Differential transcript usage across mammalian oocytes at the germinal vesicle and metaphase II stages.

Ongoing progress in mRNA-Sequencing technologies has significantly contributed to the refinement of assisted reproductive technologies. However, the prior investigations have predominantly concentrated on alterations in overall gene expression levels, thereby leaving a considerable gap in our understanding of the influence of transcript isoform expression on fundamental cellular mechanisms of oocytes. Given the efficacy of differential transcript usage (DTU) analysis to address such knowledge, we conducted comprehensive DTU analysis utilizing mRNA-Seq datasets of germinal vesicle (GV) and metaphase II (MII) oocytes across six mammalian species from the SRA database, including cow, donkey, horse, human, mouse, and pig. To further illuminate the roles of these genes, we also conducted a rigorous Gene Ontology (GO) term enrichment analysis. While the DTU analysis of each species exhibited several genes with alterations in their transcript isoform usage, referred to as DTU genes, this study focused on only ten cross-species DTU genes sharing among a minimum of five distinct species (FDR≤0.05). These cross-species DTU genes were as follows: ABCF1, CDC6, CFAP36, CNOT10, DNM3, IWS1, NBN, NDEL1, RAD50 and ZCCHC17. GO term enrichment analysis unveiled the alignment of these cross-species DTU gene functions with RNA and cell-cycle control mechanisms across diverse mammalian species, thereby suggesting their vital roles during oocyte maturation. Further exploration of the transcript isoforms of these genes hence bore the potential to uncover novel transcript isoform markers for future reproductive technologies in both human and animal contexts.

Cross-species analysis of differential transcript usage in humans and chickens with fatty liver disease.

Background and Aim: Fatty liver disease is a common condition, characterized by excess fat accumulation in the liver. It can contribute to more severe liver-related health issues, making it a critical concern in avian and human medicine. Apart from modifying the gene expression of liver cells, the disease also alters the expression of specific transcript isoforms, which might serve as new biological markers for both species. This study aimed to identify cross-species genes displaying differential expressions in their transcript isoforms in humans and chickens with fatty liver disease. Materials and Methods: We performed differential gene expression and differential transcript usage (DTU) analyses on messenger RNA datasets from the livers of both chickens and humans with fatty liver disease. Using appropriate cross-species gene identification methods, we reviewed the acquired candidate genes and their transcript isoforms to determine their potential role in fatty liver disease's pathogenesis. Results: We identified seven genes - ALG5, BRD7, DIABLO, RSU1, SFXN5, STIMATE, TJP3, and VDAC2 - and their corresponding transcript isoforms as potential candidates (false discovery rate ≤0.05). Our findings showed that these genes most likely contribute to fatty disease development and progression. Conclusion: This study successfully identified novel human-chicken DTU genes in fatty liver disease. Further research is encouraged to verify the functions and regulations of these transcript isoforms as potential diagnostic markers for fatty liver disease in humans and chickens.

Nucleoside diphosphate kinases 1 and 2 regulate a protective liver response to a high-fat diet.

The synthesis of fatty acids from acetyl-coenzyme A (AcCoA) is deregulated in diverse pathologies, including cancer. Here, we report that fatty acid accumulation is negatively regulated by nucleoside diphosphate kinases 1 and 2 (NME1/2), housekeeping enzymes involved in nucleotide homeostasis that were recently found to bind CoA. We show that NME1 additionally binds AcCoA and that ligand recognition involves a unique binding mode dependent on the CoA/AcCoA 3' phosphate. We report that Nme2 knockout mice fed a high-fat diet (HFD) exhibit excessive triglyceride synthesis and liver steatosis. In liver cells, NME2 mediates a gene transcriptional response to HFD leading to the repression of fatty acid accumulation and activation of a protective gene expression program via targeted histone acetylation. Our findings implicate NME1/2 in the epigenetic regulation of a protective liver response to HFD and suggest a potential role in controlling AcCoA usage between the competing paths of histone acetylation and fatty acid synthesis.

The Contribution of Multiplexing Single Cell RNA Sequencing in Acute Myeloid Leukemia.

Decades ago, the treatment for acute myeloid leukemia relied on cytarabine and anthracycline. However, advancements in medical research have introduced targeted therapies, initially employing monoclonal antibodies such as ant-CD52 and anti-CD123, and subsequently utilizing specific inhibitors that target molecular mutations like anti-IDH1, IDH2, or FLT3. The challenge lies in determining the role of these therapeutic options, considering the inherent tumor heterogeneity associated with leukemia diagnosis and the clonal drift that this type of tumor can undergo. Targeted drugs necessitate an examination of various therapeutic targets at the individual cell level rather than assessing the entire population. It is crucial to differentiate between the prognostic value and therapeutic potential of a specific molecular target, depending on whether it is found in a terminally differentiated cell with limited proliferative potential or a stem cell with robust capabilities for both proliferation and self-renewal. However, this cell-by-cell analysis is accompanied by several challenges. Firstly, the scientific aspect poses difficulties in comparing different single cell analysis experiments despite efforts to standardize the results through various techniques. Secondly, there are practical obstacles as each individual cell experiment incurs significant financial costs and consumes a substantial amount of time. A viable solution lies in the ability to process multiple samples simultaneously, which is a distinctive feature of the cell hashing technique. In this study, we demonstrate the applicability of the cell hashing technique for analyzing acute myeloid leukemia cells. By comparing it to standard single cell analysis, we establish a strong correlation in various parameters such as quality control, gene expression, and the analysis of leukemic blast markers in patients. Consequently, this technique holds the potential to become an integral part of the biological assessment of acute myeloid leukemia, contributing to the personalized and optimized management of the disease, particularly in the context of employing targeted therapies.

Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation.

INTRODUCTION: The application of single-cell RNA sequencing has greatly improved our understanding of various cellular and molecular mechanisms involved in physiological and pathophysiological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. MATERIALS AND METHODS: In this study, we selected two AML samples that had been fixed in methanol for 12-18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. RESULTS: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. CONCLUSIONS: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effectively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy.

Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.

Epigenetic regulation of transcription factor binding motifs promotes Th1 response in Chagas disease cardiomyopathy

Abstract: Chagas disease, caused by the protozoan Trypanosoma cruzi, is an endemic parasitic disease of Latin America, affecting 7 million people. Although most patients are asymptomatic, 30% develop complications, including the often-fatal Chronic Chagasic Cardiomyopathy (CCC). Although previous studies have demonstrated some genetic deregulations associated with CCCs, the causes of their deregulations remain poorly described. Based on bulk RNA-seq and whole genome DNA methylation data, we investigated the genetic and epigenetic deregulations present in the moderate and severe stages of CCC. Analysis of heart tissue gene expression profile allowed us to identify 1407 differentially expressed transcripts (DEGs) specific from CCC patients. A tissue DNA methylation analysis done on the same tissue has permitted the identification of 92 regulatory Differentially Methylated Regions (DMR) localized in the promoter of DEGs. An in-depth study of the transcription factors binding sites (TFBS) in the DMRs corroborated the importance of TFBS's DNA methylation for gene expression in CCC myocardium. TBX21, RUNX3 and EBF1 are the transcription factors whose binding motif appears to be affected by DNA methylation in the largest number of genes. By combining both transcriptomic and methylomic analysis on heart tissue, and methylomic analysis on blood, 4 biological processes affected by severe CCC have been identified, including immune response, ion transport, cardiac muscle processes and nervous system. An additional study on blood methylation of moderate CCC samples put forward the importance of ion transport and nervous system in the development of the disease.

A novel cross-species differential tumor classification method based on exosome-derived microRNA biomarkers established by human-dog lymphoid and mammary tumor cell lines' transcription profiles.

Background and Aim: Exosome-derived microRNA (miRNA) has been widely studied as a non-invasive candidate biomarker for tumor diagnosis in humans and dogs. Its application, however, was primarily focused on intraspecies usage for individual tumor type diagnosis. This study aimed to gain insight into its application as a cross-species differential tumor diagnostic tool; we demonstrated the process of identifying and using exosome-derived miRNA as biomarkers for the classification of lymphoid and mammary tumor cell lines in humans and dogs. Materials and Methods: Exosome-derived miRNA sequencing data from B-cell lymphoid tumor cell lines (n=13), mammary tumor cell lines (n=8), and normal mammary epithelium cultures (n=4) were pre-processed in humans and dogs. F-test and rank product (RP) analyses were used to select candidate miRNA orthologs for tumor cell line classification. The classification was carried out using an optimized support vector machine (SVM) with various kernel classifiers, including linear SVM, polynomial SVM, and radial basis function SVM. The receiver operating characteristic and precision-recall curves were used to assess the performance of all models. Results: MIR10B, MIR21, and MIR30E were chosen as the candidate orthologs from a total of 236 human-dog miRNA orthologs (p≤0.01, F-test score ≥10, and RP score ≤10). Their use of polynomial SVM provided the best performance in classifying samples from various tumor cell lines and normal epithelial culture. Conclusion: The study successfully demonstrated a method for identifying and utilizing candidate human-dog exosome-derived miRNA orthologs for differential tumor cell line classification. Such findings shed light on a novel non-invasive tumor diagnostic tool that could be used in both human and veterinary medicine in the future.

Dynamics of broad H3K4me3 domains uncover an epigenetic switch between cell identity and cancer-related genes.

Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain-associated genes in healthy human thymic T cell populations and a collection of T cell acute lymphoblastic leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation, and their varying breadth allows the distinction between normal and neoplastic cells. Although broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain-associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we showed that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain-associated genes in preleukemic cells. Finally, an H3K4me3 demethylase inhibitor differentially targets T-ALL cell lines depending on the extent and number of broad domains. Our results show that the regulation of broad H3K4me3 domains is associated with leukemogenesis, and suggest that the presence of these structures might be used for epigenetic prioritization of cancer-relevant genes, including long noncoding RNAs.

ATAD2 controls chromatin-bound HIRA turnover.

Taking advantage of the evolutionary conserved nature of ATAD2, we report here a series of parallel functional studies in human, mouse, and Schizosaccharomyces pombe to investigate ATAD2's conserved functions. In S. pombe, the deletion of ATAD2 ortholog, abo1, leads to a dramatic decrease in cell growth, with the appearance of suppressor clones recovering normal growth. The identification of the corresponding suppressor mutations revealed a strong genetic interaction between Abo1 and the histone chaperone HIRA. In human cancer cell lines and in mouse embryonic stem cells, we observed that the KO of ATAD2 leads to an accumulation of HIRA. A ChIP-seq mapping of nucleosome-bound HIRA and FACT in Atad2 KO mouse ES cells demonstrated that both chaperones are trapped on nucleosomes at the transcription start sites of active genes, resulting in the abnormal presence of a chaperone-bound nucleosome on the TSS-associated nucleosome-free regions. Overall, these data highlight an important layer of regulation of chromatin dynamics ensuring the turnover of histone-bound chaperones.

Anomaly detection in genomic catalogues using unsupervised multi-view autoencoders.

BACKGROUND: Accurate identification of Transcriptional Regulator binding locations is essential for analysis of genomic regions, including Cis Regulatory Elements. The customary NGS approaches, predominantly ChIP-Seq, can be obscured by data anomalies and biases which are difficult to detect without supervision. RESULTS: Here, we develop a method to leverage the usual combinations between many experimental series to mark such atypical peaks. We use deep learning to perform a lossy compression of the genomic regions' representations with multiview convolutions. Using artificial data, we show that our method correctly identifies groups of correlating series and evaluates CRE according to group completeness. It is then applied to the ReMap database's large volume of curated ChIP-seq data. We show that peaks lacking known biological correlators are singled out and less confirmed in real data. We propose normalization approaches useful in interpreting black-box models. CONCLUSION: Our approach detects peaks that are less corroborated than average. It can be extended to other similar problems, and can be interpreted to identify correlation groups. It is implemented in an open-source tool called atyPeak.

Metabolically controlled histone H4K5 acylation/acetylation ratio drives BRD4 genomic distribution.

In addition to acetylation, histones are modified by a series of competing longer-chain acylations. Most of these acylation marks are enriched and co-exist with acetylation on active gene regulatory elements. Their seemingly redundant functions hinder our understanding of histone acylations' specific roles. Here, by using an acute lymphoblastic leukemia (ALL) cell model and blasts from individuals with B-precusor ALL (B-ALL), we demonstrate a role of mitochondrial activity in controlling the histone acylation/acetylation ratio, especially at histone H4 lysine 5 (H4K5). An increase in the ratio of non-acetyl acylations (crotonylation or butyrylation) over acetylation on H4K5 weakens bromodomain containing protein 4 (BRD4) bromodomain-dependent chromatin interaction and enhances BRD4 nuclear mobility and availability for binding transcription start site regions of active genes. Our data suggest that the metabolism-driven control of the histone acetylation/longer-chain acylation(s) ratio could be a common mechanism regulating the bromodomain factors' functional genomic distribution.

Genetic Predisposition to the Mortality in Septic Shock Patients: From GWAS to the Identification of a Regulatory Variant Modulating the Activity of a CISH Enhancer.

The high mortality rate in septic shock patients is likely due to environmental and genetic factors, which influence the host response to infection. Two genome-wide association studies (GWAS) on 832 septic shock patients were performed. We used integrative bioinformatic approaches to annotate and prioritize the sepsis-associated single nucleotide polymorphisms (SNPs). An association of 139 SNPs with death based on a false discovery rate of 5% was detected. The most significant SNPs were within the CISH gene involved in cytokine regulation. Among the 139 SNPs associated with death and the 1311 SNPs in strong linkage disequilibrium with them, we investigated 1439 SNPs within non-coding regions to identify regulatory variants. The highest integrative weighted score (IW-score) was obtained for rs143356980, indicating that this SNP is a robust regulatory candidate. The rs143356980 region is located in a non-coding region close to the CISH gene. A CRISPR-Cas9-mediated deletion of this region and specific luciferase assays in K562 cells showed that rs143356980 modulates the enhancer activity in K562 cells. These analyses allowed us to identify several genes associated with death in patients with septic shock. They suggest that genetic variations in key genes, such as CISH, perturb relevant pathways, increasing the risk of death in sepsis patients.

OLOGRAM-MODL: mining enriched n-wise combinations of genomic features with Monte Carlo and dictionary learning.

Most epigenetic marks, such as Transcriptional Regulators or histone marks, are biological objects known to work together in n-wise complexes. A suitable way to infer such functional associations between them is to study the overlaps of the corresponding genomic regions. However, the problem of the statistical significance of n-wise overlaps of genomic features is seldom tackled, which prevent rigorous studies of n-wise interactions. We introduce OLOGRAM-MODL, which considers overlaps between n ≥ 2 sets of genomic regions, and computes their statistical mutual enrichment by Monte Carlo fitting of a Negative Binomial distribution, resulting in more resolutive P-values. An optional machine learning method is proposed to find complexes of interest, using a new itemset mining algorithm based on dictionary learning which is resistant to noise inherent to biological assays. The overall approach is implemented through an easy-to-use CLI interface for workflow integration, and a visual tree-based representation of the results suited for explicability. The viability of the method is experimentally studied using both artificial and biological data. This approach is accessible through the command line interface of the pygtftk toolkit, available on Bioconda and from https://github.com/dputhier/pygtftk.

Differential DNA methylation analysis across the promoter regions using methylated DNA immunoprecipitation sequencing profiling of porcine loin muscle.

BACKGROUND AND AIM: Pork leanness and marbling are among the essential traits of consumer preference. To acquire knowledge about universal epigenetic regulations for improving breed selection, a meta-analysis of methylated DNA immunoprecipitation sequencing (MeDIP-seq) profiling data of mixed loin muscle types was performed in this study. MATERIALS AND METHODS: MeDIP-seq profiling datasets of longissimus dorsi muscle and psoas major muscles from male and female pigs of Landrace and Tibetan breeds were preprocessed and aligned to the porcine genome. Analysis of differential methylated DNA regions (DMRs) between the breeds was performed by focusing on transcription start sites (TSSs) of known genes (-20,000-3000 bases from TSS). All associated genes were further reviewed for their functions and predicted for transcription factors (TF) possibly associated with their TSSs. RESULTS: When the methylation levels of DMRs in TSS regions of Landrace breed were compared to those of Tibetan breed, 10 DMRs were hypomethylated (Landrace < Tibetan), and 19 DMRs were hypermethylated (Landrace > Tibetan), accordingly (p≤0.001). According to the reviews about gene functions, all associated genes were pieces of evidence for their roles in a variety of muscle and lipid metabolisms. Prediction of the binding TFs revealed the six most abundant binding TFs to such DMRs-associated TSS (p≤0.0001) as follows: ZNF384, Foxd3, IRF1, KLF9, EWSR1-FLI1, HES5, and TFAP2A. CONCLUSION: Common DMRs-associated TSS between the lean-type and the marbled-type loin muscles were identified in this study. Interestingly, the genes associated with such regions were strongly evidenced for their possible roles on the muscle trait characteristics by which further novel research topics could be focused on them in the future.

Blueprint of human thymopoiesis reveals molecular mechanisms of stage-specific TCR enhancer activation.

Cell differentiation is accompanied by epigenetic changes leading to precise lineage definition and cell identity. Here we present a comprehensive resource of epigenomic data of human T cell precursors along with an integrative analysis of other hematopoietic populations. Although T cell commitment is accompanied by large scale epigenetic changes, we observed that the majority of distal regulatory elements are constitutively unmethylated throughout T cell differentiation, irrespective of their activation status. Among these, the TCRA gene enhancer (Eα) is in an open and unmethylated chromatin structure well before activation. Integrative analyses revealed that the HOXA5-9 transcription factors repress the Eα enhancer at early stages of T cell differentiation, while their decommission is required for TCRA locus activation and enforced αß T lineage differentiation. Remarkably, the HOXA-mediated repression of Eα is paralleled by the ectopic expression of homeodomain-related oncogenes in T cell acute lymphoblastic leukemia. These results highlight an analogous enhancer repression mechanism at play in normal and cancer conditions, but imposing distinct developmental constraints.

Novel classifier orthologs of bovine and human oocytes matured in different melatonin environments.

It has been demonstrated that melatonin influences the developmental competence of both in vivo and in vitro matured oocytes. It modulates oocyte-specific gene expression patterns among mammalian species. Due to differences among study systems, the identification of the classifier orthologs-the homologous genes related among mammals that could universally categorize oocytes matured in environments with varied melatonin levels is still limitedly studied. To gain insight into such orthologs, cross-species transcription profiling meta-analysis of in vitro matured bovine oocytes and in vivo matured human oocytes in low and high melatonin environments was demonstrated in the current study. RNA-Seq data of bovine and human oocytes were retrieved from the Sequence Read Archive database and pre-processed. The used datasets of bovine oocytes obtained from culturing in the absence of melatonin and human oocytes from old patients were regarded as oocytes in the low melatonin environment (Low). Datasets from bovine oocytes cultured in 10-9 M melatonin and human oocytes from young patients were considered as oocytes in the high melatonin environment (High). Candidate orthologs differentially expressed between Low and High melatonin environments were selected by a linear model, and were further verified by Zero-inflated regression analysis. Support Vector Machine (SVM) was applied to determine the potentials of the verified orthologs as classifiers of melatonin environments. According to the acquired results, linear model analysis identified 284 candidate orthologs differentially expressed between Low and High melatonin environments. Among them, only 15 candidate orthologs were verified by Zero-inflated regression analysis (FDR ≤ 0.05). Utilization of the verified orthologs as classifiers in SVM resulted in the precise classification of oocyte learning datasets according to their melatonin environments (Misclassification rates < 0.18, area under curves > 0.9). In conclusion, the cross-species RNA-Seq meta-analysis to identify novel classifier orthologs of matured oocytes under different melatonin environments was successfully demonstrated in this study-delivering candidate orthologs for future studies at biological levels. Such verified orthologs might provide valuable evidence about melatonin sufficiency in target oocytes-by which, the decision on melatonin supplementation could be implied.

IL-12 Signaling Contributes to the Reprogramming of Neonatal CD8+ T Cells.

Neonates are highly susceptible to intracellular pathogens, leading to high morbidity and mortality rates. CD8+ T lymphocytes are responsible for the elimination of infected cells. Understanding the response of these cells to normal and high stimulatory conditions is important to propose better treatments and vaccine formulations for neonates. We have previously shown that human neonatal CD8+ T cells overexpress innate inflammatory genes and have a low expression of cytotoxic and cell signaling genes. To investigate the activation potential of these cells, we evaluated the transcriptome of human neonatal and adult naïve CD8+ T cells after TCR/CD28 signals ± IL-12. We found that in neonatal cells, IL-12 signals contribute to the adult-like expression of genes associated with cell-signaling, T-cell cytokines, metabolism, and cell division. Additionally, IL-12 signals contributed to the downregulation of the neutrophil signature transcription factor CEBPE and other immaturity related genes. To validate the transcriptome results, we evaluated the expression of a series of genes by RT-qPCR and the promoter methylation status on independent samples. We found that in agreement with the transcriptome, IL-12 signals contributed to the chromatin closure of neutrophil-like genes and the opening of cytotoxicity genes, suggesting that IL-12 signals contribute to the epigenetic reprogramming of neonatal lymphocytes. Furthermore, high expression of some inflammatory genes was observed in naïve and stimulated neonatal cells, in agreement with the high inflammatory profile of neonates to infections. Altogether our results point to an important contribution of IL-12 signals to the reprogramming of the neonatal CD8+ T cells.

Integration of high-throughput reporter assays identify a critical enhancer of the Ikzf1 gene.

The Ikzf1 locus encodes the lymphoid specific transcription factor Ikaros, which plays an essential role in both T and B cell differentiation, while deregulation or mutation of IKZF1/Ikzf1 is involved in leukemia. Tissue-specific and cell identity genes are usually associated with clusters of enhancers, also called super-enhancers, which are believed to ensure proper regulation of gene expression throughout cell development and differentiation. Several potential regulatory regions have been identified in close proximity of Ikzf1, however, the full extent of the regulatory landscape of the Ikzf1 locus is not yet established. In this study, we combined epigenomics and transcription factor binding along with high-throughput enhancer assay and 4C-seq to prioritize an enhancer element located 120 kb upstream of the Ikzf1 gene. We found that deletion of the E120 enhancer resulted in a significant reduction of Ikzf1 mRNA. However, the epigenetic landscape and 3D topology of the locus were only slightly affected, highlighting the complexity of the regulatory landscape regulating the Ikzf1 locus.

RNA-Guided Genomic Localization of H2A.L.2 Histone Variant.

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.