The gene regulatory landscape driving mouse gonadal supporting cell differentiation.

Gonadal sex determination relies on tipping a delicate balance involving the activation and repression of several transcription factors and signaling pathways. This is likely mediated by numerous noncoding regulatory elements that shape sex-specific transcriptomic programs. To explore the dynamics of these in detail, we performed paired time series of transcriptomic and chromatin accessibility assays on pre-granulosa and Sertoli cells throughout their development in the embryo, making use of new and existing mouse reporter lines.

Two redundant transcription factor binding sites in a single enhancer are essential for mammalian sex determination.

Male development in mammals depends on the activity of the two SOX gene: Sry and Sox9, in the embryonic testis. As deletion of Enhancer 13 (Enh13) of the Sox9 gene results in XY male-to-female sex reversal, we explored the critical elements necessary for its function and hence, for testis and male development. Here, we demonstrate that while microdeletions of individual transcription factor binding sites (TFBS) in Enh13 lead to normal testicular development, combined microdeletions of just two SRY/SOX binding motifs can alone fully abolish Enh13 activity leading to XY male-to-female sex reversal.

Transposable elements acquire time- and sex-specific transcriptional and epigenetic signatures along mouse fetal gonad development.

Gonadal sex determination in mice is a complex and dynamic process, which is crucial for the development of functional reproductive organs. The expression of genes involved in this process is regulated by a variety of genetic and epigenetic mechanisms. Recently, there has been increasing evidence that transposable elements (TEs), which are a class of mobile genetic elements, play a significant role in regulating gene expression during embryogenesis and organ development.

Towards a "Testis in a Dish": Generation of Mouse Testicular Organoids that Recapitulate Testis Structure and Expression Profiles.

The testis is responsible for sperm production and androgen synthesis. Abnormalities in testis development and function lead to disorders of sex development and male infertility. Currently, no system exists for modelling the testis.

Epigenetic aging of mammalian gametes.

The process of aging refers to physiological changes that occur to an organism as time progresses and involves changes to DNA, proteins, metabolism, cells, and organs. Like the rest of the cells in the body, gametes age, and it is well established that there is a decline in reproductive capabilities in females and males with aging. One of the major pathways known to be involved in aging is epigenetic changes.

In vitro cellular reprogramming to model gonad development and its disorders.

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs.

SOX Genes and Their Role in Disorders of Sex Development.

SOX genesare master regulatory genes controlling development and are fundamental to the establishment of sex determination in a multitude of organisms. The discovery of the master sex-determining gene SRY in 1990 was pivotal for the understanding of how testis development is initiated in mammals. With this discovery, an entire family of SOX factors were uncovered that play crucial roles in cell fate decisions during development.

Cis-Regulatory Control of Mammalian Sex Determination.

Sex determination is the process by which an initial bipotential gonad adopts either a testicular or ovarian cell fate. The inability to properly complete this process leads to a group of developmental disorders classified as disorders of sex development (DSD). To date, dozens of genes were shown to play roles in mammalian sex determination, and mutations in these genes can cause DSD in humans or gonadal sex reversal/dysfunction in mice.

Testis formation in XX individuals resulting from novel pathogenic variants in Wilms' tumor 1 () gene.

Sex determination in mammals is governed by antagonistic interactions of two genetic pathways, imbalance in which may lead to disorders/differences of sex development (DSD) in human. Among 46,XX individuals with testicular DSD (TDSD) or ovotesticular DSD (OTDSD), testicular tissue is present in the gonad. Although the testis-determining gene is present in many cases, the etiology is unknown in most -negative patients.

The regulation of Sox9 expression in the gonad.

The bipotential nature of cell types in the early developing gonad and the process of sex determination leading to either testis or ovary differentiation makes this an interesting system in which to study transcriptional regulation of gene expression and cell fate decisions. SOX9 is a transcription factor with multiple roles during development, including being a key player in mediating testis differentiation and therefore subsequent male development. Loss of Sox9 expression in both humans and mice results in XY female development, whereas its inappropriate activation in XX embryonic gonads can give male development.

Gonadal supporting cells acquire sex-specific chromatin landscapes during mammalian sex determination.

Cis-regulatory elements are critical for the precise spatiotemporal regulation of genes during development. However, identifying functional regulatory sites that drive cell differentiation in vivo has been complicated by the high numbers of cells required for whole-genome epigenetic assays. Here, we identified putative regulatory elements during sex determination by performing ATAC-seq and ChIP-seq for H3K27ac in purified XX and XY gonadal supporting cells before and after sex determination in mice.

Sex reversal following deletion of a single distal enhancer of .

Cell fate decisions require appropriate regulation of key genes. , a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Although others are redundant, enhancer 13 (Enh13), a 557-base pair element located 565 kilobases 5' from the transcriptional start site, is essential to initiate mouse testis development; its deletion results in XY females with transcript levels equivalent to those in XX gonads.

Correction: Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

[This corrects the article DOI: 10.1371/journal.pgen.

Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone.

During mouse sex determination, transient expression of the Y-linked gene Sry up-regulates its direct target gene Sox9, via a 3.2 kb testis specific enhancer of Sox9 (TES), which includes a core 1.4 kb element, TESCO.

Homothorax plays autonomous and nonautonomous roles in proximodistal axis formation and migration of the Drosophila renal tubules.

The Drosophila Malpighian tubules (MpTs) serve as a functional equivalent of the mammalian renal tubules. The MpTs are composed of two pairs of epithelial tubes that bud from the midgut-hindgut boundary during embryogenesis. The MpT primordia grow, elongate and migrate through the body cavity to assume their final position and shape.

Antifolates in cancer therapy: structure, activity and mechanisms of drug resistance.

In the past 65 years, antifolates targeting folate metabolism played a pivotal role in drug treatment of malignant, microbial, parasitic and chronic inflammatory diseases. Drug discovery of novel antifolates with improved properties and superior activities remains an attractive strategy both in academia and in the pharmaceutical industry. Among novel antifolates are pemetrexed which primarily targets thymidylate synthase as well as pralatrexate which blocks dihydrofolate reductase, and displays enhanced transport and cellular retention properties.

The obligatory intestinal folate transporter PCFT (SLC46A1) is regulated by nuclear respiratory factor 1.

Folates are essential vitamins that play a key role as one-carbon donors in a spectrum of biosynthetic pathways including RNA and DNA synthesis. The proton-coupled folate transporter (PCFT/SLC46A1) mediates obligatory intestinal folate absorption. Loss-of-function mutations in PCFT result in hereditary folate malabsorption, an autosomal recessive disorder characterized by very low folate levels in the blood and cerebrospinal fluid.

Functional elements in the minimal promoter of the human proton-coupled folate transporter.

The proton-coupled folate transporter (PCFT) is the dominant intestinal folate transporter, however, its promoter has yet to be revealed. Hence, we here cloned a 3.1kb fragment upstream to the first ATG of the human PCFT gene and generated sequential deletion constructs evaluated in luciferase reporter assay.

PCFT/SLC46A1 promoter methylation and restoration of gene expression in human leukemia cells.

The proton-coupled folate transporter (PCFT/SLC46A1) displays optimal and prominent folate and antifolate transport activity at acidic pH in human carcinoma cells but poor activity in leukemia cells. Consistently herein, human leukemia cell lines expressed poor PCFT transcript levels, whereas various carcinoma cell lines showed substantial PCFT gene expression. We identified a CpG island with high density at nucleotides -200 through +100 and explored its role in PCFT promoter silencing.