With an understanding of
NFX1 in normal physiology, it is important then to turn to studies of
NFX1 and its variants in pathophysiology and disease. First, in central nervous system development, a human variant of
NFX1 (
NFXL1), which is a transcription factor with domain similarities to
NFX1, was found to confer increased risk for a specific language impairment [
22]. Higher expression of
NFXL1 was found in regions of the cerebellum that were associated with this diagnosis [
23], and HLA class II genes regulated by
NFX1 were also found to correlate with these specific language impairments [
24]. Second, in vascular studies, familial segregation analysis revealed that
NFX1 c.2519T>C (p.Leu840Pro) was associated with intracranial aneurysms, a cerebrovascular disorder; this
NFX1 point mutation was found in only cases and was absent among unaffected family members [
25], linking
NFX1 as a genetic risk factor for these familial intracranial aneurysms. Third, in gastrointestinal development, an
NFX1 novel de novo variant c.1723G>A (p.Val575Met) was found to be one of the deleterious variants in human esophageal atresia, the most common malformation of the upper digestive tract [
26], suggesting a functional significance to
NFX1 expression in upper digestive tract development and disease. Lastly, in metabolism and endocrinopathies, greater NFX1, with decreased HLA-DRA gene expression, was observed in obese adolescent individuals with insulin resistance compared to those who were insulin sensitive [
27]. These studies highlight the normal function that
NFX1, and
NFXL1, may play in development, metabolism, and cellular or organ system physiology that can be disrupted by a modulation in expression or de novo novel variant gene expression.
3.1. NFX1, High-Risk Human Papillomavirus, and Cancer Studies
All of the NFX1 studies summarized above were conducted across species assuming normal biology, although exogenous environmental stressors were included. What is interesting to consider is how infectious agents could modulate the cell or organism, and how NFX1 may participate in the dysregulation of cellular genes and pathways due to these infections. One infectious agent, human papillomavirus (HPV), has been researched in exquisite detail in epithelial cells, and many of the NFX1 studies over the past 15 years have been performed in epithelial cells in the context of HPV. These seminal papers have led to a greater understanding of the splice variants of NFX1 expressed in human epithelial cells, their conserved protein domains, their normal functions in cells, and the direct roles in supporting the viral life cycle of HPV and the oncogenic potential of high-risk (HR) HPV infections.
Human papillomaviruses infect epithelial cells, and those that are defined as high-risk are so based on their epidemiologic association with cervical cancer [
28]. Of the more than a dozen HR HPV types causally linked to cancer, HPV type 16 causes half of all cervical cancers worldwide [
28]. All cancers caused by HR HPVs universally express the viral oncogenes E6 and E7; however, neither of these genes have an enzymatic function. Therefore, they must partner with host cell proteins to drive oncogenesis. More than 15 years ago, a study to discover novel proteins that collaborate with, and bind directly to, the HR HPV type 16 E6 (16E6) viral oncogene identified
NFX1 [
29]. Two isoforms of
NFX1 are expressed in epithelial cells, and they both were found to bind to the 16E6 oncoprotein [
29,
30].
3.2. Two Human NFX1 Isoforms in Epithelial Cells: NFX1-91 and NFX1-123
The two splice variants of
NFX1 that are expressed in epithelial cells are named NFX1-91 and NFX1-123, based on their respective kilodalton masses [
29]. NFX1-91 and NFX1-123 share a common N-terminus and central domain, but they have unique C-termini ().
Figure 1. Model of NFX1 splice variants in humans. NFX1-123 and NFX1-91 share a common N-terminus, with a PAM2 motif, and Central Domain, with a PHD/RING domain and six cysteine-rich zinc-like fingers. NFX1-91 has a truncated C-terminus that is lysine-rich. NFX1-123 has a unique C-terminus with two additional zinc-like fingers and an R3H domain.
In the N-terminus, there is a PAM2 motif. This motif is necessary for proteins to bind to cytoplasmic poly(A) binding proteins. In the central domain, there is a PHD/RING domain that has an E3 ubiquitin ligase function. There are also six cysteine-rich zinc-like fingers, which are required for DNA binding and are conserved from the murine homolog. For NFX1-91, its C-terminus is short and is lysine-rich. For NFX1-123, its unique C-terminus includes two additional zinc-like fingers and an R3H domain, and this domain has putative single-stranded nucleic acid binding capabilities [
31].
The shorter splice variant, NFX1-91, is rapidly targeted for ubiquitin-mediated degradation by 16E6 and the E3 ubiquitin ligase E6 Associated Protein (E6AP). 16E6 and E6AP polyubiquitinate the NFX1-91 protein at its lysine-rich unique C-terminus and target it for 26S proteasomal degradation; meanwhile, the longer isoform of
NFX1, NFX1-123, is not [
29]. These two isoforms of
NFX1 have distinct, and at times opposing, functions in gene regulation, driven by their protein domains and subcellular location. Those are described in detail below, as are how HR HPV disrupts and co-opts those functions of
NFX1 during oncogenesis.
3.3. NFX1-91: A Transcriptional Regulator Destabilized by HR HPV
The NFX1-91 isoform is a nuclear protein that binds to an X1 box
cis-element in the proximal
hTERT promoter via its central domain.
hTERT is the catalytic subunit of telomerase, and telomerase, as a key regulator of cellular immortalization, is universally activated in HPV-associated cancers [
29]. NFX1-91 is a constitutive transcriptional repressor of
hTERT, and it sits in complex with PKC-delta, a cellular senescence-inducing factor, and with the co-repressor complex mSin3A and histone deacetylase 1 at the
hTERT promoter [
32,
33]. 16E6, and other E6 proteins from the beta HPV genus that can cause skin cancer, partner with E6AP to degrade NFX1-91, removing it from the
hTERT promoter. This derepresses
hTERT expression during an HPV infection [
33] and extends the lifespan of epithelial cells in culture [
34].
At the
hTERT promoter, NFX1-91 functions as a transcriptional repressor, but NFX1-91 can also function as a transcriptional activator for other genes. Knockdown of NFX1-91 led to a reduction of NF-kB inhibitors such as p105, driving an induction of NF-kB-responsive genes [
35]. The authors of this study offer a yet-to-be-tested hypothesis that different transcriptional co-regulators, partnering with NFX1-91, may drive this difference in activation versus repression of genes. It highlights the importance of the protein-partnerships between NFX1-91 and other transcriptional regulators. This holds true in protein partnerships and functions of the longer splice variant of
NFX1, NFX1-123.
3.4. NFX1-123: A Post-Transcriptional Regulator Stabilized by HR HPV
Since 16E6 and E6AP mediate the removal of NFX1-91, the more stable, longer splice variant of
NFX1, NFX1-123, rises in importance during HR HPV infections. As NFX1-123 is not targeted by 16E6 and E6AP for rapid ubiquitin-mediated degradation like NFX1-91, and because NFX1-123 has opposing effects to NFX1-91 in HPV associated cancers, more recent studies have focused on defining and understanding the role NFX1-123 plays in cancers caused by HR HPV. A paper published in 2019 demonstrated that the deubiquitinase USP9X interacted with and stabilized the NFX1-123 protein through its efficient deubiquitination [
36]. USP9X was increased in HPV-associated cancers, and specifically by 16E6 [
37]; as such, preserving greater NFX1-123 expression, through augmented USP9X, may be an important function during cancer development and progression. These roles of NFX1-123, in the context of HR HPV and 16E6 specifically, are described in further detail below.
In 2007, we were the first to identify that
NFX1 indeed had a conserved PAM2 motif in its N-terminus () [
30]. We determined that the NFX1-123 protein-bound cytoplasmic poly(A) binding proteins (PABPCs) via this PAM2 motif, and together NFX1-123 and PABPCs synergistically augmented hTERT expression and telomerase activity in 16E6 expressing cells [
30]. We then demonstrated that NFX1-123 colocalized with PABPCs in the cytoplasm but did not shuttle between nucleus and cytoplasm with them [
38], so NFX1-123 played no direct role in transcriptional regulation of RNA translocation. Rather, we noted that NFX1-123 contained both a PAM2 motif and a unique R3H domain, and these protein domains were required to post-transcriptionally increase hTERT through binding and stabilization of its mRNA. The 5′ UTR of the hTERT mRNA was a required
cis-element for NFX1-123 to maintain the post-transcriptional upregulation of hTERT [
38] and to further increase hTERT and telomerase activity over time [
39]. Therefore, NFX1-123 and its protein partnership with PABPCs led to increased hTERT expression and telomerase activity in HR HPV positive, or 16E6 positive, cells. This extension of growth and drive towards cellular immortalization by
NFX1, during an HR HPV infection, echoes the extension of lifespan seen with the
stc homolog in Drosophila.
3.5. NFX1-123: Increased in Epithelial Differentiation and Drives Differentiation Pathways
We discovered that NFX1-123 was not only normally expressed in epithelial cells but NFX1-123 itself increased during epithelial cell differentiation [
40]. That increase is further augmented by 16E6, and together NFX1-123 and 16E6 upregulate downstream differentiation pathways and targets [
40,
41]. One example of this is Notch1.
Notch1, an important regulator of cell growth and differentiation, was found to be increased by NFX1-123 and 16E6; like hTERT, this increase depended on the PAM2 and R3H domains of NFX1-123 [
42]. NFX1-123, with 16E6, increased expression of the Notch1 canonical pathway genes Hes1 and Hes5, and the increase in these genes by NFX1-123 required the presence and activation of the Notch1 receptor. Expression of the keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased by NFX1-123 and 16E6, but their upregulation was not directly linked to Notch1 receptor stimulation like Hes1 and Hes5 were. More intriguingly, the increase in keratinocyte differentiation induced by NFX1-123 with 16E6 was uncoupled from the growth arrest, increase in p21, and decrease in proliferative factor Ki67 typically seen during differentiation [
41]. These findings led to the thesis that NFX1-123 normally regulates differentiation in epithelial cells and keratinocytes, and NFX1-123 itself is increased during differentiation as well [
40]. However, the regulation of differentiation pathways can be co-opted by 16E6. Described in detail below, greater NFX1-123 has been shown to permit differentiation with continued growth and protection of longevity in the context of 16E6 co-expression—all of which are fundamental to the HPV viral life cycle and the oncogenesis of HPV associated cancers.
3.6. NFX1-123 Increased in Cervical Cancers and Co-Regulates Differentiation and Longevity
High expression of NFX1-123 has been demonstrated in HPV-positive cervical cancer cell lines [
41] and in primary cervical cancers, which are nearly universally HPV-positive [
39,
43]. In normal keratinocytes expressing 16E6, greater expression of NFX1-123 was associated with extended longitudinal active cellular growth and augment hTERT expression along with telomerase activity [
39]. These are all key steps (longevity and immortalization) to support the initiation and progression of HPV-associated cancers.
In addition, HPV as a virus requires cellular differentiation to maintain a productive and long-term infection. NFX1-123 expression in keratinocytes mediated augmented activation of epithelial differentiation indirectly through Notch1 [
41] and directly through the JNK signaling pathway [
40] while still protecting their growth [
41]. In cells with episomal HPV 16 genome, greater NFX1-123 correlated with greater expression of HPV 16 L1, the major capsid protein of HPV that is induced by the host cell’s differentiation [
40]. These studies collectively demonstrated the link between cellular gene regulation (hTERT, Notch1, JNK/ERK) and cellular pathway regulation (growth, immortalization, differentiation). NFX1-123 and 16E6 support both the HPV lifecycle in a keratinocyte [
40] and oncogenesis over time [
39]. All of these genes and pathways are linked by the continued, and increasing expression, of NFX1-123.