Our recent work has demonstrated that the somatic cyst cells that enclose the germ cells play key roles at several decision points in specifying that germ cells progress to differentiation. Local signals that regulate self-renewal vs differentiation is important in tumor biology as well, as aberrant signals from supporting stromal cells are now known to play an important role in maintaining proliferating tumor cells. We are currently using the cyst, composed of germ cells enclosed by squamous epithelia-like somatic support cells that co-differentiate dependent on each other, as a model to study how close-range signals and cell-cell interactions guide the coordinated development of organs.

a. Kiger, A., H. White-Cooper, and M.T. Fuller (2000) Somatic support cells restrict germline stem cell self- renewal and promote differentiation. Nature 407: 750-754.

b. Brantley, S.E., and M.T. Fuller (2019) Somatic support cells regulate germ cell survival through the Baz/aPKC/Par-6 complex. Development 146: dev169342. doi:10.1242/dev.169342. PMCID: PMC6503986.

c. Papagiannouli, F., C.W. Berry, and M. T. Fuller (2019) The Dlg-module and clathrin-mediated endocytosis regulate EGFR signaling and cyst cell-germline coordination in the Drosophila testis. Stem Cell Reports 12: 1024-1040. DOI: https://doi/org/10/1016/j.stemcr.2019.03.008. PMCID: PMC6523063.

The switch from transit amplifying (TA) cell proliferation to onset of terminal differentiation is a key regulatory point in adult stem cell lineages. Failure to limit TA divisions can lead to cancer: indeed some leukemias, glioblastomas, and medulloblastomas have been shown to originate from transit amplifying cells that fail to stop dividing. Using Drosophila spermatogenesis as a model, we showed that the number of rounds of spermatogonial TA divisions is set by accumulation of a key regulator, Bag-of-marbles (Bam) to a critical threshold, and that Bam and its binding partner, Bgcn, required for spermatogonia to stop division and enter the spermatocyte program in Drosophila, act as translational repressors, directly binding to target mRNAs. We are now investigating the targets of Bam that lead to the switch from proliferation to differentiation.

We identified the mouse Bgcn homolog, made null mutant mice, and showed that in both sexes, mitotic germ cells proliferate, but die soon after they attempt to enter meiosis. We found that the core cell cycle control protein Cyclin A2, which is normally expressed in spermatogonia but absent in spermatocytes, instead continues to be expressed in young spermatocytes from the null mutant mice and the spermatocytes attempt a premature mitotic rather than meiotic division, confirming that function of the mouse Bgcn homolog is required to clear the mitotic program to allow cells to execute the extended G2 of meiotic prophase. These findings suggest deep conservation of a posttranscriptional mechanism regulating the switch from mitotic proliferation to onset of meiosis and differentiation.

We recently discovered that alternative processing of nascent mRNAs plays important roles in establishing functional differences between proliferating precursors and cells initiating differentiation. We found that alternative 3’ end processing of nascent mRNAs regulates the proteins expressed in spermatogonia vs spermatocytes. 3’ end Seq revealed developmentally regulated differences in the choice of where to make the 3’ end cut that terminates nascent transcripts (termed alternative polyadenylation, or APA) that results in production of transcripts with long 3’UTRs in spermatogonia but short 3’UTRs in spermatocytes from specific genes. In some cases, where antibodies or GFP-tagged genomic constructs were available, we found that the change in 3’UTR length led to abrupt switching of protein expression from on in spermatogonia to off in spermatocytes or vice versa. Thus a single developmentally regulated molecular event, choice of a different 3’ end cut site, can produce dramatic changes in expression of a large number of target proteins, with the some switching on and others switching off, presumably depending on the particular cis-regulatory sequences present in the long version of the 3’UTR. This mechanism may be especially important for a clean switch from one developmental state (proliferation) to the next (differentiation) in a cell lineage because APA takes place on nascent mRNAs at the last step of transcription, so changing to a new suite of proteins does not need to await opening of chromatin, formation of a preinitiation complex, or elongation of transcripts across the exons and introns of a gene body. We have also uncovered several instances where cell-type-specific alternative splicing of nascent transcripts led to production of a spermatocyte-specific protein isoform that plays an important role in regulating differentiation in the male germ line stem cell lineage.

In work on the switch from proliferation to differentiation in mammalian brain development, two graduate students in the lab analyzed cerebellar granule neuron precursors (GNPs), which proliferate extensively perinatally, then stop dividing and grow characteristic T-shaped processes to become the most abundant neuronal cell type in the brain. Failure of GNPs to stop dividing can lead to formation of the Sonic hedgehog (Shh) dependent form of medulloblastoma, a severe childhood brain cancer. Analysis of cerebellar granule neuron precursors at peak proliferation vs onset of differentiation by quantitative phosphoproteomics revealed that the kinase CK2 plays key roles in terminal steps of the Hh signal transduction pathway essential for proliferation of GNPs and Shh medulloblastoma cells. Treatment with a CK2 inhibitor cured mouse Shh medulloblastoma in vivo and prevented growth of human medulloblastoma cells in culture. Based on these results, children with recurrent Shh medulloblastoma are now being enrolled in a clinical trial to test whether the CK2 inhibitor can cure Shh medulloblastoma in humans.

a. Insco, M, A Leon, D McKearin, MT Fuller (2009) Accumulation of a differentiation regulator specifies transit amplifying division number in an adult stem cell lineage. PNAS 106: 22311-22316. PMCID: PMC2799733.

b. Insco*, M., A. S. Bailey*, J. Kim, G. H. Olivares, O. Wapinsky, C. H. Tam, and M. T. Fuller (2012) A self-limiting switch based on translational control regulates the transition from proliferation to differentiation in an adult stem cell lineage. Cell Stem Cell 11: 689-700. PMCID: PMC3833810.

c. Bailey, A., S., P. Batista, R. S. Gold, Y. G. Chen, D. G. de Rooij, H. Y. Chang, and M. T. Fuller (2017) The conserved RNA helicase YTHDC2 regulates the transition from proliferation to differentiation in the germline. eLife DOI: 10.7554/eLife.26116. PMCID: PMC5703642.

d. Purzner, T., J. Purzner, T. Buckstaff, G. Cozza, S., Gholamin, J. M. Rusert, T. Hartl, J. Sanders, N. Conley, X. Ge, M. Langan, V. Ramaswamy, L. Ellis, U. Litzenburger, S. Bolin, J. Theruvath, R. Nitta, L. Qi, X.-N. Li, G. Li, M. D. Taylor, R. J. Wechsler-Reya, L. Pinna, Y.-J. Cho*, M. T. Fuller*, J. Elias, and M. P. Scott (2018) Developmental phosphoproteomics identifies CK2 kinase as a driver of Hedgehog transduction and a therapeutic target in medulloblastoma. Science Signaling 11: eaau5147. PMCID: PMC6475502.

In the Drosophila male germ line cell lineage, onset of the spermatocyte program is marked by dramatic changes in transcriptional behavior and chromatin state. Many genes expressed in precursor cells are turned off in spermatocytes, many genes are transcribed in spermatocytes but not in precursor cells, and genes expressed in both cell types are transcribed from a different promoter in spermatocytes than in other cells. The spermatocyte transcription program involves at least two different gene activation waves: expression of genes that program the special cell cycle, cell growth and morphological characteristics of spermatocytes, and transcription of genes required for subsequent spermatid differentiation. The transcription program for spermatid differentiation requires sequential activity of two cell type specific protein complexes. The tMAC complex, a spermatocyte-specific alternate form of the Mip/dREAM transcriptional repressor, is required to activate transcription of over 1000 genes in spermatocytes. The tTAFs, five tissue specific homologs of components of the “general” transcription factor TFIID expressed only in spermatocytes, are required to upregulate transcription of many of these genes in spermatocytes. We discovered that action of tMAC is required for recruitment of the coactivator Mediator, which in turn helps recruit the tTAFs to chromatin to fully activate the terminal differentiation genes.

We devised a protocol to induce proliferating spermatogonia to switch to spermatocyte state synchronously in vivo and mapped in temporal detail the transcriptional changes as male germ cells switch from mitosis to meiosis. We discovered that one of the earliest transcripts turned on in spermatocytes encodes a testis specific multiple Zn Finger protein (Kumgang) required in spermatocytes to prevent expression of genes normally only expressed in differentiated somatic tissues. ChIP-Seq revealed that Kmg protein is located along the gene bodies of active genes, where it recruits the chromatin remodeler Mi-2. RNA-Seq revealed that function of Kmg and Mi-2 is required in spermatocytes to prevent firing of cryptic promoters scattered at many sites throughout the genome. Firing of these cryptic promoters depends on function of the tMAC subunit Aly and drives expression of aberrant transcripts from genes that would normally only be expressed in differentiated somatic tissues. ChIP-Seq for Aly suggested recruitment of Aly to the promoters of its proper targets – spermatocyte and spermatid differentiation genes – by Kmg may prevent Aly from acting at cryptic promoters.

We found by ChIP-Seq that Aly binds at transcription start sites of the genes it regulates and that function of Aly is required to open chromatin at the target promoters. ATAC-Seq and CAGE analysis revealed that transcripts that turn on in spermatocytes fire from novel core promoters that differ in sequence from the two core promoter types, TATA/DPE or DRE, previously characterized for Drosophila and utilized for transcripts expressed in precursor cells but turned down with differentiation. These surprising new results show that promoter proximal elements, acted upon by tMAC, rather than distal enhancers control one of the most robust cell type specific differential gene expression programs in Drosophila.

a. Chen, X., M. Hiller, Y. Sancak, and M. T. Fuller (2005) Tissue specific TAFs counteract Polycomb to turn on terminal differentiation. Science 310: 869-873.

b. Chen, X., C. Lu, J.R. Morillo Prado, S.H. Eun, and M.T. Fuller (2011) Sequential changes at differentiation gene promoters as they become active in a stem cell lineage. Development 138: 2441-2450. PMCID: PMC3100706.

c. Lu, C., and M.T. Fuller (2015) Recruitment of Mediator complex by cell type and stage-specific factors required for tissue-specific TAF dependent gene activation in an adult stem cell lineage. PLoS Genetics 11: e1005701. doi: 10.1371/journal.pgen.1005701 PMCID: PMC4666660.

d. Kim, J., C. Lu*, S. Srinivasan*, S. Awe, A. Brehm, and M. T. Fuller (2017) Blocking promiscuous activation at cryptic promoters directs selective gene expression. Science 356: 717-721. PMCID:28522526.

e. Lu, D., H.S. Sin, C. Lu, and M.T. Fuller (2019). Developmental regulation of cell type-specific transcription by novel promoter-proximal sequence elements. Genes and Development 34: 663-677. PMCID: PMC7197356.

The developmental program must impose regulatory mechanisms upon the core cell cycle machinery to ensure proper execution of cell-type and stage-specific cell cycles in multicellular animals. Although these developmental controls are crucial for embryonic development, adult cell differentiation, tissue renewal and repair and are prime targets for defects leading to cancer, the molecular regulatory mechanisms are not well understood. Regulation of cell cycle progression is a central target of the switch from transit amplifying spermatogonia to the spermatocyte differentiation program: spermatogonia execute a mitotic program, while spermatocytes enter into the specialized cell cycle of meiosis. A crucial feature of the meiotic cell cycle is the extended G2 phase known as meiotic prophase. It is during this cell cycle stage that the dramatic gene expression program that prepares germ cells for subsequent spermatid differentiation takes place.

We have found that the delay in the G2/M transition that allows time for meiotic prophase is specified by the developmental program through an additional layer of regulatory mechanisms that control translation of core cell cycle machinery components. At least two pathways act during meiotic prophase to delay the G2/M transition: 1) Translational repression via the cyclin B (cycB) 3’ UTR delays expression of CycB protein. 2) Expression of the cell cycle regulatory phosphatase cdc25/twine is translationally repressed until just prior to the G2/M transition, when expression of Twine protein is activated by the DAZ homolog Boule. We discovered that some of the earliest transcripts upregulated when spermatogonia become spermatocytes encode cell-type-specific translational regulators that control the specialized cell cycle of meiosis in the male. We found that a spermatocyte-specific homolog of the translation initiation complex core component eIF4G is required for translation of core cell cycle regulators such as CycB in spermatocytes. In a phenotype conserved from yeast sporulation to mouse spermatogenesis, we found that loss of function of the major polyubiquitin locus causes meiosis I maturation arrest infertility. We discovered that early spermatocytes express two cell-type-specific translational repressors, Rbp4 and Fest, that together prevent premature expression of CycB during the extended G2 phase of meiotic prophase. We have now found that a spermatocyte-specific isoform of the hnRNP-R/Q Syncrip, generated by alterative splicing, is required to activate translation of cycB in mature spermatocytes for the G2/M transition of meiosis I. We found that cycB mRNA is expressed with a long 3’UTR of over 700nt in spermatogonia, but a short 3’UTR of only 130nt in spermatocytes. Rbp4 and Fest bind to this short 3’UTR, as does the Syp protein required for activation of cycB translation of in mature spermatocytes. Together our results show that both cell-type-specific mRNA processing to make alternative transcript isoforms and cell-type-specific trans-acting factors that control translation of specific core cell cycle machinery components shape the specialized cell cycle of meiosis.

a. Baker, C.C., and M.T. Fuller (2007) Translational control of meiotic cell cycle progression and spermatid differentiation in male germ cells by a novel eIF4G homolog. Development 134:2863-2869. PMCID: PMC4620998.

b. Lu, C., J. Kim, and M.T. Fuller (2013) The D. melanogaster polyubiquitin gene magn/Ubi-p63 is required for male meiotic cell cycle progression and germ cell differentiation. Development 140: 3541-3551. PMCID: PMC3742140.

c. Baker, C.C., B.S. Gim, and M.T. Fuller (2015) Cell-type specific translational repression of Cyclin B during meiosis in males. Development 142: 3394-3402. PMCID: PMC4631753.