DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools.

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation as well as the identification of exogenously placed DNA N6-methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as coprocessing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools, a state-of-the-art toolkit that features a semisupervised convolutional neural network for fast and accurate identification of m6A-marked bases using PacBio single-molecule long-read sequencing, as well as the coprocessing of long-read genetic and epigenetic data produced using either PacBio or Oxford Nanopore sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kilobase long DNA molecules with a ~1,000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions.

Functional categorization of gene regulatory variants that cause Mendelian conditions.

Much of our current understanding of rare human diseases is driven by coding genetic variants. However, non-coding genetic variants play a pivotal role in numerous rare human diseases, resulting in diverse functional impacts ranging from altered gene regulation, splicing, and/or transcript stability. With the increasing use of genome sequencing in clinical practice, it is paramount to have a clear framework for understanding how non-coding genetic variants cause disease. To this end, we have synthesized the literature on hundreds of non-coding genetic variants that cause rare Mendelian conditions via the disruption of gene regulatory patterns and propose a functional classification system. Specifically, we have adapted the functional classification framework used for coding variants (i.e., loss-of-function, gain-of-function, and dominant-negative) to account for features unique to non-coding gene regulatory variants. We identify that non-coding gene regulatory variants can be split into three distinct categories by functional impact: (1) non-modular loss-of-expression (LOE) variants; (2) modular loss-of-expression (mLOE) variants; and (3) gain-of-ectopic-expression (GOE) variants. Whereas LOE variants have a direct corollary with coding loss-of-function variants, mLOE and GOE variants represent disease mechanisms that are largely unique to non-coding variants. These functional classifications aim to provide a unified terminology for categorizing the functional impact of non-coding variants that disrupt gene regulatory patterns in Mendelian conditions.

DNA-m6A calling and integrated long-read epigenetic and genetic analysis with fibertools.

Long-read DNA sequencing has recently emerged as a powerful tool for studying both genetic and epigenetic architectures at single-molecule and single-nucleotide resolution. Long-read epigenetic studies encompass both the direct identification of native cytosine methylation as well as the identification of exogenously placed DNA N6-methyladenine (DNA-m6A). However, detecting DNA-m6A modifications using single-molecule sequencing, as well as co-processing single-molecule genetic and epigenetic architectures, is limited by computational demands and a lack of supporting tools. Here, we introduce fibertools, a state-of-the-art toolkit that features a semi-supervised convolutional neural network for fast and accurate identification of m6A-marked bases using PacBio single-molecule long-read sequencing, as well as the co-processing of long-read genetic and epigenetic data produced using either PacBio or Oxford Nanopore sequencing platforms. We demonstrate accurate DNA-m6A identification (>90% precision and recall) along >20 kilobase long DNA molecules with a ~1,000-fold improvement in speed. In addition, we demonstrate that fibertools can readily integrate genetic and epigenetic data at single-molecule resolution, including the seamless conversion between molecular and reference coordinate systems, allowing for accurate genetic and epigenetic analyses of long-read data within structurally and somatically variable genomic regions.

Androgen receptor positively regulates gonadotropin-releasing hormone receptor in pituitary gonadotropes.

Within pituitary gonadotropes, the gonadotropin-releasing hormone receptor (GnRHR) receives hypothalamic input from GnRH neurons that is critical for reproduction. Previous studies have suggested that androgens may regulate GnRHR, although the mechanisms remain unknown. In this study, we demonstrated that androgens positively regulate Gnrhr mRNA in mice. We then investigated the effects of androgens and androgen receptor (AR) on Gnrhr promoter activity in immortalized mouse LßT2 cells, which represent mature gonadotropes. We found that AR positively regulates the Gnrhr proximal promoter, and that this effect requires a hormone response element (HRE) half site at -159/-153 relative to the transcription start site. We also identified nonconsensus, full-length HREs at -499/-484 and -159/-144, which are both positively regulated by androgens on a heterologous promoter. Furthermore, AR associates with the Gnrhr promoter in ChIP. Altogether, we report that GnRHR is positively regulated by androgens through recruitment of AR to the Gnrhr proximal promoter.

Distal Enhancer Potentiates Activin- and GnRH-Induced Transcription of FSHB.

FSH is critical for fertility. Transcription of FSHB, the gene encoding the beta subunit, is rate-limiting in FSH production and is regulated by both GnRH and activin. Activin signals through SMAD transcription factors. Although the mechanisms and importance of activin signaling in mouse Fshb transcription are well-established, activin regulation of human FSHB is less well understood. We previously reported a novel enhancer of FSHB that contains a fertility-associated single nucleotide polymorphism (rs10031006) and requires a region resembling a full (8 base-pair) SMAD binding element (SBE). Here, we investigated the role of the putative SBE within the enhancer in activin and GnRH regulation of FSHB. In mouse gonadotrope-derived LßT2 cells, the upstream enhancer potentiated activin induction of both the human and mouse FSHB proximal promoters and conferred activin responsiveness to a minimal promoter. Activin induction of the enhancer required the SBE and was blocked by the inhibitory SMAD7, confirming involvement of the classical SMAD signaling pathway. GnRH induction of FSHB was also potentiated by the enhancer and dependent on the SBE, consistent with known activin/GnRH synergy regulating FSHB transcription. In DNA pull-down, the enhancer SBE bound SMAD4, and chromatin immunoprecipitation demonstrated SMAD4 enrichment at the enhancer in native chromatin. Combined activin/GnRH treatment elevated levels of the active transcriptional histone marker, histone 3 lysine 27 acetylation, at the enhancer. Overall, this study indicates that the enhancer is directly targeted by activin signaling and identifies a novel, evolutionarily conserved mechanism by which activin and GnRH can regulate FSHB transcription.

FSHB Transcription is Regulated by a Novel 5' Distal Enhancer With a Fertility-Associated Single Nucleotide Polymorphism.

The pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone, signal the gonads to regulate male and female fertility. FSH is critical for female fertility as it regulates oocyte maturation, ovulation, and hormone synthesis. Multiple genome-wide association studies (GWAS) link a 130 Kb locus at 11p14.1, which encompasses the FSH beta-subunit (FSHB) gene, with fertility-related traits that include polycystic ovary syndrome, age of natural menopause, and dizygotic twinning. The most statistically significant single nucleotide polymorphism from several GWAS studies (rs11031006) resides within a highly conserved 450 bp region 26 Kb upstream of the human FSHB gene. Given that sequence conservation suggests an important biological function, we hypothesized that the region could regulate FSHB transcription. In luciferase assays, the conserved region enhanced FSHB transcription and gel shifts identified a binding site for Steroidogenic factor 1 (SF1) contributing to its function. Analysis of mouse pituitary single-cell ATAC-seq demonstrated open chromatin at the conserved region exclusive to a gonadotrope cell-type cluster. Additionally, enhancer-associated histone markers were identified by immunoprecipitation of chromatin from mouse whole pituitary and an immortalized mouse gonadotrope-derived LßT2 cell line at the conserved region. Furthermore, we found that the rs11031006 minor allele upregulated FSHB transcription via increased SF1 binding to the enhancer. All together, these results identify a novel upstream regulator of FSHB transcription and indicate that rs11031006 can modulate FSH levels.