Oligomeganephronia (OMN) is a rare congenital renal hypoplasia reported more often in children than in adults. The diagnosis of OMN relies on renal biopsy and exhibits a significant reduction in the number of glomeruli and pronounced glomerular hypertrophy. Here, we report the case of an 8-year-old boy with recurrent proteinuria and abnormal external ears. A renal biopsy revealed large and rare glomeruli. The histological findings confirmed the diagnosis of OMN. Whole-exome sequencing of the patient revealed a new pathogenic variant in PBX1 (hg19, NM_002585, c.262delA, p.Thr88Glnfs*3). The PBX1 gene encodes a transcription factor whose pathogenic variants can result in congenital renal and urinary system anomalies, with or without hearing loss, abnormal ears, and developmental retardation (CAKUTED). This is the first report to detect PBX1 pathogenic variants in children with OMN, a novel phenotype of human PBX1 pathogenic variants. We performed functional prediction analyses of deletions in the corresponding structural domains. We summarized 27 cases of PBX1 single pathogenic variants reported between 2003 and 2023 in terms of truncating and missense pathogenic variants, which can deepen our understanding of the PBX1 structural domain and expand our knowledge of the PBX1 genotype and phenotype.

Organization of protein sequences into domain families is a foundation for cataloging and investigating protein functions. However, long-standing strategies based on primary amino acid sequences are blind to the possibility that proteins with dissimilar sequences could have comparable tertiary structures. Building on our recent findings that in silico structural predictions of BEN family DNA-binding domains closely resemble their experimentally determined crystal structures, we exploited the AlphaFold2 database for comprehensive identification of BEN domains. Indeed, we identified numerous novel BEN domains, including members of new subfamilies. For example, while no BEN domain factors had previously been annotated in C. elegans, this species actually encodes multiple BEN proteins. These include key developmental timing genes of orphan domain status, sel-7 and lin-14, the latter being the central target of the founding miRNA lin-4. We also reveal that the domain of unknown function 4806 (DUF4806), which is widely distributed across metazoans, is structurally similar to BEN and comprises a new subtype. Surprisingly, we find that BEN domains resemble both metazoan and non-metazoan homeodomains in 3D conformation and preserve characteristic residues, indicating that despite their inability to be aligned by conventional methods, these DNA-binding modules are probably evolutionarily related. Finally, we broaden the application of structural homology searches by revealing novel human members of DUF3504, which exists on diverse proteins with presumed or known nuclear functions. Overall, our work strongly expands this recently identified family of transcription factors and illustrates the value of 3D structural predictions to annotate protein domains and interpret their functions.

The choroid plexus is an important blood barrier that secretes cerebrospinal fluid, which essential for embryonic brain development and adult brain homeostasis. The OTX2 homeoprotein is a transcription factor that is critical for choroid plexus development and remains highly expressed in adult choroid plexus. Through RNA sequencing analyses of constitutive and conditional knockdown adult mouse models, we reveal putative functional roles for OTX2 in adult choroid plexus function, including cell signaling and adhesion, and show that OTX2 regulates the expression of factors that are secreted into the cerebrospinal fluid, notably transthyretin. We also show that Otx2 expression impacts choroid plexus immune and stress responses, and affects splicing, leading to changes in the mRNA isoforms of proteins that are implicated in the oxidative stress response and DNA repair. Through mass spectrometry analysis of OTX2 protein partners in the choroid plexus, and in known non-cell-autonomous target regions, such as the visual cortex and subventricular zone, we identify putative targets that are involved in cell adhesion, chromatin structure, and RNA processing. Thus, OTX2 retains important roles for regulating choroid plexus function and brain homeostasis throughout life.

Two-cell-like (2C-like) embryonic stem cells (ESCs) are a small group of ESCs that spontaneously express zygotic genome activation (ZGA) genes and repeats, such as Zscan4 and murine endogenous retrovirus with leucine (MERVL), and are specifically expressed in 2-cell-stage mouse embryos. Although numerous types of treatment and agents elevate the transition of ESCs to 2C-like ESCs, Dux serves as a critical factor in this transition by increasing the expression of Zscan4 and MERVL directly. However, the loss of Dux did not impair the birth of mice, suggesting that Dux may not be the primary transitioning factor in fertilized embryos. It has been reported that for 2-cell embryos derived from somatic cell nuclear transfer (SCNT) and whose expression of ZGA genes and repeats was aberrant, Dux improved the reprogramming efficiency by correcting aberrant H3K9ac modification via its C-terminal domain. We confirmed that the overexpression of full-length Dux mRNA in SCNT embryos improved the efficiency of preimplantation development (62.16% vs. 41.26% with respect to controls) and also increased the expression of Zscan4 and MERVL. Furthermore, we found that the N-terminal double homeodomains of Dux were indispensable for Dux localization and function. The intermediate region was essential for MERVL and Zscan4 activation, and the C-terminal domain was important for elevating level of H3K27ac. Mutant Dux mRNA containing N-terminal double homeodomains with the intermediate region or the C-terminal domain also improved the preimplantation development of SCNT embryos. This is the first report focusing on distinguishing functional domains of Dux in embryos derived from SCNT.

The transcription factor nuclear factor interleukin-3-regulated protein (NFIL3, also called E4BP4) is crucial for commitment of natural killer (NK) cells from common lymphoid progenitors (CLPs). However, the identity of the factor that can regulate NFIL3 directly during the NK-cell development is not known. Here, we reveal that pre-B-cell leukemia transcription factor 1 (PBX1) can upregulate the NFIL3 expression directly. We used conditional knockout mice in which PBX1 in hematopoietic cells was specifically absent. The number of NK-committed progenitor pre-NKP cells and rNKP cells was reduced significantly in the absence of PBX1, which was consistent with NFIL3 deficiency. Also, the NFIL3 expression in NK cells was decreased if PBX1 was absent. We demonstrated that PBX1 was bound directly to the promoter of Nfil3 and facilitated transcription. Upon knockout of the binding site of PBX1 in the Nfil3 promoter, mice showed fewer NK-precursor cells and NK cells, just like that observed in Nfil3 knockout mice. Furthermore, asparagine N286 in the homeodomain of PBX1 controlled the binding of PBX1 to the Nfil3 promoter. Collectively, these findings demonstrate that the transcription factor PBX1 promotes the early development of NK cells by upregulating the Nfil3 expression directly.

UNASSIGNED: Sexual development in Flammulina velutipes is controlled by two different mating type loci (HD and PR). The HD locus contains homeodomain (Hd) genes on two separate HD subloci: HD-a and HD-b. While the functionality of the HD-b sublocus has been largely confirmed, the status and content of the HD-a sublocus has remained unclear.
UNASSIGNED: To examine the function of the HD-a sublocus, genome sequences of a series of F. velutipes strains were analyzed and tested through series of amplification by specific primer sets. Furthermore, activity of di-allelic HD-a locus was confirmed by crossing strains with different combinations of HD-a and HD-b subloci.
UNASSIGNED: Sublocus HD-b contained a large variety of fixed Hd1/Hd2 gene pairs, while the HD-a sublocus either contained a conserved Hd2 gene or, a newly discovered Hd1 gene that was also conserved. Identification of whole HD loci, that is, the contents of HD-a and HD-b subloci in a strain, revealed that strains with similar HD-b subloci could still form normal dikaryons if the two genes at the HD-a sublocus differed. At least di-allelic HD-a sublocus, is thus indicated to be actively involved in mating type compatibility.
UNASSIGNED: HD-a sublocus is active and di-allelic. Using the new information on the HD subloci, primers sets were developed that specifically amplify HD-a or HD-b subloci in the majority of F. velutipes strains. In this way, unknown HD mating types of F. velutipes can now be quickly identified, and HD mating type compatibility conferred by HD-a or HD-b can be confirmed by PCR.

A number of homeobox transcription factors (TFs) play critical role in regulating developmental processes of fungi. However, studies on TFs in fruiting body development of mushroom forming species, Volvariella volvacea, are still at initial stage. Here, we report homeobox TFs in the whole-genomic sequence of V. volvacea and expression analysis of the homeobox TFs during a series of developmental stages. Homeobox TFs were identified using InterPro terms and Fungal Transcription Factor Database (FTFD) from the genome of V. volvacea and quantitative real-time PCR were used for gene expression analysis. Based on phylogenetic analysis, the homeobox TFs of V. volvacea were divided into two groups and showed close relationships with the TFs of other Basidiomycetes. Eight differentially expressed homeobox TFs were selected by digital gene expression analysis from 47 putative homeobox TFs, including five up-regulated genes in primordia and three down-regulated genes in fruiting elongation stage of V. volvacea. VvHox1, VvHox2, and VvHox3 might be participating in fruiting body elongation. It can be assumed that VvHox3 might be involved in volva development. Moreover, five TFs (VvHox4-VvHox8) might be contributing in primordia formation. Results indicated that differentially expressed homeobox TFs are significant candidates for fruiting body development study in V. volvacea.

Nkx2-2 homeoprotein is essential for the development of the central nervous system and pancreas. Although the nuclear localization signals of Nkx2-2 have been identified, the responsible transport receptor is still unknown. Here, we demonstrate that imp α1 not only interacts with Nkx2-2 but also transports it into the nucleus in vitro by acting together with imp β1. However, the nuclear import of Nkx2-2 in cells was not inhibited in response to knockdown expression of endogenous imp β1 or over-expression of Bimax2. Furthermore, imp β1 and imp 13, but not imp 4, directly interact with Nkx2-2 and are capable of transporting Nkx2-2 in an in vitro import assay. By GST pull-down assay, we demonstrate that mutation of NLS1 or NLS2 has no effect on interaction with imp α1 or imp 13, but significantly reduced binding to imp β1. Thus, the nuclear import of Nkx2-2 is mediated not only by the classical import pathway but also directly by imp β1 or imp 13.

The initiation of sexual development in the important edible and medicinal mushroom Flammulina velutipes is controlled by special genes at two different, independent, mating type (MAT) loci: HD and PR. We expanded our understanding of the F. velutipes mating type system by analyzing the MAT loci from a series of strains. The HD locus of F. velutipes houses homeodomain genes (Hd genes) on two separated locations: sublocus HD-a and HD-b. The HD-b subloci contained strain-specific Hd1/Hd2 gene pairs, and crosses between strains with different HD-b subloci indicated a role in mating. The function of the HD-a sublocus remained undecided. Many, but not all strains contained the same conserved Hd2 gene at the HD-a sublocus. The HD locus usually segregated as a whole, though we did detect one new HD locus with a HD-a sublocus from one parental strain, and a HD-b sublocus from the other. The PR locus of F. velutipes contained pheromone receptor (STE3) and pheromone precursor (Pp) genes at two locations, sublocus PR-a and PR-b. PR-a and PR-b both contained sets of strain-specific STE3 and Pp genes, indicating a role in mating. PR-a and PR-b cosegregated in our experiments. However, the identification of additional strains with identical PR-a, yet different PR-b subloci, demonstrated that PR subloci can recombine within the PR locus. In conclusion, at least three of the four MAT subloci seem to participate in mating, and new HD and PR loci can be generated through intralocus recombination in F. velutipes.

NKX2.5 plays important roles in heart development. Being a transcription factor, NKX2.5 exerts its biological functions in nucleus. However, the sequence motif that localize NKX2.5 into nucleus is still not clear. Here, we found a R/K-rich sequence motif from Q187 to R197 (QNRRYKCKRQR) was required for exclusive nuclear localization of NKX2.5. Eight truncated plasmids (E109X, Q149X, Q170X, Q187X, Q198X, Y256X, Y259X, and C264X) which were associated with congenital heart disease (CHD) were constructed. Compared with the wild type NKX2.5, the proteins E109X, Q149X, Q170X, Q187X without intact homeodomain (HD) showed no transcriptional activity while Q198X, Y256X, Y259X and C264X with intact HD showed 50 to 66% transcriptional activity. E109X, Q149X, Q170X, Q187X without intact HD localized in the cytoplasm and nucleus simultaneously and Q198X, Y256X, Y259X and C264X with intact HD localized completely in nucleus. These results inferred the indispensability of 187QNRRYKCKRQR197 in exclusive nucleus localization. Additionally, this sequence motif was very conservative among human, mouse and rat, indicating this motif was important for NKX2.5 function. Thus, we concluded that R/K-rich sequence motif 187QNRRYKCKRQR197 played a central role for NKX2.5 nuclear localization. Our findings provided a clue to understand the mechanisms between the truncated NKX2.5 mutants and CHD.