Phenotypic continuum and poor intracytoplasmic sperm injection intracytoplasmic sperm injection prognosis in patients harboring HENMT1 variants.

BACKGROUND: Small RNAs interacting with PIWI (piRNAs) play a crucial role in regulating transposable elements and translation during spermatogenesis and are essential in male germ cell development. Disruptions in the piRNA pathway typically lead to severe spermatogenic defects and thus male infertility. The HENMT1 gene is a key player in piRNAs primary biogenesis and dysfunction of HENMT1 protein in meiotic and haploid germ cells resulted in the loss of piRNA methylation, piRNA instability, and TE de-repression. Henmt1-knockout mice exhibit a severe oligo-astheno-teratozoospermia (OAT) phenotype, whereas patients with HENMT1 variants display more severe azoospermia phenotypes, ranging from meiotic arrest to hypospermatogenesis. Through whole-exome sequencing (WES) of infertile patient cohorts, we identified two new patients with variants in the HENMT1 gene presenting spermatozoa in their ejcaulate, providing us the opportunity to study spermatozoa from these patients. OBJECTIVES: Investigate the spermatozoa of two patients harboring an HENMT1 variant to determine whether or not these scarce spermatozoa could be used with assisted reproductive technologies. MATERIALS AND METHODS: HENMT1 variants identified by WES were validated through Sanger sequencing. Comprehensive semen analysis was conducted, and sperm cells were subjected to transmission electron microscopy for structural examination, in situ hybridization for aneuploidy assessment, and aniline blue staining for DNA compaction status. Subsequently, we assessed their suitability for in vitro fertilization using intracytoplasmic sperm injection (IVF-ICSI). RESULTS: Our investigations revealed a severe OAT phenotype similar to knockout mice, revealing altered sperm concentration, mobility, morphology, aneuploidy and nuclear compaction defects. Multiple IVF-ICSI attempts were also performed, but no live births were achieved. DISCUSSION: We confirm the crucial role of HENMT1 in spermatogenesis and highlight a phenotypic continuum associated with HENMT1 variants. Unfortunately, the clinical outcome of these genetic predispositions remains unfavorable, regardless of the patient's phenotype. CONCLUSION: The presence of spermatozoa is insufficient to anticipate ICSI pregnancy success in HENMT1 patients.

Human histone pre-mRNA assembles histone or canonical mRNA-processing complexes by overlapping 3'-end sequence elements.

Human pre-mRNA processing relies on multi-subunit macromolecular complexes, which recognize specific RNA sequence elements essential for assembly and activity. Canonical pre-mRNA processing proceeds via the recognition of a polyadenylation signal (PAS) and a downstream sequence element (DSE), and produces polyadenylated mature mRNAs, while replication-dependent (RD) histone pre-mRNA processing requires association with a stem-loop (SL) motif and a histone downstream element (HDE), and produces cleaved but non-polyadenylated mature mRNAs. H2AC18 mRNA, a specific H2A RD histone pre-mRNA, can be processed to give either a non-polyadenylated mRNA, ending at the histone SL, or a polyadenylated mRNA. Here, we reveal how H2AC18 captures the two human pre-mRNA processing complexes in a mutually exclusive mode by overlapping a canonical PAS (AAUAAA) sequence element with a HDE. Disruption of the PAS sequence on H2AC18 pre-mRNA prevents recruitment of the canonical complex in vitro, without affecting the histone machinery. This shows how the relative position of cis-acting elements in histone pre-mRNAs allows the selective recruitment of distinct human pre-mRNA complexes, thereby expanding the capability to regulate 3' processing and polyadenylation.

Dual role of histone variant H3.3B in spermatogenesis: positive regulation of piRNA transcription and implication in X-chromosome inactivation.

The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions.

The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity.

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

Reinforcement of STAT3 activity reprogrammes human embryonic stem cells to naive-like pluripotency.

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.

Forced expression of LIM homeodomain transcription factor 1b enhances differentiation of mouse embryonic stem cells into serotonergic neurons.

The LIM homeodomain transcription factor 1b (Lmx1b) is a key factor in the specification of the serotonergic neurotransmitter phenotype. Here, we explored the capacity of Lmx1b to direct differentiation of mouse embryonic stem (mES) cells into serotonergic neurons. mES cells stably expressing human Lmx1b were generated by lentiviral vector infection. Clones expressing Lmx1b at a low level showed increased neurogenesis and elevated production of neurons expressing serotonin, serotonin transporter, tryptophan hydroxylase 2, and transcription factor Pet1, the landmarks of serotonergic differentiation. To explore the role of Lmx1b in the specification of the serotonin neurotransmission phenotype further, a conditional system making use of a floxed inducible vector targeted into the ROSA26 locus and a hormone-dependent Cre recombinase was engineered. This novel strategy was tested with the reporter gene encoding human placental alkaline phosphatase, and demonstrated its capacity to drive transgene expression in nestin(+) neural progenitors (NPs) and in Tuj1(+) neurons. When it was applied to inducible expression of human Lmx1b, it resulted in elevated expression of serotonergic markers. Treatment of neural precursors with the floor plate signal Sonic hedgehog further enhanced differentiation of Lmx1b-overexpressing NPs into neurons expressing 5-HT, serotonin transporter, tryptophan hydroxylase 2, and Pet1, when compared with Lmx1b-nonexpressing progenitors. Together, our results demonstrate the capacity of Lmx1b to specify a serotonin neurotransmitter phenotype when overexpressed in mES cell-derived NPs.

Effect of genomic long-range correlations on DNA persistence length: from theory to single molecule experiments.

Sequence dependency of DNA intrinsic bending properties has been emphasized as a possible key ingredient to in vivo chromatin organization. We use atomic force microscopy (AFM) in air and liquid to image intrinsically straight (synthetic), uncorrelated (hepatitis C RNA virus) and persistent long-range correlated (human) DNA fragments in various ionic conditions such that the molecules freely equilibrate on the mica surface before being captured in a particular conformation. 2D thermodynamic equilibrium is experimentally verified by a detailed statistical analysis of the Gaussian nature of the DNA bend angle fluctuations. We show that the worm-like chain (WLC) model, commonly used to describe the average conformation of long semiflexible polymers, reproduces remarkably well the persistence length estimates for the first two molecules as consistently obtained from (i) mean square end-to-end distance measurement and (ii) mean projection of the end-to-end vector on the initial orientation. Whatever the operating conditions (air or liquid, concentration of metal cations Mg(2+) and/or Ni(2+)), the persistence length found for the uncorrelated viral DNA underestimates the value obtained for the straight DNA. We show that this systematic difference is the signature of the presence of an uncorrelated structural intrinsic disorder in the hepatitis C virus (HCV) DNA fragment that superimposes on local curvatures induced by thermal fluctuations and that only the entropic disorder depends upon experimental conditions. In contrast, the WLC model fails to describe the human DNA conformations. We use a mean-field extension of the WLC model to account for the presence of long-range correlations (LRC) in the intrinsic curvature disorder of human genomic DNA: the stronger the LRC, the smaller the persistence length. The comparison of AFM imaging of human DNA with LRC DNA simulations confirms that the rather small mean square end-to-end distance observed, particularly for G+C-rich human DNA molecules, more likely results from a large-scale intrinsic curvature due to a persistent distribution of DNA curvature sites than from some increased flexibility.

Atomic force microscopy imaging of SWI/SNF action: mapping the nucleosome remodeling and sliding.

We propose a combined experimental (atomic force microscopy) and theoretical study of the structural and dynamical properties of nucleosomes. In contrast to biochemical approaches, this method allows us to determine simultaneously the DNA-complexed length distribution and nucleosome position in various contexts. First, we show that differences in the nucleoproteic structure observed between conventional H2A and H2A.Bbd variant nucleosomes induce quantitative changes in the length distribution of DNA-complexed with histones. Then, the sliding action of remodeling complex SWI/SNF is characterized through the evolution of the nucleosome position and wrapped DNA length mapping. Using a linear energetic model for the distribution of DNA-complexed length, we extract the net-wrapping energy of DNA onto the histone octamer and compare it to previous studies.