We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.

The transforming growth factor ␤s (TGF-␤s) are a group of multifunctional growth factors that inhibit cell cycle progression in many cell types. The TGF-␤-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-␤ on both the levels and activities of the G 1 cyclins and their cyclin-dependent kinase partners. The ability of TGF-␤ to inhibit the activity of these kinase complexes derives in part from its regulatory effects on the cyclin-dependent kinase inhibitors, p21/WAF1/Cip1, p27 Kip1, and p15. Upon treatment of cells with TGF-␤, these three inhibitors bind to and block the activities of specific cyclin-cyclin-dependent kinase complexes to cause cell cycle arrest. Little is known, however, on the mechanism through which TGF-␤ activates these cyclin-dependent kinase inhibitors. In the case of p21, TGF-␤ treatment leads to an increase in p21 mRNA. This increase in p21 mRNA is partly due to transcriptional activation of the p21 promoter by TGF-␤. To further define the signaling pathways through which TGF-␤ induces p21, we have performed a detailed functional analysis on the p21 promoter. Through both deletion and mutation analysis of the p21 promoter, we have defined a 10-base pair sequence that is required for the activation of the p21 promoter by TGF-␤. In addition, this sequence is sufficient to drive TGF-␤-mediated transcription from a previously nonresponsive promoter. Preliminary gel shift assays demonstrate that this TGF-␤ responsive element binds specifically to several proteins in vitro. Two of these proteins are the transcription factors Sp-1 and Sp-3. These studies represent the initial steps toward defining the signaling pathways involved in TGF-␤-mediated transcriptional activation of p21.The transforming growth factor ␤s (TGF-␤s), 1 a group of protein hormones that regulate many cellular functions, inhibit cell proliferation by causing growth arrest in the G 1 phase of the cell cycle (1-4). Progression through G 1 is dependent on the sequential formation, activation, and subsequent inactivation of the G 1 cyclin-cyclin-dependent kinase complexes, primarily cyclin D-cyclin-dependent kinase 4 and cyclin E-cyclin-dependent kinase 2 complexes (5, 6). The TGF-␤-induced G 1 cell cycle arrest has been attributed to the regulatory effects of TGF-␤ on both the levels and activities of these G 1 cyclins and cyclin-dependent kinases (7-9). The inhibition of G 1 cyclin-cyclin-dependent kinase complex activity by TGF-␤ is mediated in part through several members of a recently described family of low molecular weight cyclin dependent kinase inhibitors. These cyclin-dependent kinase inhibitors, which include p21/WAF1/ Cip1, p27 Kip1 , p57 Kip2 , p18, p16, and p15, physically associate with their target cyclins, cyclin-dependent kinases, or cyclincyclin-dependent kinase complexes to inhibit their activities (reviewed in Refs. 10 -13). TGF-␤ regulates the activities of three of these cyclin-dependent kinase inhibitor family members: p27 Kip1 , p15, and p21 (reviewed in Refs. 10...

Purpose: The existence of cancer stem cells (CSCs) in breast cancer has profound implications for cancer prevention. In this study, we evaluated sulforaphane, a natural compound derived from broccoli/broccoli sprouts, for its efficacy to inhibit breast CSCs and its potential mechanism.Experimental Design: Aldefluor assay and mammosphere formation assay were used to evaluate the effect of sulforaphane on breast CSCs in vitro. A nonobese diabetic/severe combined immunodeficient xenograft model was used to determine whether sulforaphane could target breast CSCs in vivo, as assessed by Aldefluor assay, and tumor growth upon cell reimplantation in secondary mice. The potential mechanism was investigated using Western blotting analysis and β-catenin reporter assay.Results: Sulforaphane (1-5 μmol/L) decreased aldehyde dehydrogenase-positive cell population by 65% to 80% in human breast cancer cells (P < 0.01) and reduced the size and number of primary mammospheres by 8-to 125-fold and 45% to 75% (P < 0.01), respectively. Daily injection with 50 mg/kg sulforaphane for 2 weeks reduced aldehyde dehydrogenase-positive cells by >50% in nonobese diabetic/ severe combined immunodeficient xenograft tumors (P = 0.003). Sulforaphane eliminated breast CSCs in vivo, thereby abrogating tumor growth after the reimplantation of primary tumor cells into the secondary mice (P < 0.01). Western blotting analysis and β-catenin reporter assay showed that sulforaphane downregulated the Wnt/β-catenin self-renewal pathway.Conclusions: Sulforaphane inhibits breast CSCs and downregulates the Wnt/β-catenin self-renewal pathway. These findings support the use of sulforaphane for the chemoprevention of breast cancer stem cells and warrant further clinical evaluation. Clin Cancer Res; 16(9); 2580-90. ©2010 AACR.

Summary Mutations in the RNA binding protein FUS cause ALS, a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder SMA. The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of SMA patient fibroblasts. Here, we report that FUS associates with the SMN complex, an interaction mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions between SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, adding strong new support to the view that these motor neuron diseases are related.