Inherited defects of piRNA biogenesis cause transposon de-repression, impaired spermatogenesis, and human male infertility.

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.

Mild Cognitive Impairment Subtype Performance in Comparison to Healthy Older Controls on the NIH Toolbox and Cogstate.

BACKGROUND: Early detection is necessary for the treatment of dementia. Computerized testing has become more widely used in clinical trials; however, it is unclear how sensitive these measures are to early signs of neurodegeneration. We investigated the use of the NIH Toolbox-Cognition (NIHTB-CB) and Cogstate-Brief computerized neuropsychological batteries in the identification of mild cognitive impairment (MCI) versus healthy older adults [healthy control (HC)] and amnestic (aMCI) versus nonamnestic MCI (naMCI). Exploratory analyses include investigating potential racial differences. METHODS: Two hundred six older adults were diagnosed as aMCI (n = 58), naMCI (n = 15), or cognitively healthy (HC; n = 133). RESULTS: The NIH Toolbox-CB subtests of Flanker, Picture Sequence Memory, and Picture Vocabulary significantly differentiated MCI from HC. Further, subtests from both computerized batteries differentiated patients with aMCI from those with naMCI. Although the main effect of race differences was noted on tests and in diagnostic groups was significant, there were no significant race-by-test interactions. CONCLUSIONS: Computer-based subtests vary in their ability to help distinguish MCI subtypes, though these tests provide less expensive and easier-to-administer clinical screeners to help identify patients early who may qualify for more comprehensive evaluations. Further work is needed, however, to refine computerized tests to achieve better precision in distinguishing impairment subtypes.

WGS Revealed Novel BBS5 Pathogenic Variants, Missed by WES, Causing Ciliary Structure and Function Defects.

Bardet-Biedl syndrome (BBS) is an autosomal recessive ciliopathy that affects multiple organs, leading to retinitis pigmentosa, polydactyly, obesity, renal anomalies, cognitive impairment, and hypogonadism. Until now, biallelic pathogenic variants have been identified in at least 24 genes delineating the genetic heterogeneity of BBS. Among those, BBS5 is a minor contributor to the mutation load and is one of the eight subunits forming the BBSome, a protein complex implied in protein trafficking within the cilia. This study reports on a European BBS5 patient with a severe BBS phenotype. Genetic analysis was performed using multiple next-generation sequencing (NGS) tests (targeted exome, TES and whole exome, WES), and biallelic pathogenic variants could only be identified using whole-genome sequencing (WGS), including a previously missed large deletion of the first exons. Despite the absence of family samples, the biallelic status of the variants was confirmed. The BBS5 protein's impact was confirmed on the patient's cells (presence/absence and size of the cilium) and ciliary function (Sonic Hedgehog pathway). This study highlights the importance of WGS and the challenge of reliable structural variant detection in patients' genetic explorations as well as functional tests to assess a variant's pathogenicity.

Phosphorylation of XPD drives its mitotic role independently of its DNA repair and transcription functions.

The helicase XPD is known as a key subunit of the DNA repair/transcription factor TFIIH. However, here, we report that XPD, independently to other TFIIH subunits, can localize with the motor kinesin Eg5 to mitotic spindles and the midbodies of human cells. The XPD/Eg5 partnership is promoted upon phosphorylation of Eg5/T926 by the kinase CDK1, and conversely, it is reduced once Eg5/S1033 is phosphorylated by NEK6, a mitotic kinase that also targets XPD at T425. The phosphorylation of XPD does not affect its DNA repair and transcription functions, but it is required for Eg5 localization, checkpoint activation, and chromosome segregation in mitosis. In XPD-mutated cells derived from a patient with xeroderma pigmentosum, the phosphomimetic form XPD/T425D or even the nonphosphorylatable form Eg5/S1033A specifically restores mitotic chromosome segregation errors. These results thus highlight the phospho-dependent mitotic function of XPD and reveal how mitotic defects might contribute to XPD-related disorders.

Statistical Approach of the Role of the Conserved CSB-PiggyBac Transposase Fusion Protein (CSB-PGBD3) in Genotype-Phenotype Correlation in Cockayne Syndrome Type B.

Cockayne syndrome is a rare condition that encompasses a very wide spectrum of clinical severity. Mutations upstream of a transposon called PiggyBac Transposable Element Derived 3 in intron 5 of the CSB/ERCC6 gene could bring about less severe forms than mutations located downstream of that transposon insertion. Our aim was to study genotype-phenotype correlation by determining whether the position of each mutation of the CSB/ERCC6 gene has an impact on the phenotype. A hundred and forty-seven Cockayne patients, who had two pathogenic mutations in the CSB/ERCC6 gene and for whom clinical data was available, were retrospectively selected and included in the study. Data analysis was performed under the Bayesian paradigm. Analysis of the proportion of the different subtypes of Cockayne syndrome according to the position of the mutations was done using an ordinal logistic regression model. Using a vague prior, the risk of developing a more severe subtype when exposed to 2 mutations downstream compared to 2 mutations upstream was 2.0 [0.9-4.5]. Estimations varied through the sensitivity analysis. We could reasonably conclude that a relationship between the number of downstream mutations and the Cockayne syndrome clinical expression exists but it is still difficult to give a precise estimate of this relationship. The real effect could be more complex that the one described in the initial model and other genetic factors might be taken into consideration together with the mutation site to better explain clinical variability.

Growth charts in Cockayne syndrome type 1 and type 2.

Cockayne syndrome (CS) is a multisystem degenerative disorder divided in 3 overlapping subtypes, with a continuous phenotypic spectrum: CS2 being the most severe form, CS1 the classical form and CS3 the late-onset form. Failure to thrive and growth difficulties are among the most consistent features of CS, leaving affected individuals vulnerable to numerous medical complications, including adverse effects of undernutrition, abrupt overhydration and overfeeding. There is thus a significant need for specific growth charts. We retrospectively collected growth parameters from genetically-confirmed CS1 and CS2 patients, used the GAMLSS package to construct specific CS growth charts compared to healthy children from WHO and CDC databases. Growth data were obtained from 88 CS patients with a total of 1626 individual growth data points. 49 patients were classified as CS1 and 39 as CS2 with confirmed mutations in CSB/ERCC6, CSA/ERCC8 or ERCC1 genes. Individuals with CS1 initially have normal growth parameters; microcephaly occurs from 2 months whereas onset of weight and height restrictions appear later, between 5 and 22 months. In CS2, growth parameters are already below standard references at birth or drop below the 5th percentile before 3 months. Microcephaly is the first parameter to show a delay, appearing around 2 months in CS1 and at birth in CS2. Height and head circumference are more severely affected in CS2 compared to CS1 whereas weight curves are similar in CS1 and CS2 patients. These new growth charts will serve as a practical tool to improve the nutritional management of children with CS.

Activation of P-TEFb by RBM7: To Live or Let Die.

In this issue of Molecular Cell, Bugai et al. (2019) unveil that a key step of the pro-survival cellular response to a genotoxic attack is the activation of P-TEFb by RBM7. This crucial step triggers RNA polymerase II release from promoter-proximal pausing and expression of DNA damage response genes.

Functional interplay between TFIIH and KAT2A regulates higher-order chromatin structure and class II gene expression.

The TFIIH subunit XPB is involved in combined Xeroderma Pigmentosum and Cockayne syndrome (XP-B/CS). Our analyses reveal that XPB interacts functionally with KAT2A, a histone acetyltransferase (HAT) that belongs to the hSAGA and hATAC complexes. XPB interacts with KAT2A-containing complexes on chromatin and an XP-B/CS mutation specifically elicits KAT2A-mediated large-scale chromatin decondensation. In XP-B/CS cells, the abnormal recruitment of TFIIH and KAT2A to chromatin causes inappropriate acetylation of histone H3K9, leading to aberrant formation of transcription initiation complexes on the promoters of several hundred genes and their subsequent overexpression. Significantly, this cascade of events is similarly sensitive to KAT2A HAT inhibition or to the rescue with wild-type XPB. In agreement, the XP-B/CS mutation increases KAT2A HAT activity in vitro. Our results unveil a tight connection between TFIIH and KAT2A that controls higher-order chromatin structure and gene expression and provide new insights into transcriptional misregulation in a cancer-prone DNA repair-deficient disorder.

Transcription without XPB Establishes a Unified Helicase-Independent Mechanism of Promoter Opening in Eukaryotic Gene Expression.

Transcription starts with the assembly of pre-initiation complexes on promoters followed by their opening. Current models suggest that class II gene transcription requires ATP and the TFIIH XPB subunit to open a promoter. Here, we observe that XPB depletion surprisingly leaves transcription virtually intact. In contrast, inhibition of XPB ATPase activity affects transcription, revealing that mRNA expression paradoxically accommodates the absence of XPB while being sensitive to the inhibition of its ATPase activity. The XPB-depleted TFIIH complex is recruited to active promoters and contributes to transcription. We finally demonstrate that the XPB ATPase activity is only used to relieve a transcription initiation block imposed by XPB itself. In the absence of this block, transcription initiation can take place without XPB ATPase activity. These results suggest that a helicase is dispensable for mRNA transcription, thereby unifying the mechanism of promoter DNA opening for the three eukaryotic RNA polymerases.

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes.

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.

TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals.

Poly (ADP-ribose) glycohydrolase regulates retinoic acid receptor-mediated gene expression.

Poly-(ADP-ribose) glycohydrolase (PARG) is a catabolic enzyme that cleaves ADP-ribose polymers synthesized by poly-(ADP-ribose) polymerases. Here, transcriptome profiling and differentiation assay revealed a requirement of PARG for retinoic acid receptor (RAR)-mediated transcription. Mechanistically, PARG accumulates early at promoters of RAR-responsive genes upon retinoic acid treatment to promote the formation of an appropriate chromatin environment suitable for transcription. Silencing of PARG or knockout of its enzymatic activity maintains the H3K9me2 mark at the promoter of the RAR-dependent genes, leading to the absence of preinitiation complex formation. In the absence of PARG, we found that the H3K9 demethylase KDM4D/JMJD2D became PARsylated. Mutation of two glutamic acids located in the Jumonji N domain of KDM4D inhibited PARsylation. PARG becomes dispensable for ligand-dependent transcription when either a PARP inhibitor or a non-PARsylable KDM4D/JMJD2D mutant is used. Our results define PARG as a coactivator regulating chromatin remodeling during RA-dependent gene expression.

XPG and XPF endonucleases trigger chromatin looping and DNA demethylation for accurate expression of activated genes.

Nucleotide excision repair factors, initially characterized as part of DNA repair, have been shown to participate in the transcriptional process in the absence of genotoxic attack. However, their molecular function when recruited at the promoters of activated genes together with the transcription machinery remained obscure. Here we show that the NER factors XPG and XPF are essential for establishing CTCF-dependent chromatin looping between the promoter and terminator of the activated RARß2 gene. Silencing XPG and/or XPF endonucleases, or mutations in their catalytic sites, prevents CTCF recruitment, chromatin loop formation, and optimal transcription of RARß2. We demonstrated that XPG endonuclease promotes DNA breaks and DNA demethylation at promoters allowing the recruitment of CTCF and gene looping, which is further stabilized by XPF. Our results highlight a timely orchestrated activity of the NER factors XPG and XPF in the formation of the active chromatin hub that controls gene expression.

Antiviral escape strategies developed by bunyaviruses pathogenic for humans.

New or re-emerging pathogens for humans have emerged outside of their usual endemic range during the last decade originating severe public health concern and economical losses. Climate changes have played a significant role in the emergence or re-emergence of arboviruses. Among these pathogens, several viruses belong to the Bunyaviridae family. This family is composed of RNA viruses grouped into five genera Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus characterized by their antigenic, genetic and ecological properties. These viruses use cellular proteins to promote their own replication/transcription and reciprocally the host induces, in response, an important transcriptional reprogramming to activate antiviral defences including the interferon type I pathways. The virulence of the pathogenic bunyaviruses is directly linked to the roles of viral virulence factors and their capacity to counteract the host pathways. This review summarizes the various strategies developed by the different genera of the Bunyaviridae family to overcome and escape the innate immune response and eventually other cellular functions.

The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process.

In response to hormonal stimuli, a cascade of hierarchical post-translational modifications of nuclear receptors are required for the correct expression of target genes. Here, we show that the transcription factor TFIIH, via its cdk7 kinase, phosphorylates the androgen receptor (AR) at position AR/S515. Strikingly, this phosphorylation is a key step for an accurate transactivation that includes the cyclic recruitment of the transcription machinery, the MDM2 E3 ligase, the subsequent ubiquitination of AR at the promoter of target genes and its degradation by the proteasome machinery. Impaired phosphorylation disrupts the transactivation, as observed in cells either overexpressing the non-phosphorylated AR/S515A, isolated from xeroderma pigmentosum patient (bearing a mutation in XPD subunit of TFIIH), or in which cdk7 kinase was silenced. Indeed, besides affecting the cyclic recruitment of the transcription machinery, the AR phosphorylation defect favourizes to the recruitment of the E3 ligase CHIP instead of MDM2, at the PSA promoter, that will further attract the proteasome machinery. These observations illustrate how the TFIIH phosphorylation might participate to the transactivation by regulating the nuclear receptors turnover.

True lies: the double life of the nucleotide excision repair factors in transcription and DNA repair.

Nucleotide excision repair (NER) is a major DNA repair pathway in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation or bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to three genetic disorders that result in predisposition to cancers, accelerated aging, neurological and developmental defects. During NER, more than 30 polypeptides cooperate to recognize, incise, and excise a damaged oligonucleotide from the genomic DNA. Recent papers reveal an additional and unexpected role for the NER factors. In the absence of a genotoxic attack, the promoters of RNA polymerases I- and II-dependent genes recruit XPA, XPC, XPG, and XPF to initiate gene expression. A model that includes the growth arrest and DNA damage 45alpha protein (Gadd45alpha) and the NER factors, in order to maintain the promoter of active genes under a hypomethylated state, has been proposed but remains controversial. This paper focuses on the double life of the NER factors in DNA repair and transcription and describes the possible roles of these factors in the RNA synthesis process.

NER factors are recruited to active promoters and facilitate chromatin modification for transcription in the absence of exogenous genotoxic attack.

Upon gene activation, we found that RNA polymerase II transcription machinery assembles sequentially with the nucleotide excision repair (NER) factors at the promoter. This recruitment occurs in absence of exogenous genotoxic attack, is sensitive to transcription inhibitors, and depends on the XPC protein. The presence of these repair proteins at the promoter of activated genes is necessary in order to achieve optimal DNA demethylation and histone posttranslational modifications (H3K4/H3K9 methylation, H3K9/14 acetylation) and thus efficient RNA synthesis. Deficiencies in some NER factors impede the recruitment of others and affect nuclear receptor transactivation. Our data suggest that there is a functional difference between the presence of the NER factors at the promoters (which requires XPC) and the NER factors at the distal regions of the gene (which requires CSB). While the latter may be a repair function, the former is a function with respect to transcription unveiled in the current study.

RNA polymerase I-mediated expression of viral RNA for the rescue of infectious virulent and avirulent Rift Valley fever viruses.

Rift Valley fever virus (RVFV, Bunyaviridae, Phlebovirus) is a mosquito-transmitted arbovirus that causes human and animal diseases in sub-Saharan Africa and was introduced into the Arabian Peninsula in 2000. Here, we describe a method of reverse genetics to recover infectious RVFV from transfected plasmids based on the use of the cellular RNA polymerase I promoter to synthesize viral transcripts. We compared its efficiency with a system using T7 RNA polymerase and found that both are equally efficient for the rescue of RVFV generating titers of approx. 10(7) to 10(8) pfu/ml. We used the RNA polymerase I-based system to rescue both attenuated MP12 and virulent ZH548 strains as well as chimeric MP12-ZH548 viruses, and in addition RVFV expressing reporter proteins.

A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells.

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon beta (IFN-beta) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-beta gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-beta promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-beta promoter chromatin structure.

The N terminus of Rift Valley fever virus nucleoprotein is essential for dimerization.

Rift Valley fever virus (RVFV) is a Phlebovirus in the Bunyaviridae family. The nucleoprotein N is the most abundant component of the virion; numerous copies of N associate with the viral RNA genome and form pseudohelicoidal ribonucleoproteins (RNPs) circularized by a panhandle structure formed by the base-paired RNA sequences at the 3' and 5' termini. These structures play a central role in transcription and replication. We investigated the intermolecular interactions of the RVFV N protein and found that after chemical cross-linking treatment, the nucleoprotein from purified RNPs migrates mainly as dimers. The N-N interaction was studied using the yeast two-hybrid system, the GST pull-down method, and mutational analysis. We demonstrated that the N terminus from residue 1 to 71, and particularly Tyr 4 and Phe 11, which are conserved among phlebovirus N sequences, are involved in the interaction. The C-terminal region did not seem to be essential for the N-N interaction. Moreover, we showed that N(TOS), the N protein of the related Toscana phlebovirus, interacts with itself and forms heterodimers with N(RVF), suggesting that the dimeric form of N may be a conserved feature in phlebovirus RNPs.