Divergent transcriptional and transforming properties of PAX3-FOXO1 and PAX7-FOXO1 paralogs

The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma.

Dnmt1 has de novo activity targeted to transposable elements

DNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.

HDAC3 controls male fertility through enzyme-independent transcriptional regulation at the meiotic exit of spermatogenesis

The transition from meiotic spermatocytes to postmeiotic haploid germ cells constitutes an essential step in spermatogenesis. The epigenomic regulatory mechanisms underlying this transition remain unclear. Here, we find a prominent transcriptomic switch from the late spermatocytes to the early round spermatids during the meiotic-to-postmeiotic transition, which is associated with robust histone acetylation changes across the genome. Among histone deacetylases (HDACs) and acetyltransferases, we find that HDAC3 is selectively expressed in the late meiotic and early haploid stages. Three independent mouse lines with the testis-specific knockout of HDAC3 show infertility and defects in meiotic exit with an arrest at the late stage of meiosis or early stage of round spermatids. Stage-specific RNA-seq and histone acetylation ChIP-seq analyses reveal that HDAC3 represses meiotic/spermatogonial genes and activates postmeiotic haploid gene programs during meiotic exit, with associated histone acetylation alterations. Unexpectedly, abolishing HDAC3 catalytic activity by missense mutations in the nuclear receptor corepressor (NCOR or SMRT) does not cause infertility, despite causing histone hyperacetylation as HDAC3 knockout, demonstrating that HDAC3 enzyme activity is not required for spermatogenesis. Motif analysis of the HDAC3 cistrome in the testes identified SOX30, which has a similar spatiotemporal expression pattern as HDAC3 during spermatogenesis. Depletion of SOX30 in the testes abolishes the genomic recruitment of the HDAC3 to the binding sites. Collectively, these results establish the SOX30/HDAC3 signaling as a key regulator of the transcriptional program in a deacetylase-independent manner during the meiotic-to-postmeiotic transition in spermatogenesis.

Bcl11b prevents catastrophic autoimmunity by controlling multiple aspects of a regulatory T cell gene expression program

Foxp3 and its protein partners establish a regulatory T (Treg) cell transcription profile and promote immunological tolerance. However, molecular features contributing to a Treg-specific gene expression program are still incompletely understood. We find that the transcription factor Bcl11b is a prominent Foxp3 cofactor with multifaceted functions in Treg biology. Optimal genomic recruitment of Foxp3 and Bcl11b is critically interdependent. Genome-wide occupancy studies coupled with gene expression profiling reveal that Bcl11b, in association with Foxp3, is primarily responsible in establishing a Treg-specific gene activation program. Furthermore, Bcl11b restricts misdirected recruitment of Foxp3 to sites, which would otherwise result in an altered Treg transcriptome profile. Consequently, Treg-specific ablation of Bcl11b results in marked breakdown of immune tolerance, leading to aggressive systemic autoimmunity. Our study provides previously underappreciated mechanistic insights into molecular events contributing to basic aspects of Treg function. Furthermore, it establishes a therapeutic target with potential implications in autoimmunity and cancer.