The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.

Transcription of long noncoding RNAs (lncRNAs) within gene regulatory elements can modulate gene activity in response to external stimuli, but the scope and functions of such activity are not known. Here we use an ultra-high density array that tiles the promoters of 56 cell cycle genes to interrogate 108 samples representing diverse perturbations. We identify 216 transcribed regions that encode putative lncRNAs--many with RT-PCR-validated periodic expression during the cell cycle, show altered expression in human cancers, and are regulated in expression by specific oncogenic stimuli, stem cell differentiation, or DNA damage. DNA damage induces five lncRNAs from the CDKN1A promoter, and one such lncRNA, named PANDA, is induced in a p53- dependent manner. PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes; PANDA depletion markedly sensitized human fibroblasts to apoptosis by doxorubicin. These findings suggest potentially widespread roles for promoter lncRNAs in cell growth control.

BackgroundScleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.Methodology and FindingsWe analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of ‘intrinsic’ genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud's phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc.Conclusions and SignificanceGenome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.

When normal tissue and tumour samples are compared by microarray analysis, the biggest differences most often occur in the expression levels of genes that control cell proliferation. However, this difference is detected whenever mRNA samples that are taken from two cell populations with different proliferation rates are compared. Although the exact genes that comprise this 'proliferation signature' often differ, they are almost always genes that are involved in the fundamental process of cell proliferation. Can the proliferation signature be used to improve our understanding of the cell cycle and cancer pathogenesis, as well as being used as a biomarker for cancer diagnosis and prognosis?

We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20 ؉ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.

Objective Accumulation of myofibroblasts in fibrotic skin is a hallmark of systemic sclerosis (SSc; scleroderma), but the origins of these cells remain unknown. Because loss of intradermal adipose tissue is a consistent feature of cutaneous fibrosis, we sought to examine the hypothesis that myofibroblasts populating fibrotic dermis derive from adipocytic progenitors. Methods We performed genetic fate mapping studies to investigate the loss of intradermal adipose tissue and its potential role in fibrosis in mice with bleomycin-induced scleroderma. Modulation of adipocytic phenotypes ex vivo was investigated in adipose tissue–derived cells in culture. Results A striking loss of intradermal adipose tissue and its replacement with fibrous tissue were consistently observed in mice with bleomycin-induced fibrosis. Loss of adipose tissue and a decline in the expression of canonical adipogenic markers in lesional skin preceded the onset of dermal fibrosis and expression of fibrogenic markers. Ex vivo, subcutaneous adipocytes were driven by transforming growth factor β to preferentially undergo fibrogenic differentiation. Cell fate mapping studies in mice with the adiponectin promoter–driven Cre recombinase transgenic construct indicated that adiponectin-positive progenitors that are normally confined to the intradermal adipose tissue compartment were distributed throughout the lesional dermis over time, lost their adipocytic markers, and expressed myofibroblast markers in bleomycin-treated mice. Conclusion These observations establish a novel link between intradermal adipose tissue loss and dermal fibrosis and demonstrate that adiponectin-positive intradermal progenitors give rise to dermal myofibroblasts. Adipose tissue loss and adipocyte–myofibroblast transition might be primary events in the pathogenesis of cutaneous fibrosis that represent novel potential targets for therapeutic intervention.

The expression of the replication-dependent histone mRNAs is tightly regulated during the cell cycle. As cells progress from G 1 to S phase, histone mRNA levels increase 35-fold, and they decrease again during G 2 phase. Replication-dependent histone mRNAs are the only metazoan mRNAs that lack polyadenylated tails, ending instead in a conserved stem-loop. Much of the cell cycle regulation is posttranscriptional and is mediated by the 3 stem-loop. A 31-kDa stem-loop binding protein (SLBP) binds the 3 end of histone mRNA. The SLBP is necessary for pre-mRNA processing and accompanies the histone mRNA to the cytoplasm, where it is a component of the histone messenger RNP. We used synchronous CHO cells selected by mitotic shakeoff and HeLa cells synchronized at the G 1 /S or the M/G 1 boundary to study the regulation of SLBP during the cell cycle. In each system the amount of SLBP is regulated during the cell cycle, increasing 10-to 20-fold in the late G 1 and then decreasing in the S/G 2 border. SLBP mRNA levels are constant during the cell cycle. SLBP is regulated at the level of translation as cells progress from G 1 to S phase, and the protein is rapidly degraded as they progress into G 2 . Regulation of SLBP may account for the posttranscriptional component of the cell cycle regulation of histone mRNA.The replication-dependent histone mRNAs are tightly regulated during the cell cycle, increasing 35-fold as cells progress from G 1 to S phase (18). There is only a three-to five-fold increase in the rate of transcription of the histone genes (7,19), indicating that much of the regulation is posttranscriptional. The posttranscriptional component of cell cycle regulation is mediated by the 3Ј end of the histone mRNA (32, 35), which is a highly conserved stem-loop (34). The only processing step necessary for formation of the mature histone mRNA is an endonucleolytic cleavage to form the 3Ј end (14). Cleavage is directed by two cis-acting elements, the stem-loop which binds to the hairpin binding factor (37, 38), and a purine-rich sequence about 10 nucleotides (nt) 3Ј of the cleavage site that binds the 5Ј end of U7 snRNA (3,39,55). Hairpin binding factor is composed of a single 31-kDa protein, the stem-loop binding protein (SLBP) (10, 60) or hairpin binding protein (33), which is required for processing in vivo (44) and which remains with the mature mRNA as a component of the cytoplasmic messenger RNP (mRNP) (9, 17). At least one additional factor, a heat-labile factor, is required for processing, but this factor has not been well defined biochemically (15).Two posttranscriptional regulatory steps contribute to the cell cycle regulation of histone mRNA concentrations. Processing is regulated as cells progress from G 1 to S phase, and the half-life of histone mRNA is reduced to about 10 min at the end of S phase (18), resulting in the destruction of histone mRNA prior to mitosis. We (60) and others (33) recently cloned the cDNA for the SLBP from several species using the yeast three-hybrid system (50). Here we show tha...

Replication-dependent histone mRNAs are not polyadenylated but end in a conserved 26-nucleotide structure that contains a stem-loop. Much of the cell cycle regulation of histone mRNA is post-transcriptional and is mediated by the 3' end of histone mRNA. The stem-loop binding protein (SLBP) that binds the 3' end of histone mRNA is a candidate for the factor that participates in most, if not all, of the post-transcriptional regulatory events. We have cloned the cDNA for the SLBP from humans, mice, and frogs, using the recently developed yeast three-hybrid system. The human SLBP is a 31-kD protein and contains a novel RNA-binding domain, which has been mapped to a 73-amino-acid region of the protein. The cloned SLBP is the protein bound to the 3' end of histone mRNA as antibodies specific for the SLBP remove all specific binding activity from nuclear and polyribosomal extracts. These depleted extracts do not cleave histone pre-mRNA efficiently, demonstrating that the SLBP is required for efficient histone pre-mRNA processing.