Margaret Tatnall Fuller

Polycomb chromatin domains are chromosomal regions decorated with histone H2A monoubiquitination at lysine 119 (H2Aub1) and histone H3 trimethylation at lysine 27 (H3K27me3). These domains are dynamically shaped through the actions of different Polycomb group protein complexes to control gene expression during development. To assess how different Polycomb group subcomplexes contribute to these histone modification profiles in Drosophila embryos, we used mutants that abrogate their function. Canonical Polycomb repressive complex (PRC) 1 deposits low levels of H2Aub1 solely at Polycomb target genes, whereas variant PRC1 generates the bulk of H2Aub1 genome-wide. In late-stage embryos, PR-DUB-mediated deubiquitination effectuates a uniform low-level H2Aub1 profile across the genome. The combined activities of PRC2.1 and PRC2.2 drive the formation and maintenance of most H3K27me3 domains, but PRC2.1 is the limiting enzyme for creating such domains at HOX genes. Surprisingly, reduction in the H3K27me3 level and repression defects caused by removing PRC2.1 were largely rescued in animals also lacking PR-DUB, which showed extensive H2Aub1 accumulation at Polycomb targets that promoted compensatory H3K27me3 deposition by PRC2.2. Diversification of Polycomb protein complexes combined with feedback loop mechanisms involving histone modification cross-talk equips the system with the plasticity, adaptability, and buffering capacity needed to safeguard cell fate decisions during development.

Regulators of chromatin accessibility play key roles in cell fate transitions, triggering the onset of novel transcription programs as cells differentiate. In the Drosophila male germline stem cell lineage, tMAC, a master regulator of spermatocyte differentiation that binds thousands of loci, is required for local opening of chromatin, allowing activation of spermatocyte-specific promoters. Here we show that a cell type-specific surveillance system involving the multiple zinc finger protein Kmg and the pipsqueak domain protein Dany dampens transcriptional output from weak tMAC-dependent promoters and counteracts tMAC binding at thousands of additional cryptic promoters, thus preventing massive expression of aberrant protein-coding transcripts. ChIP-seq showed Kmg enriched at the tMAC-bound promoters that it repressed, consistent with direct action. In contrast, Kmg and Dany did not repress highly expressed tMAC-dependent genes, where they colocalized with their binding partner, the chromatin remodeler Mi-2 (NuRD), along the transcribed regions rather than at the promoter. We discuss a model where Kmg, together with Dany and Mi-2, dampens expression from weak or ectopic promoters while allowing robust transcription from highly expressed Aly-dependent genes.

Stage specific increased expression of PCF11 and Cbc, the two components of Cleavage Factor complex II, contribute to developmentally regulated 3’UTR shortening due to alternate polyadenylation of nascent RNA molecules in Drosophila spermatocytes compared to spermatogonia. Here, we show that both Cbc and PCF11 change subnuclear localization during male germ cell differentiation, from homogeneous in the nucleus in spermatogonia and early spermatocytes to concentrated around the nucleolus in later spermatocyte stages.

The switch from precursor cell proliferation to onset of differentiation in adult stem cell lineages must be carefully regulated to produce sufficient progeny to maintain and repair tissues, yet prevent overproliferation that may enable oncogenesis. In the Drosophila male germ cell lineage, spermatogonia produced by germ line stem cells undergo a limited number of transit amplifying mitotic divisions before switching to the spermatocyte program that sets up meiosis and eventual spermatid differentiation. The number of transit amplifying divisions is set by accumulation of the bag-of-marbles (Bam) protein to a critical threshold. In bam mutants, spermatogonia proliferate through several extra rounds of mitosis and then die without becoming spermatocytes. Here, we show that a key role of Bam for the mitosis to differentiation switch is repressing expression of Held Out Wings ( how ), homolog of mammalian Quaking. Knockdown of how in germ cells was sufficient to allow spermatogonia mutant for bam or its partner benign gonial cell neoplasm to differentiate, while forced expression of nuclear-targeted How protein in spermatogonia wild-type for bam resulted in continued proliferation at the expense of differentiation. Our findings suggest that Bam targets how RNA for degradation by acting as an adapter to recruit the CCR4-NOT deadenylation complex via binding its subunit, Caf40. As How is itself an RNA-binding protein with roles in RNA processing, our findings reveal that the switch from proliferation to meiosis and differentiation in the Drosophila male germ line adult stem cell lineage is regulated by a cascade of RNA-binding proteins.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, remains difficult to cure upon relapse, with only 12.4% of patients surviving five years post-recurrence. While specific quiescent tumor cell populations are known to contribute to treatment-resistance, the molecular mechanisms that maintain quiescence remain poorly defined. Here, we identify the cell cycle inhibitor p57 as a regulator of quiescence and chemotherapy resistance in Sonic Hedgehog (SHH) MB. Nuclear p57 was enriched in Sox2+ and Nestin+ stem-like MB cells compared to proliferative Atoh1+ cells. Inducing p57 expression in SHH MB cells led to a six-fold increase in G ₀ -phase cells and conferred resistance to the frontline chemotherapeutic vincristine. Clinically, dexamethasone is a glucocorticoid given to nearly all MB patients to manage cerebral edema and is administered with wide variability in timing and dosing. We show that dexamethasone significantly increased nuclear p57 levels and expanded the G ₀ population in both Ptch1 ^+/− and Ptch1 ^+/− ; Trp53 ^−/− SHH MB mouse models. Pre-treatment with dexamethasone reduced vincristine sensitivity in SHH MB cells. Together, our findings reveal a clinically relevant and previously unrecognized mechanism of treatment resistance, whereby dexamethasone, despite its benefits in managing edema, may inadvertently contribute to tumor persistence or recurrence by driving a quiescent, drug-resistant state. Addressing the lack of standardization in steroid use or targeting p57 may improve treatment response and reduce recurrence in patients diagnosed with SHH MB.

Medulloblastoma (MB) is the most common malignant brain tumour in children. The Sonic Hedgehog (SHH)-medulloblastoma subtype arises from the cerebellar granule neuron lineage. Terminally differentiated neurons are incapable of undergoing further cell division, so an effective treatment for this tumour could be to force neuronal differentiation. Differentiation therapy provides a potential alternative for patients with medulloblastoma who harbor mutations that impair cell death pathways (TP53), which is associated a with high mortality. To this end, our goal was to explore epigenetic regulation of cerebellar granule neuron differentiation in medulloblastoma cells. Key regulators were discovered using chromatin immunoprecipitation with high-throughput sequencing. DNA-bound protein and chromatin protein modifications were investigated across all genes. We discovered that Ezh2-mediated tri-methylation of the H3 histone (H3K27me3), occurred on more than half of the 787 genes whose transcription normally increases as granule neurons terminally differentiate. Conditional knockout of Ezh2 led to early initiation of differentiation in granule neuron precursors (GNPs), but only after cell cycle exit had occurred. Similarly, in MB cells, neuronal differentiation could be induced by preventing H3K27me3 modifications using an Ezh2 inhibitor (UNC1999), but only when UNC1999 was combined with forced cell cycle exit driven by a CDK4/6 inhibitor (Palbociclib). Ezh2 emerges as a powerful restraint upon post-mitotic differentiation during normal GNP development and combination of Ezh2 inhibition with cell cycle exit leads to MB cell differentiation.

Regulators of chromatin accessibility play key roles in cell fate transitions, triggering onset of novel transcription programs as cells differentiate. In the Drosophila male germ line stem cell lineage, tMAC, a master regulator of spermatocyte differentiation that binds thousands of loci, is required for local opening of chromatin, allowing activation of spermatocyte-specific promoters. Here we show that a cell-type specific surveillance system involving the multiple zinc finger protein Kmg and the pipsqueak domain protein Dany dampens transcriptional output from weak tMAC dependent promoters and blocks tMAC binding at thousands of additional cryptic promoters, thus preventing massive expression of aberrant protein-coding transcripts. ChIP-seq showed Kmg enriched at the tMAC-bound promoters it repressed, consistent with direct action. In contrast, Kmg and Dany did not repress highly expressed tMAC dependent genes, where they colocalized with their binding partner, the chromatin modeler Mi-2 (NuRD), along the transcribed regions rather than at the promoter. Mi-2 has been shown to preferentially bind RNA over chromatin (Ullah et al . 2022). We propose that at highly expressed genes binding of Mi-2 to the abundant nascent RNA pulls the Kmg/Dany complex away from promoters, providing a mechanism to effectively repress ectopic promoters while protecting robust transcription.

Medulloblastoma (MB) is the most common malignant brain tumour in children. The Sonic Hedgehog (SHH)-medulloblastoma subtype arises from the cerebellar granule neuron lineage. Terminally differentiated neurons are incapable of undergoing further cell division, so an effective treatment for this tumour could be to force neuronal differentiation. Differentiation therapy provides a potential alternative for patients with medulloblastoma who harbor mutations that impair cell death pathways (TP53), which is associated a with high mortality. To this end, our goal was to explore epigenetic regulation of cerebellar granule neuron differentiation in medulloblastoma cells. Key regulators were discovered using chromatin immunoprecipitation with high-throughput sequencing. DNA-bound protein and chromatin protein modifications were investigated across all genes. We discovered that Ezh2-mediated tri-methylation of the H3 histone (H3K27me3), occurred on more than half of the 787 genes whose transcription normally increases as granule neurons terminally differentiate. Conditional knockout of Ezh2 led to early initiation of differentiation in granule neuron precursors (GNPs), but only after cell cycle exit had occurred. Similarly, in MB cells, neuronal differentiation could be induced by preventing H3K27me3 modifications using an Ezh2 inhibitor (UNC1999), but only when UNC1999 was combined with forced cell cycle exit driven by a CDK4/6 inhibitor (Palbociclib). Ezh2 emerges as a powerful restraint upon post-mitotic differentiation during normal GNP development and combination of Ezh2 inhibition with cell cycle exit leads to MB cell differentiation.

Posttranscriptional regulation of gene expression by RNA-binding proteins can enhance the speed and robustness of cell state transitions by controlling RNA stability, localization, or if, when, or where mRNAs are translated. The RNA helicase YTHDC2 is required to shut down components of the mitotic program to facilitate a proper switch from mitosis to meiosis in mouse germ cells. Here, we show that YTHDC2 has a second essential role in promoting meiotic progression in late spermatocytes. Inducing conditional knockout of Ythdc2 during the first wave of spermatogenesis, after initiation of meiotic prophase, allowed YTHDC2 -deficient germ cells to advance to the pachytene stage and properly express many meiotic markers. However, the YTHDC2 -deficient spermatocytes mis-expressed a number of genes, some up-regulated and some down-regulated, failed to transition to the diplotene stage, and then quickly died. Coimmunoprecipitation experiments revealed that YTHDC2 interacts with several RNA-binding proteins in early or late spermatocytes, with many of the interacting proteins, including MEIOC, localizing to granules, similar to YTHDC2. Our findings suggest that YTHDC2 collaborates with other RNA granule components to facilitate proper progression of germ cells through multiple steps of meiosis via mechanisms influencing posttranscriptional regulation of RNAs.

The switch from precursor cell proliferation to onset of differentiation in adult stem cell lineages must be carefully regulated to produce sufficient progeny to maintain and repair tissues, yet prevent overproliferation that may enable oncogenesis. In the Drosophila male germ cell lineage, spermatogonia produced by germ line stem cells undergo a limited number of transit amplifying mitotic divisions before switching to the spermatocyte program that sets up meiosis and eventual spermatid differentiation. The number of transit amplifying divisions is set by accumulation of the bag-of-marbles (Bam) protein to a critical threshold. In bam mutants, spermatogonia proliferate through several extra rounds of mitosis then die without becoming spermatocytes. Here we show that a key role of Bam for the mitosis to differentiation switch is repressing expression of Held Out Wings ( how ), homolog of mammalian Quaking. Knockdown of how in germ cells was sufficient to allow spermatogonia mutant for bam or its partner benign gonial cell neoplasm ( bgcn ) to differentiate, while forced expression of nuclear-targeted How protein in spermatogonia wild-type for bam resulted in continued proliferation at the expense of differentiation. Our findings suggest that Bam targets how RNA for degradation by acting as an adapter to recruit the CCR4-NOT deadenylation complex via binding its subunit, Caf40. As How is itself an RNA binding protein with roles in RNA processing, our findings reveal that the switch from proliferation to meiosis and differentiation in the Drosophila male germ line adult stem cell lineage is regulated by a cascade of RNA-binding proteins.