the lamprey (Lethenteron reissneri) database. Evolutionary relationships of NFAT and RBPJ genes among different species were determined through molecular phylogenetic analysis. Motif, genetic structure, and tertiary structure analyses showed that NFATs and RBPJ are conserved and contain RHD and IPT domains. Moreover, synteny analysis showed that the neighbourhood genes of Lr-NFATs and Lr-RBPJ have undergone significant changes compared to jawed vertebrates. Real-time quantitative results demonstrated that the RHR gene family plays a significant role in immune defence. This study provides a new understanding of the origin and evolution of the RHR gene family in different species.

Introduction

Transcription factors are a class of proteins that regulate gene transcription and expression by binding to gene-specific sequences and play an essential role in regulating the biological activities of cells (Lambert et al., 2018). Rel homology region (RHR) transcription factors, also known as Rel homology domain (RHD), belong to the immunoglobulin fold (IF) transcription factor superfamily (Wingender et al., 2018). The RHR family of transcription factors mainly consists of four members: nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT), early B-cell factor (EBF), and recombination signal binding protein (RBP), which contain a conserved Rel domain and immunoglobulin-like fold (IPT) domain.

NFAT is a transcription factor that binds to the interleukin-2 (IL-2) promoter in activated T cells (Shaw et al., 1988). The NFAT family consists of five members, including NFAT1 (NFATp or NFATc2), NFAT2 (NFATc or NFATc1), NFAT3 (NFATc4), NFAT4 (NFATx or NFATc3), and NFAT5 (TonE-BP or NFATL1) (Macian, 2005). Four of these are regulated by calcium signalling. NFAT5, the only protein not regulated by calcium ions, is a primitive member of the NFAT family. NFAT5 first evolved from the transcription factor family containing the RHD and appears in Nematostella vectensis (Sullivan et al., 2007). These proteins participate in non-immune cells, T cells, and other immune system cells by regulating the expression of many genes, such as immune response, immune tolerance, immune metabolism, developmental apoptosis, and angiogenesis (Vaeth and Feske, 2018). NFAT family proteins share an N-terminal NFAT-homology region (NHR), a highly conserved RHD, and a C-terminal immunoglobulin-like fold domain (IPT) (Qin et al., 2017). The NHR is moderately conserved among NFAT family members and contains several serine-rich regions (SRRs) that can be phosphorylated by a variety of kinases and a transactivation domain (Lee et al., 2018; Pan et al., 2013). All NFAT members that are responsive to calcium (NFAT1-NFAT4) contain NHR, which has an effective transactivation domain and a calcineurin docking site (Luo et al., 1996). However, NFAT5 lacks the calcineurin docking site and is completely insensitive to calcium and calcineurin (Lopez-Rodríguez et al., 1999). NFAT5 can be activated in response to osmotic stress (Miyakawa et al., 1999). The NHR domain also contains a nuclear localisation sequence (NLS) and nuclear export sequence (NES), which control NFAT subcellular localisation through phosphorylation and dephosphorylation of NFAT proteins in SRRs (Okamura et al., 2000; Beals et al., 1997). At rest, many serine residues of NFAT proteins are highly phosphorylated, which allows NFAT to remain in the cytoplasm. During cell activation, it is dephosphorylated by calcium/calmodulin-dependent phosphatase calcineurin, exposing its NLS, thus triggering the transport of NFAT proteins from the cytoplasm to the nucleus.

RBP family proteins are involved in the development of the nervous system, differentiation of the haematopoietic system (Pursglove and Mackay, 2005), and the regulation of the immune response (Strug et al., 2018; Diéguez-Hurtado et al., 2019). There are two subtypes of RBP proteins: recombination signal binding protein for immunoglobulin kappa J region (RBPJκ/RBPJ), also called Suppressor of Hairless [Su(H)] (Bartkuhn et al., 2019), which is a crucial downstream effector of Notch signalling that is involved in cell proliferation, apoptosis, and the maintenance of stem cells (Xiao et al., 2019); and recombination signal binding protein for immunoglobulin kappa J region like (RBPL), whose function does not depend on the Notch signalling pathway (Prevorovský et al., 2007). RBP is composed of three domains: an N-terminal domain (NTD); a β-trefoil domain (BTD); and a C-terminal domain (CTD). The NTD and CTD share structural similarities with the RHR family of transcription factors (Wilson and Kovall, 2006). The CTD is similar to the IPT/RHR-C domain, whereas the NTD closely resembles the RHR-N domain. However, the CTD of RBPJ is not similar to the RHR-C domain in function; it does not promote dimerization and does not contact DNA. There is a unique hydrophobic pocket on the surface of the BTD, which contributes to specific and non-specific DNA-binding (Kovall and Hendrickson, 2004).

Lampreys and hagfish are the most primitive vertebrates still living today, belonging to the class Cyclostomata, which has no upper or lower jaws (Zhu et al., 2020; Shimeld and Donoghue, 2012). Study of lampreys has revealed deep insight into the evolution of early vertebrates; they are an ideal model organism in the fields of immunology, genetics, developmental neurobiology, animal physiology, etc. (Xu et al., 2016; Cooper and Alder, 2006). Previous studies have identified and analysed homologs of EBF and NF-κB in lampreys (Lara-Ramírez et al., 2017; Su et al., 2017). However, little is known about the existence and potential role of the two other members of the RHR transcription factor family, NFAT and RBPJ genes, in jawless vertebrates. Therefore, to gain more insight into the evolutionary relationship of the gene families, we identified and cloned NFAT and RBPJ genes (NFAT3, NFAT4, NFAT5, and RBPJ) in lampreys (Lethenteron reissneri) using the L. reissneri genome database (Zhu et al., 2021). Subsequently, motifs, synthetic relationships, and gene and domain structure analyses were used to investigate the evolutionary origin of the NFAT and RBPJ gene families. Moreover, we examined the expression of NFAT and RBPJ genes in lampreys in response to pathogens. Our study not only provides new insights into the evolution and functional diversity of NFAT and RBPJ genes, but also lays a foundation for research on the evolutionary relationship of the RHR gene family.