Chromatin remodeler Dmp18 regulates apoptosis by controlling H2Av incorporation in Drosophila imaginal disc development.

Programmed Cell Death (PCD) or apoptosis is a highly conserved biological process and plays essential roles both in the development and stress context. In Drosophila, expression of pro-apoptotic genes, including reaper (rpr), head involution defective (hid), grim, and sickle (skl), is sufficient to induce cell death. Here, we demonstrate that the chromatin remodeler Dmp18, the homolog of mammalian Znhit1, plays a crucial role in regulating apoptosis in eye and wing development. We showed that loss of Dmp18 disrupted eye and wing development, up-regulated transcription of pro-apoptotic genes, and induced apoptosis. Inhibition of apoptosis suppressed the eye defects caused by Dmp18 deletion. Furthermore, loss of Dmp18 disrupted H2Av incorporation into chromatin, promoted H3K4me3, but reduced H3K27me3 modifications on the TSS regions of pro-apoptotic genes. These results indicate that Dmp18 negatively regulates apoptosis by mediating H2Av incorporation and histone H3 modifications at pro-apoptotic gene loci for transcriptional regulation. Our study uncovers the role of Dmp18 in regulating apoptosis in Drosophila eye and wing development and provides insights into chromatin remodeling regulating apoptosis at the epigenetic levels.

Chromatin remodeler Znhit1 controls bone morphogenetic protein signaling in embryonic lung tissue branching.

Branching morphogenesis is a key process essential for lung and other organ development in which cellular and tissue architecture branch out to maximize surface area. While this process is known to be regulated by differential gene expression of ligands and receptors, how chromatin remodeling regulates this process remains unclear. Znhit1 (zinc finger HIT-type containing 1), acting as a chromatin remodeler, has previously been shown to control the deposition of the histone variant H2A.Z. Here, we demonstrate that Znhit1 also plays an important role in regulating lung branching. Using Znhit1 conditional KO mice, we show that Znhit1 deficiency in the embryonic lung epithelium leads to failure of branching morphogenesis and neonatal lethality, which is accompanied by reduced cell proliferation and increased cell apoptosis of the epithelium. The results from the transcriptome and the chromatin immunoprecipitation assay reveal that this is partially regulated by the derepression of Bmp4, encoding bone morphogenetic protein (BMP) 4, which is a direct target of H2A.Z. Furthermore, we show that inhibition of BMP signaling by the protein inhibitor Noggin rescues the lung branching defects of Znhit1 mutants ex vivo. Taken together, our study identifies the critical role of Znhit1/H2A.Z in embryonic lung morphogenesis via the regulation of BMP signaling.

A novel posttranslational modification of histone, H3 S-sulfhydration, is down-regulated in asthenozoospermic sperm.

Oxidative stress is one of the major causes leading to male infertility including asthenozoospermia. Hydrogen sulfide (H2S) has been widely recognized to be a potent antioxidant whose role is partially implemented by protein S-sulfhydration. However, protein S-sulfhydration has not been reported in germ cells. Therefore, we investigated whether asthenozoospermia could be associated with sperm protein S-sulfhydration. S-sulfhydrated proteins in human sperm were enriched via biotin-switch assay and analyzed using LC-MS/MS spectrometry. Two hundred forty-four S-sulfhydrated proteins were identified. Importantly, we validated that sperm histones H3.1 and H3.3 were the S-sulfhydrated proteins. Their S-sulfhydrated amino acid residue was Cysteine111. Abundances of S-sulfhydrated H3 (sH3) and S-sulfhydrated H3.3 (sH3.3) were significantly down-regulated in asthenozoospermic sperm, compared with the fertile controls, and were significantly correlated with progressive motility. Retinoic acid (RA) up-regulated level of sH3.3 in primary round spermatids and the C18-4 cells (a mouse spermatogonial stem cell line). Overexpression of the mutant H3.3 (Cysteine111 was replaced with serine) affected expression of 759 genes and raised growth rate of C18-4 cells. For the first time, S-sulfhydration H3 and H3.3 were demonstrated in the present study. Our results highlight that aberrant S-sulfhydration of H3 is a new pathophysiological basis in male infertility.

Epithelial heparan sulfate regulates Sonic Hedgehog signaling in lung development.

The tree-like structure of the mammalian lung is generated from branching morphogenesis, a reiterative process that is precisely regulated by numerous factors. How the cell surface and extra cellular matrix (ECM) molecules regulate this process is still poorly understood. Herein, we show that epithelial deletion of Heparan Sulfate (HS) synthetase Ext1 resulted in expanded branching tips and reduced branching number, associated with several mesenchymal developmental defects. We further demonstrate an expanded Fgf10 expression and increased FGF signaling activity in Ext1 mutant lungs, suggesting a cell non-autonomous mechanism. Consistent with this, we observed reduced levels of SHH signaling which is responsible for suppressing Fgf10 expression. Moreover, reactivating SHH signaling in mutant lungs rescued the tip dilation phenotype and attenuated FGF signaling. Importantly, the reduced SHH signaling activity did not appear to be caused by decreased Shh expression or protein stability; instead, biologically active form of SHH proteins were reduced in both the Ext1 mutant epithelium and surrounding wild type mesenchymal cells. Together, our study highlights the epithelial HS as a key player for dictating SHH signaling critical for lung morphogenesis.

Chronic exposure to diesel exhaust particulate matter impairs meiotic progression during spermatogenesis in a mouse model.

Exposure to ambient PM2.5 may correlate with the decline of semen quality, and the underlying biological mechanism has not been fully understood. In the present study, mice were intratracheally instilled with diesel exhaust PM2.5 (DEP), and its effects on the spermatogenic process as well as the alterations of testicular gene expression profile were assessed. Our results showed that chronic exposure to DEP impaired the fertility of male mice without influencing their libido. Compared with Vehicle-exposed group, the sperm count and motility from DEP-exposed mice were significantly decreased. In addition, immunohistological staining of γH2AX and DMC1, biomarkers for meiotic double strand breaks (DSBs), demonstrated that chronic exposure to DEP comprised the repair of meiotic DSBs, thus disrupting the spermatogenesis. Deep RNA sequencing test showed altered expressions of testicular genes including the GnRH signaling pathway. In summary, our research demonstrated that chronic exposure to DEP may disrupt spermatogenesis through targeting the meiotic recombination, providing a new perspective for the research on the male reproductive system damage caused by air pollution.

Znhit1 controls meiotic initiation in male germ cells by coordinating with Stra8 to activate meiotic gene expression.

The switch from mitosis to meiosis ensures the successive formation of gametes. However, it remains unclear how meiotic initiation occurs within the context of chromatin. Recent studies have shown that zinc finger HIT-type containing 1 (Znhit1), a subunit of the SRCAP chromatin remodeling complex, plays essential roles in modulating the chromatin structure. Herein, we report that the germline-conditional deletion of Znhit1 in male mice specifically blocks meiotic initiation. We show that Znhit1 is required for meiotic prophase events, including synapsis, DNA double-strand break formation, and meiotic DNA replication. Mechanistically, Znhit1 controls the histone variant H2A.Z deposition, which facilitates the expression of meiotic genes, such as Meiosin, but not the expression of Stra8. Interestingly, Znhit1 deficiency disrupts the transcription bubbles of meiotic genes. Thus, our findings identify the essential role of Znhit1-dependent H2A.Z deposition in allowing activation of meiotic gene expression, thereby controlling the initiation of meiosis.

Lgr4 Regulates Oviductal Epithelial Secretion Through the WNT Signaling Pathway.

The WNT signaling pathway plays a crucial role in oviduct/fallopian development. However, the specific physiological processes regulated by the WNT pathway in the fallopian/oviduct function remain obscure. Benefiting from the Lgr4 knockout mouse model, we report the regulation of oviduct epithelial secretion by LGR4. Specifically, the loss of Lgr4 altered the mouse oviduct size and weight, severely reduced the number of oviductal epithelial cells, and ultimately impaired the epithelial secretion. These alterations were mediated by a failure of CTNNB1 protein accumulation in the oviductal epithelial cytoplasm, by the modulation of WNT pathways, and subsequently by a profound change of the gene expression profile of epithelial cells. In addition, selective activation of the WNT pathway triggered the expression of steroidogenic genes, like Cyp11a1 and 3ß-Hsd1, through the activation of the transcriptional factor NR5A2 in an oviduct primary cell culture system. As demonstrated, the LGR4 protein modulates a WNT-NR5A2 signaling cascade facilitating epithelial secretory cell maturation and steroidogenesis to safeguard oviduct development and function in mice.

Chromatin remodeler Znhit1 preserves hematopoietic stem cell quiescence by determining the accessibility of distal enhancers.

Hematopoietic stem cell (HSC) utilizes its quiescence feature to combat exhaustion for lifetime blood cell supply. To date, how certain chromatin architecture and subsequent transcription profile permit HSC quiescence remains unclear. Here, we show an essential role of chromatin remodeler zinc finger HIT-type containing 1 (Znhit1) in maintaining HSC quiescence. We find that loss of Znhit1 leads to exhaustion of stem cell pool and impairment of hematopoietic function. Mechanically, Znhit1 determines the chromatin accessibility at distal enhancers of HSC quiescence genes, including Pten, Fstl1, and Klf4, for sustained transcription and consequent PI3K-Akt signaling inhibition. Moreover, Znhit1-Pten-PI3K-Akt axis also participates in controlling myeloid expansion and B-lymphoid specification. Our findings therefore identify a dominant role of Znhit1-mediated chromatin remodeling in preserving HSC function for hematopoietic homeostasis.

UHRF1-repressed 5'-hydroxymethylcytosine is essential for the male meiotic prophase I.

5'-hydroxymethylcytosine (5hmC), an important 5'-cytosine modification, is altered highly in order in male meiotic prophase. However, the regulatory mechanism of this dynamic change and the function of 5hmC in meiosis remain largely unknown. Using a knockout mouse model, we showed that UHRF1 regulated male meiosis. UHRF1 deficiency led to failure of meiosis and male infertility. Mechanistically, the deficiency of UHRF1 altered significantly the meiotic gene profile of spermatocytes. Uhrf1 knockout induced an increase of the global 5hmC level. The enrichment of hyper-5hmC at transcriptional start sites (TSSs) was highly associated with gene downregulation. In addition, the elevated level of the TET1 enzyme might have contributed to the higher 5hmC level in the Uhrf1 knockout spermatocytes. Finally, we reported Uhrf1, a key gene in male meiosis, repressed hyper-5hmC by downregulating TET1. Furthermore, UHRF1 facilitated RNA polymerase II (RNA-pol2) loading to promote gene transcription. Thus our study demonstrated a potential regulatory mechanism of 5hmC dynamic change and its involvement in epigenetic regulation in male meiosis.

Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation.

Lgr5+ stem cells are crucial to gut epithelium homeostasis; however, how these cells are maintained is not fully understood. Zinc finger HIT-type containing 1 (Znhit1) is an evolutionarily conserved subunit of the SRCAP chromosome remodeling complex. Currently, the function of Znhit1 in vivo and its working mechanism in the SRCAP complex are unknown. Here we show that deletion of Znhit1 in intestinal epithelium depletes Lgr5+ stem cells thus disrupts intestinal homeostasis postnatal establishment and maintenance. Mechanistically, Znhit1 incorporates histone variant H2A.Z into TSS region of genes involved in Lgr5+ stem cell fate determination, including Lgr5, Tgfb1 and Tgfbr2, for subsequent transcriptional regulation. Importantly, Znhit1 promotes the interaction between H2A.Z and YL1 (H2A.Z chaperone) by controlling YL1 phosphorylation. These results demonstrate that Znhit1/H2A.Z is essential for Lgr5+ stem cell maintenance and intestinal homeostasis. Our findings identified a dominant role of Znhit1/H2A.Z in controlling mammalian organ development and tissue homeostasis in vivo.