A cell atlas of adult muscle precursors uncovers early events in fibre-type divergence in Drosophila.

In Drosophila, the wing disc-associated muscle precursor cells give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single-cell RNA-sequencing experiments and built a cell atlas of myoblasts associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM myoblasts that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of myoblasts, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM myoblasts at various states of differentiation by immunofluorescence and genetic cell-tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene, Amalgam which is functionally important in muscle development. Our work provides a framework for leveraging scRNA-seq for gene discovery and details a strategy that can be applied to other scRNA-seq datasets.

Differential effects of exogenous and autocrine growth hormone on LNCaP prostate cancer cell proliferation and survival.

The prostate gland is regulated by multiple hormones and growth factors that may also affect prostate tumorigenesis. Growth hormone (GH) contributes to prostate development and function, but the direct effects of GH on prostate cancer cells are not well understood. The expression of endogenous GH in prostate cancer cell lines has also been observed, suggesting the potential for an effect of autocrine GH. In the present study, we measure the levels of GH and GH receptor (GHR) mRNA in multiple prostate cancer and normal prostate-derived cell lines, and compare the effects of exogenous and autocrine GH on LNCaP prostate cancer cell proliferation and apoptosis, and the associated signal transduction pathways. We found that GHR and GH expression were higher in the prostate cancer cell lines, and that exogenous GH increased LNCaP cell proliferation, but had no effect on apoptosis. In contrast, autocrine GH overexpression reduced LNCaP cell proliferation and increased apoptosis. The distinct actions of exogenous and autocrine GH were accompanied by differences in the involvement of GHR-associated signal transduction pathways, and were paralleled by an alteration in the subcellular localization of GHR, in which autocrine GH appeared to sequester GHR in the Golgi and endoplasmic reticulum. This alteration of GHR trafficking may underlie a distinct mode of GH-mediated signaling associated with the effect of autocrine GH. These findings clarify the potential effects of GH on prostate cancer cell function, and indicate that the activity of autocrine GH may be distinct from that of endocrine GH in prostate cancer cells.

Identification of motor protein cargo by yeast 2-hybrid and affinity approaches.

Identification of the molecular composition of the cargo transported by individual kinesin motors is critical to an understanding of both motor function and regulation of the proper intracellular placement of numerous cellular components including proteins, RNA, and organelles. In this chapter, we describe methods to identify the motor tail sequences responsible for cargo binding by expression of green fluorescent protein (GFP)-motor tail fusion proteins in mammalian cells. In addition, we detail two complementary approaches to identify specific proteins associated with these targeting sequences: a yeast 2-hybrid screen and affinity chromatography.

POU1F1-mediated activation of hGH-N by deoxyribonuclease I hypersensitive site II of the human growth hormone locus control region.

The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.

Expression of Pit-1 in nonsomatotrope cell lines induces human growth hormone locus control region histone modification and hGH-N transcription.

The POU domain transcription factor Pit-1 is expressed in somatotropes, lactotropes, and thyrotropes of the anterior pituitary. Pit-1 is essential for the establishment of these lineages during development and regulates the expression of genes encoding the peptide hormones secreted by each cell type, including the growth hormone gene expressed in somatotropes. In contrast to rodent growth hormone loci, the human growth hormone (hGH) locus is regulated by a distal locus control region (LCR), which is required in cis for the proper expression of the hGH gene cluster in transgenic mice. The hGH LCR mediates a domain of histone acetylation targeted to the hGH locus that is associated with distal hGH-N activation, and the discrete determinants of this activity coincide with DNaseI hypersensitive site (HS) I of the LCR. The identification of three in vitro Pit-1 binding sites within the HS-I region suggested a model in which Pit-1 binding at HS-I initiates the chromatin modification mechanism associated with hGH LCR activity. To test this hypothesis directly and to determine whether Pit-1 expression is sufficient to confer hGH locus histone acetylation and activate hGH-N transcription from an inactive locus, we expressed Pit-1 in nonpituitary cell types. We show that Pit-1 expression established a domain of histone hyperacetylation at the LCR and hGH-N promoter in these cells similar to that observed in pituitary chromatin. This was accompanied by the activation of hGH-N transcription and an increase in intergenic and CD79b transcripts proximal to HS-I. These effects were coincident with Pit-1 occupancy at HS-I and the hGH-N promoter and were observed irrespective of the basal histone modification status of HS-I in the heterologous cell line. These findings are consistent with a role for Pit-1 as an initiating factor in hGH locus activation during somatotrope ontogeny, acting through binding sites at HS-I of the hGH LCR.

A dominant role for p53-dependent cellular senescence in radiosensitization of human prostate cancer cells.

Because p53 inactivation may limit the effectiveness of radiation therapy for localized prostate cancer, it is important to understand how this gene regulates clonogenic survival after an exposure to ionizing radiation. Here, we show that premature cellular senescence is the principal mode of cell death accounting for the radiosensitivity of human prostate cancer cell lines retaining p53 function. Alternative stress response pathways controlled by this tumor suppressor, including cell cycle arrest, DNA damage repair, mitotic catastrophe and apoptosis, contributed significantly less to radiation-induced clonogenic death. Using a dominant negative C-terminal fragment of p53, we present the first evidence that a complete loss of endogenous p53 function is sufficient to limit the irradiation-induced senescence and clonogenic death of prostate cancer cells. Conversely, inheritance of wild-type p53 by prostate cancer cells lacking a functional allele of this gene (i.e., DU145) significantly increases clonogenic death through p53-dependent cellular senescence and apoptotic pathways. Our data provide evidence that mutations of even one p53 allele may be sufficient to alter their clonogenic fate. In addition, they support the idea that the p53 pathway can be used as a specific target for enhancing the radiosensitivity of prostate cancer cells. Activation of p53 by the drug nutlin-3 is shown to be an effective radiosensitizer of prostate cancer cells retaining functional alleles of p53 and this effect was entirely attributable to an increased induction of p53-dependent cellular senescence.

The molecular motor KIFC1 associates with a complex containing nucleoporin NUP62 that is regulated during development and by the small GTPase RAN.

KIFC1 is a C-terminal kinesin motor associated with the nuclear membrane and acrosome in round and elongating spermatids. This location in developing spermatids is consistent with possible roles in acrosome elongation and manchette motility or both. Here we describe the association of the KIFC1 motor with a complex containing the nucleoporin NUP62. Formation of this complex is developmentally regulated, being absent before puberty and appearing only after nuclear elongation has begun. In addition, the integrity of this complex is dependent on GTP hydrolysis and the GTP state of the small GTPase RAN. Concomitant with the association of this motor with the NUP62-containing complex is an apparent reorganization of the nuclear pore with loss of NUP62 from larger complexes containing other nucleoporins. The association of KIFC1 with a component of the nuclear membrane is more consistent with a role for this motor in acrosome/manchette transport along the nuclear membrane than for a role for this motor in transport of vesicles along the outer face of the manchette.