We compared the transcriptomes of marrow-derived mesenchymal stem cells (MSCs) with differentiated adipocytes, osteocytes, and chondrocytes derived from these MSCs. Using global gene-expression profiling arrays to detect RNA transcripts, we have identified markers that are specific for MSCs and their differentiated progeny. Further, we have also identified pathways that MSCs use to differentiate into adipogenic, chondrogenic, and osteogenic lineages. We identified activin-mediated transforming growth factor (TGF)-␤ signaling, platelet-derived growth factor (PDGF) signaling and fibroblast growth factor (FGF) signaling as the key pathways involved in MSC differentiation. The differentiation of MSCs into these lineages is affected when these pathways are perturbed by inhibitors of cell surface receptor function. Since growth and differentiation are tightly linked processes, we also examined the importance of these 3 pathways in MSC growth. These 3 pathways were necessary and sufficient for MSC growth. Inhibiting any of these pathways slowed MSC growth, whereas a combination of TGF-␤, PDGF, and ␤-FGF was sufficient to grow MSCs in a serum-free medium up to 5 passages. Thus, this study illustrates it is possible to predict signaling pathways active in cellular differentiation and growth using microarray data and experimentally verify these predictions. (Blood. 2008;112:295-307)

SummaryThe complex anatomy of the epidermis contains multiple adult stem cell populations, but the extent to which they functionally overlap during homeostasis, wound healing, and tumor initiation remains poorly defined. Here, we demonstrate that Lrig1+ve cells are highly proliferative epidermal stem cells. Long-term clonal analysis reveals that Lrig1+ve cells maintain the upper pilosebaceous unit, containing the infundibulum and sebaceous gland as independent compartments, but contribute to neither the hair follicle nor the interfollicular epidermis, which are maintained by distinct stem cell populations. In contrast, upon wounding, stem cell progeny from multiple compartments acquire lineage plasticity and make permanent contributions to regenerating tissue. We further show that oncogene activation in Lrig1+ve cells drives hyperplasia but requires auxiliary stimuli for tumor formation. In summary, our data demonstrate that epidermal stem cells are lineage restricted during homeostasis and suggest that compartmentalization may constitute a conserved mechanism underlying epithelial tissue maintenance.

SUMMARY To provide a lifelong supply of blood cells, hematopoietic stem cells (HSCs) need to carefully balance both self-renewing cell divisions and quiescence. Although several regulators that control this mechanism have been identified, we demonstrate that the transcription factor PU.1 acts upstream of these regulators. So far, attempts to uncover PU.1’s role in HSC biology have failed because of the technical limitations of complete loss-of-function models. With the use of hypomorphic mice with decreased PU.1 levels specifically in phenotypic HSCs, we found reduced HSC long-term repopulation potential that could be rescued completely by restoring PU.1 levels. PU.1 prevented excessive HSC division and exhaustion by controlling the transcription of multiple cell-cycle regulators. Levels of PU.1 were sustained through autoregulatory PU.1 binding to an upstream enhancer that formed an active looped chromosome architecture in HSCs. These results establish that PU.1 mediates chromosome looping and functions as a master regulator of HSC proliferation.

CODEX (http://codex.stemcells.cam.ac.uk/) is a user-friendly database for the direct access and interrogation of publicly available next-generation sequencing (NGS) data, specifically aimed at experimental biologists. In an era of multi-centre genomic dataset generation, CODEX provides a single database where these samples are collected, uniformly processed and vetted. The main drive of CODEX is to provide the wider scientific community with instant access to high-quality NGS data, which, irrespective of the publishing laboratory, is directly comparable. CODEX allows users to immediately visualize or download processed datasets, or compare user-generated data against the database's cumulative knowledge-base. CODEX contains four types of NGS experiments: transcription factor chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), histone modification ChIP-Seq, DNase-Seq and RNA-Seq. These are largely encompassed within two specialized repositories, HAEMCODE and ESCODE, which are focused on haematopoiesis and embryonic stem cell samples, respectively. To date, CODEX contains over 1000 samples, including 221 unique TFs and 93 unique cell types. CODEX therefore provides one of the most complete resources of publicly available NGS data for the direct interrogation of transcriptional programmes that regulate cellular identity and fate in the context of mammalian development, homeostasis and disease.

Mutations in the parkin gene, which encodes a ubiquitin ligase, are a major genetic cause of parkinsonism. Interestingly, parkin also plays a role in cancer as a putative tumor suppressor, and the gene is frequently targeted by deletion and inactivation in human malignant tumors. Here, we investigated a potential tumor suppressor role for parkin in gliomas. We found that parkin expression was dramatically reduced in glioma cells. Restoration of parkin expression promoted G 1 phase cell-cycle arrest and mitigated the proliferation rate of glioma cells in vitro and in vivo. Notably, parkin-expressing glioma cells showed a reduction in levels of cyclin D1, but not cyclin E, and a selective downregulation of Akt serine-473 phosphorylation and VEGF receptor levels. In accordance, cells derived from a parkin-null mouse model exhibited increased levels of cyclin D1, VEGF receptor, and Akt phosphorylation, and divided significantly faster when compared with wild-type cells, with suppression of these changes following parkin reintroduction. Clinically, analysis of parkin pathway activation was predictive for the survival outcome of patients with glioma. Taken together, our study provides mechanistic insight into the tumor suppressor function of parkin in brain tumors and suggests that measurement of parkin pathway activation may be used clinically as a prognostic tool in patients with brain tumor. Cancer Res; 72(10); 2543-53. Ó2012 AACR.

Despite major advances in the generation of genome-wide binding maps, the mechanisms by which transcription factors (TFs) regulate cell type identity have remained largely obscure. Through comparative analysis of 10 key haematopoietic TFs in both mast cells and blood progenitors, we demonstrate that the largely cell type-specific binding profiles are not opportunistic, but instead contribute to cell type-specific transcriptional control, because (i) mathematical modelling of differential binding of shared TFs can explain differential gene expression, (ii) consensus binding sites are important for cell type-specific binding and (iii) knock-down of blood stem cell regulators in mast cells reveals mast cell-specific genes as direct targets. Finally, we show that the known mast cell regulators Mitf and c-fos likely contribute to the global reorganisation of TF binding profiles. Taken together therefore, our study elucidates how key regulatory TFs contribute to transcriptional programmes in several distinct mammalian cell types.

Treatment with anti–PD-1 and anti-PD-L1 therapies has shown durable clinical benefit in non–small cell lung cancer (NSCLC). However, patients with NSCLC with epidermal growth factor receptor (EGFR) mutations do not respond as well to treatment as patients without an EGFR mutation. We show that EGFR-mutated NSCLC expressed higher levels of CD73 compared with EGFR WT tumors and that CD73 expression was regulated by EGFR signaling. EGFR-mutated cell lines were significantly more resistant to T cell killing compared with WT cell lines through suppression of T cell proliferation and function. In a xenograft mouse model of EGFR-mutated NSCLC, neither anti–PD-L1 nor anti-CD73 antibody alone inhibited tumor growth compared with the isotype control. In contrast, the combination of both antibodies significantly inhibited tumor growth, increased the number of tumor-infiltrating CD8 + T cells, and enhanced IFN-γ and TNF-α production of these T cells. Consistently, there were increases in gene expression that corresponded to inflammation and T cell function in tumors treated with the combination of anti–PD-L1 and anti-CD73. Together, these results further support the combination of anti-CD73 and anti–PD-L1 therapies in treating EGFR-mutated NSCLC, while suggesting that increased T cell activity may play a role in response to therapy.

Combinatorial transcription factor (TF) binding is essential for cell-type-specific gene regulation. However, much remains to be learned about the mechanisms of TF interactions, including to what extent constrained spacing and orientation of interacting TFs are critical for regulatory element activity. To examine the relative prevalence of the ‘enhanceosome’ versus the ‘TF collective’ model of combinatorial TF binding, a comprehensive analysis of TF binding site sequences in large scale datasets is necessary. We developed a motif-pair discovery pipeline to identify motif co-occurrences with preferential distance(s) between motifs in TF-bound regions. Utilizing a compendium of 289 mouse haematopoietic TF ChIP-seq datasets, we demonstrate that haematopoietic-related motif-pairs commonly occur with highly conserved constrained spacing and orientation between motifs. Furthermore, motif clustering revealed specific associations for both heterotypic and homotypic motif-pairs with particular haematopoietic cell types. We also showed that disrupting the spacing between motif-pairs significantly affects transcriptional activity in a well-known motif-pair—E-box and GATA, and in two previously unknown motif-pairs with constrained spacing—Ets and Homeobox as well as Ets and E-box. In this study, we provide evidence for widespread sequence-specific TF pair interaction with DNA that conforms to the ‘enhanceosome’ model, and furthermore identify associations between specific haematopoietic cell-types and motif-pairs.