A Novel WT1 Mutation Identified in a 46,XX Testicular/Ovotesticular DSD Patient Results in the Retention of Intron 9

The 46,XX testicular DSD (disorder/difference of sexual development) and 46,XX ovotesticular DSD (46,XX TDSD and 46,XX OTDSD) phenotypes are caused by genetic rearrangements or point mutations resulting in imbalance between components of the two antagonistic, pro-testicular and pro-ovarian pathways; however, the genetic causes of 46,XX TDSD/OTDSD are not fully understood, and molecular diagnosis for many patients with the conditions is unavailable. Only recently few mutations in the WT1 (WT1 transcription factor; 11p13) gene were described in a group of 46,XX TDSD and 46,XX OTDSD individuals. The WT1 protein contains a DNA/RNA binding domain consisting of four zinc fingers (ZnF) and a three-amino acid (KTS) motif that is present or absent, as a result of alternative splicing, between ZnF3 and ZnF4 (±KTS isoforms). Here, we present a patient with 46,XX TDSD/OTDSD in whom whole exome sequencing revealed a heterozygous de novo WT1 c.1437A>G mutation within an alternative donor splice site which is used for −KTS WT1 isoform formation. So far, no mutation in this splice site has been identified in any patient group. We demonstrated that the mutation results in the retention of intron 9 in the mature mRNA of the 46,XX TDSD/OTDSD patient. In cases when the erroneous mRNA is translated, exclusively the expression of a truncated WT1 +KTS protein lacking ZnF4 and no −KTS protein occurs from the mutated allele of the patient. We discuss potential mechanisms and pathways which can be disturbed upon two conditions: Absence of Zn4F and altered +KTS/−KTS ratio.

Ataxia with Ocular Apraxia Type 1 (AOA1) (APTX, W279* Mutation): Neurological, Neuropsychological, and Molecular Outlining of a Heterogenous Phenotype in Four Colombian Siblings

Hereditary ataxias are a group of devastating neurological disorders that affect coordination of gait and are often associated with poor coordination of hands, speech, and eye movements. Ataxia with Ocular Apraxia type 1 (AOA1) (OMIM: 606350.0006) is characterized by slowly progressive symptoms of childhood-onset and pathogenic mutations in APTX; the only known cause underpinning AOA1. APTX encodes the protein Aprataxin, composed of three domains sharing homology with proteins involved in DNA damage, signaling, and repair. We present four siblings from an endogamic family in a rural, isolated town of Colombia with ataxia and ocular apraxia of childhood-onset and confirmed molecular diagnosis of AOA1, homozygous for the W279* p.Trp279Ter mutation. We predicted the mutated APTX with Alpha Fold to demonstrate the effects of this stop-gain mutation that deletes three beta helices encoded by amino acid 270 to 339 rescinding the C2H2-type zinc fingers (Znf) (C2H2 Znf) DNA-binding and DNA-repair domain and the whole tridimensional structure of the APTX. All siblings exhibited different ages of onset (4, 6, 8, and 11 y/o) and heterogeneous patterns of dysarthria (ranging from absence to mild-moderate dysarthria). Neuropsychological evaluation showed no neurocognitive impairment in three siblings, but one sibling showed temporospatial disorientation, semantic and phonologic fluency impairment, episodic memory affection, constructional apraxia, moderate anomia, low executive function, and symptoms of depression. This heterogeneous phenotype suggests genetic interactions can shape the natural history of AOA1. To our knowledge, this report represents the most extensive series of siblings affected with AOA1 in Latin America, and the genetic analysis completed adds important knowledge to outline this family’s disease and general complex phenotype of hereditary ataxias.

Nuclear import of BCL11B is mediated by a classical nuclear localization signal and not the Krüppel-like zinc fingers

The Krüppel-like transcription factor BCL11B is characterized by wide tissue distribution and crucial functions in key developmental and cellular processes and various pathologies including cancer or HIV infection. Although basics of BCL11B activity and relevant interactions with other proteins were uncovered, how this exclusively nuclear protein localizes to its compartment remained unclear. Here, we demonstrate that unlike other KLFs, BCL11B does not require the C-terminal DNA-binding domain to pass through the nuclear envelope but encodes an independent, previously unidentified nuclear localization signal (NLS) which is located distantly from the zinc finger domains and fulfills the essential criteria of an autonomous NLS. First, it can redirect a heterologous cytoplasmic protein to the nucleus. Second, its mutations cause aberrant localization of the protein of origin. Finally, we provide experimental and in silico evidences of the direct interaction with importin alpha. The relative conservation of this motif allows formulating a consensus sequence (K/R)K-X13-14-KR+K++ which can be found in all BCL11B orthologues among vertebrates and in the closely related protein BCL11A.

Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

Neural network modeling of differential binding between wild-type and mutant CTCF reveals putative binding preferences for zinc fingers 1-2

Background: Many transcription factors (TFs), such as multi zinc-finger (ZF) TFs, have multiple DNA binding domains (DBDs) with multiple components, and deciphering the DNA binding motifs of individual components is a major challenge. One example of such a TF is CCCTC-binding factor (CTCF), a TF with eleven ZFs that plays a variety of roles in transcriptional regulation, most notably anchoring DNA loops. Previous studies found that CTCF zinc fingers (ZFs) 3-7 bind CTCF's core motif and ZFs 9-11 bind a specific upstream motif, but the motifs of ZFs 1-2 have yet to be identified. Results: We developed a new approach to identifying the binding motifs of individual DBDs of a TF through analyzing chromatin immunoprecipitation sequencing (ChIP-seq) experiments in which a single DBD is mutated: we train a deep convolutional neural network to predict whether wild-type TF binding sites are preserved in the mutant TF dataset and interpret the model. We applied this approach to mouse CTCF ChIP-seq data and, in addition to identifying the known binding preferences of CTCF ZFs 3-11, we identified a GAG binding motif for ZF1 and a weak ATT binding motif for ZF2. We analyzed other CTCF datasets to provide additional evidence that ZFs 1-2 interact with the motifs we identified, and we found that the presence of the motif for ZF1 is associated with Ctcf peak strength. Conclusions: Our approach can be applied to any TF for which in vivo binding data from both the wild-type and mutated versions of the TF are available, and our findings provide an unprecedently comprehensive understanding of the binding preferences of CTCF's DBDs.

Supratentorial non-RELA, ZFTA-fused ependymomas: a comprehensive phenotype genotype correlation highlighting the number of zinc fingers in ZFTA-NCOA1/2 fusions

The cIMPACT-NOW Update 7 has replaced the WHO nosology of “ependymoma, RELA fusion positive” by “Supratentorial-ependymoma, C11orf95-fusion positive”. This modification reinforces the idea that supratentorial-ependymomas exhibiting fusion that implicates the C11orf95 (now called ZFTA) gene with or without the RELA gene, represent the same histomolecular entity. A hot off the press molecular study has identified distinct clusters of the DNA methylation class of ZFTA fusion-positive tumors. Interestingly, clusters 2 and 4 comprised tumors of different morphologies, with various ZFTA fusions without involvement of RELA. In this paper, we present a detailed series of thirteen cases of non-RELA ZFTA-fused supratentorial tumors with extensive clinical, radiological, histopathological, immunohistochemical, genetic and epigenetic (DNA methylation profiling) characterization. Contrary to the age of onset and MRI aspects similar to RELA fusion-positive EPN, we noted significant histopathological heterogeneity (pleomorphic xanthoastrocytoma-like, astroblastoma-like, ependymoma-like, and even sarcoma-like patterns) in this cohort. Immunophenotypically, these NFκB immunonegative tumors expressed GFAP variably, but EMA constantly and L1CAM frequently. Different gene partners were fused with ZFTA: NCOA1/2, MAML2 and for the first time MN1. These tumors had epigenetic homologies within the DNA methylation class of ependymomas-RELA and were classified as satellite clusters 2 and 4. Cluster 2 (n = 9) corresponded to tumors with classic ependymal histological features (n = 4) but also had astroblastic features (n = 5). Various types of ZFTA fusions were associated with cluster 2, but as in the original report, ZFTA:MAML2 fusion was frequent. Cluster 4 was enriched with sarcoma-like tumors. Moreover, we reported a novel anatomy of three ZFTA:NCOA1/2 fusions with only 1 ZFTA zinc finger domain in the putative fusion protein, whereas all previously reported non-RELA ZFTA fusions have 4 ZFTA zinc fingers. All three cases presented a sarcoma-like morphology. This genotype/phenotype association requires further studies for confirmation. Our series is the first to extensively characterize this new subset of supratentorial ZFTA-fused ependymomas and highlights the usefulness of ZFTA FISH analysis to confirm the existence of a rearrangement without RELA abnormality.

Comparative transcriptomics of leaves of five mulberry accessions and cataloguing structural and expression variants for future prospects

Bombyx mori, a monophagous insect, prefers leaves of the certain species of Morus more than others. The preference has been attributed to morphological and anatomical features and biochemical compounds. In the present manuscript a comparison has been made among the transcriptome of leaves of the two preferred cultivated varieties and three wild types species. While assembling, high quality transcriptomes of five genotypes were constructed with a total of 100930, 151245, 89724, 181761 and 102908 transcripts from ML, MN, MS, K2 and V1 samples respectively. Further, to compare them, orthologs were identified from these assembled transcriptome. A total of 22462, 23413, 23685, 24371, 18362, 22326, 20058, 18049, 17567 and 20518 clusters of orthologs were found in one to one comparison in KvsN, KvsL, KvsS, KvsV, LvsN, LvsS, LvsV, NvsS, NvsV, and SvsV respectively. 4236 orthologs with algebraic connectivity of 1.0 were then used to compare and to find out differentially expressed transcripts from all the genotypes. A total of 1037 transcripts expressed that include some of the important morphology, anatomy and biochemical pathways regulating transcription factors (AP2/ERFs and C2H2 Zinc fingers) and signalling components were identified to express differentially. Further, these transcriptomes were used find out markers (SSR) and variants and a total of 1101013, 537245, 970877, 310437, 675772, 338400, 581189, 751477, 514999 and 257107 variants including SNP, MNP, Insertions and deletions were found in one to one comparisons. Taken together, our data could be highly useful for mulberry community worldwide as it could be utilized in mulberry breeding programs.

Structural analysis of viral ExoN domains reveals polyphyletic hijacking events

Nidoviruses and arenaviruses are the only known RNA viruses encoding a 3’-5’ exonuclease domain (ExoN). The proofreading activity of the ExoN domain has played a key role in the growth of nidoviral genomes, while in arenaviruses this domain partakes in the suppression of the host innate immune signaling. Sequence and structural homology analyses suggest that these proteins have been hijacked from cellular hosts many times. Analysis of the available nidoviral ExoN sequences reveals a high conservation level comparable to that of the viral RNA-dependent RNA polymerases (RdRp), which are the most conserved viral proteins. Two highly preserved zinc fingers are present in all nidoviral exonucleases, while in the arenaviral protein only one zinc finger can be identified. This is in sharp contrast with the reported lack of zinc fingers in cellular ExoNs, and opens the possibility of therapeutic strategies in the struggle against COVID-19.