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.

Distinct Waves from the Hemogenic Endothelium Give Rise to Layered Lymphoid Tissue Inducer Cell Ontogeny.

During embryogenesis, lymphoid tissue inducer (LTi) cells are essential for lymph node organogenesis. These cells are part of the innate lymphoid cell (ILC) family. Although their earliest embryonic hematopoietic origin is unclear, other innate immune cells have been shown to be derived from early hemogenic endothelium in the yolk sac as well as the aorta-gonad-mesonephros. A proper model to discriminate between these locations was unavailable. In this study, using a Cxcr4-CreERT2 lineage tracing model, we identify a major contribution from embryonic hemogenic endothelium, but not the yolk sac, toward LTi progenitors. Conversely, embryonic LTi cells are replaced by hematopoietic stem cell-derived cells in adults. We further show that, in the fetal liver, common lymphoid progenitors differentiate into highly dynamic alpha-lymphoid precursor cells that, at this embryonic stage, preferentially mature into LTi precursors and establish their functional LTi cell identity only after reaching the periphery.

A critical regulator of Bcl2 revealed by systematic transcript discovery of lncRNAs associated with T-cell differentiation.

Normal T-cell differentiation requires a complex regulatory network which supports a series of maturation steps, including lineage commitment, T-cell receptor (TCR) gene rearrangement, and thymic positive and negative selection. However, the underlying molecular mechanisms are difficult to assess due to limited T-cell models. Here we explore the use of the pro-T-cell line P5424 to study early T-cell differentiation. Stimulation of P5424 cells by the calcium ionophore ionomycin together with PMA resulted in gene regulation of T-cell differentiation and activation markers, partially mimicking the CD4-CD8- double negative (DN) to double positive (DP) transition and some aspects of subsequent T-cell maturation and activation. Global analysis of gene expression, along with kinetic experiments, revealed a significant association between the dynamic expression of coding genes and neighbor lncRNAs including many newly-discovered transcripts, thus suggesting potential co-regulation. CRISPR/Cas9-mediated genetic deletion of Robnr, an inducible lncRNA located downstream of the anti-apoptotic gene Bcl2, demonstrated a critical role of the Robnr locus in the induction of Bcl2. Thus, the pro-T-cell line P5424 is a powerful model system to characterize regulatory networks involved in early T-cell differentiation and maturation.

Hes1 in the somatic cells of the murine ovary is necessary for oocyte survival and maturation.

The Notch pathway plays an important role in ovary development in invertebrates like Drosophila. However its role for the mammalian ovary is unclear. Mammalian Hes genes encode transcriptional factors that mediate many of the activities of the Notch pathway. Here, we have studied the function of Hes1 during embryonic development of the mouse ovary. We find that Hes1 protein is present in somatic cells and oocyte cytoplasm and decreases between E15.5 and P0. Conventional Hes1 knock-out (KO), Hes1 conditional KO in the ovarian somatic, and chemical inhibition of Notch signaling decrease the total number, size and maturation of oocytes and increase the number of pregranulosa cells at P0. These defects correlate with abnormal proliferation and enhanced apoptosis. Expression of the proapoptotic gene Inhbb is increased, while the levels of the antiapoptotic and oocyte maturation marker Kit are decreased in the Hes1 KO ovaries. Conversely, overactivation of the Notch pathway in ovarian somatic cells increases the number of mature oocytes and decreases the number of pregranulosa cells. Fertility is also reduced by either Hes1 deletion or Notch pathway overactivation. In conclusion, our data suggest that the Notch-Hes1 pathway regulates ovarian somatic cell development, which is necessary for oocyte survival and maturation.

Control of Hes7 expression by Tbx6, the Wnt pathway and the chemical Gsk3 inhibitor LiCl in the mouse segmentation clock.

The mouse segmentation is established from somites, which are iteratively induced every two hours from the presomitic mesoderm (PSM) by a system known as the segmentation clock. A crucial component of the segmentation clock is the gene Hes7, which is regulated by the Notch and Fgf/Mapk pathways, but its relation to other pathways is unknown. In addition, chemical alteration of the Wnt pathway changes the segmentation clock period but the mechanism is unclear.To clarify these questions, we have carried out Hes7 promoter analysis in transgenic mouse embryos and have identified an essential 400 bp region, which contains binding sites of Tbx6 and the Wnt signaling effector Lef1. We have found that the Hes7 promoter is activated by Tbx6, and normal activity of the Hes7 promoter in the mouse PSM requires Tbx6 binding sites. Our results demonstrate that Wnt pathway molecules activate the Hes7 promoter cooperatively with Tbx6 in cell culture and are necessary for its proper expression in the mouse PSM. Furthermore, it is shown that the chemical Gsk3 inhibitor LiCl lengthens the oscillatory period of Hes7 promoter activity.Our data suggest that Tbx6 and the Wnt pathway cooperatively regulate proper Hes7 expression. Furthermore, proper Hes7 promoter activity and expression is important for the normal pace of oscillation.

Aging alters histone H4 acetylation and CDC2A in mouse germinal vesicle stage oocytes.

The reproductive potential of mammals decreases with aging, until reaching infertility. One reason for aging-related infertility is the decrease of the reproductive capability of old oocytes. It was found previously that gene expression, histone acetylation, and protein function are altered by aging in metaphase II (MII) stage oocytes. MII oocytes develop from germinal vesicle (GV)-stage oocytes. Here, we hypothesized that the defects of old MII oocytes arise at the GV stage. To prove this hypothesis, we examined the acetylations of histone H4 at lysines 5 (H4K5), 8 (H4K8), 12 (H4K12), and 16 (H4K16) in old GV and MII oocytes. We found that acetylation of H4K12 and H4K16 decreased in old GV oocytes. Acetylation of H4K12 later increased in old MII oocytes. We also examined expression of Cdc2a, a gene related to H4K12 acetylation. Cdc2a expression increased in old nonsurrounded nucleolus (NSN) oocytes but decreased in old MII oocytes. On the other hand, the protein and kinase activities of CDC2A decreased in both GV and MII old oocytes. Finally, we showed that correction of the histone deacetylation of old oocytes at the GV stage restores younglike levels of H4K12 acetylation and CDC2A protein at the MII stage. These data support our hypothesis that abnormalities of histone acetylation at the GV stage are the cause of alterations at the MII stage. Our study provides evidence for strategies targeting the GV stage of oocytes to overcome aging-induced infertility.

Proacrosin/acrosin quantification as an indicator of acrosomal integrity in fresh and frozen dog spermatozoa.

The scope of the present study was to evaluate the presence and activation of proacrosin/acrosin as a tool to determine the acrosomal status of fresh and frozen/thawed dog spermatozoa. Monoclonal antibody C5F11, directed against human acrosin, cross-reacted with dog spermatozoa and labeled the acrosome of both fresh and frozen/thawed dog spermatozoa. Frozen/thawed spermatozoa had a lesser proportion of labeled spermatozoa than fresh spermatozoa (P<0.05). When live spermatozoa were labeled with soybean trypsin inhibitor conjugated with Alexa 488 (SBTI-Alexa 488), the proportion of acrosome-labeled fresh spermatozoa was less than frozen/thawed spermatozoa (P<0.05). By using Western blots and enzymatic activity, frozen/thawed spermatozoa had a greater proportion of active acrosin than fresh spermatozoa. In addition, beta 1,4-galactosyl-transferase (GalT), a plasma membrane bound protein, remained attached to frozen/thawed spermatozoa. Proacrosin is activated during freezing/thawing of dog spermatozoa, and that proacrosin/acrosin may be a good indicator of acrosomal integrity of frozen/thawed spermatozoa.