DNA chip technology enables simultaneous examination of how Ϸ6,200 Saccharomyces cerevisiae gene transcript levels, representing the entire genome, respond to environmental change. By using chips bearing oligonucleotide arrays, we show that, after exposure to the alkylating agent methyl methanesulfonate, Ϸ325 gene transcript levels are increased and Ϸ76 are decreased. Of the 21 genes that already were known to be induced by a DNA-damaging agent, 18 can be scored as inducible in this data set, and surprisingly, most of the newly identified inducible genes are induced even more strongly than these 18. We examined 42 responsive and 8 nonresponsive ORFs by conventional Northern blotting, and 48 of these 50 ORFs responded as they did by DNA chip analysis, with magnitudes displaying a correlation coefficient of 0.79. Responsive genes fall into several expected and many unexpected categories. Evidence for the induction of a program to eliminate and replace alkylated proteins is presented.

Exposure to carcinogenic alkylating agents, oxidizing agents, and ionizing radiation modulates transcript levels for over one third of Saccharomyces cerevisiae's 6,200 genes. Computational analysis delineates groups of coregulated genes whose upstream regions bear known and novel regulatory sequence motifs. One group of coregulated genes contain a number of DNA excision repair genes (including the MAG1 3-methyladenine DNA glycosylase gene) and a large selection of protein degradation genes. Moreover, transcription of these genes is modulated by the proteasome-associated protein Rpn4, most likely via its binding to MAG1 upstream repressor sequence 2-like elements, that turn out to be almost identical to the recently identified proteasome-associated control element (G. Mannhaupt, R. Schnall, V. Karpov, I. Vetter, and H. Feldmann, FEBS Lett. 450:27-34, 1999). We have identified a large number of genes whose transcription is influenced by Rpn4p.Biological processes depend upon the structural integrity of the molecules that comprise living organisms. The structural integrity of the genome is particularly important because molecular alterations in the genetic material, usually DNA, can lead to permanent inheritable changes, i.e., mutations. However, the structural integrity of other cellular molecules, such as proteins, RNA, carbohydrates, and lipids, is also important, because the precise three-dimensional shape and the detailed chemistry of these molecules orchestrate the biochemical processes vital for life. Most biomolecules are inherently reactive, and as such their structural integrity is constantly challenged by reactive chemical and physical agents in the environment. It should therefore come as no surprise that all cells can sense and respond to unfavorable molecular alterations. Indeed, it is well known that cells sense and respond to damaged DNA and proteins, and such responses are exemplified by the SOS and heat shock responses that have been well characterized in Escherichia coli and other organisms (11,12,28).Here we explore the transcriptional response of Saccharomyces cerevisiae to a wide range of chemical and physical damaging agents. Specifically, we explore how transcript levels for every S. cerevisiae gene and open reading frame (ORF) respond when cellular molecules are damaged by a selection of environmentally and clinically relevant chemical and physical carcinogens. The global transcriptional response of this budding yeast to these damaging agents turns out to be far more extensive than anticipated. However, computational analysis of almost 200,000 data points reveals patterns in the data that allow us to define novel regulatory networks. We find that the responses of S. cerevisiae to each of six damaging agents are markedly different and that, for at least one agent, the response is dramatically affected by the cell's position in the cell cycle at the time of exposure. Computational clustering of the data and subsequent searching for common sequence motifs in promoter regions reveal nine such m...

Maturation of spermatozoa, including the acquisition of motility and the ability to undergo capacitation, occurs during transit through the dynamic environment of the epididymis. The microenvironments created along the length of the epididymal tubule are essential to the molecular modifications of spermatozoa that result in fertile gametes. The secretory and resorptive processes of the epithelial cells that line this tubule generate these microenvironments. In the current study, 10 morphologically distinct segments of the mouse epididymis were identified by microdissection. We hypothesized that the changing environments of the epididymal lumen are established by differential gene expression among these segments. RNA isolated from each of the 10 segments was analyzed by microarray analysis. More than 17,000 genes are expressed in the mouse epididymis, compared with about 12,000 genes identified from whole epididymal samples. Screening a panel of normal mouse tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, this study identified 2168 genes that are up-regulated or down-regulated by greater than 4-fold between at least two different segments. The expression patterns of these genes identify distinct patterns of segmental regulation. Using principal component analysis, we determined that the 10 segments form 6 different transcriptional units. These analyses elucidate the changes in gene expression along the length of the epididymis for 17,000 expressed transcripts and provide a powerful resource for the research community in future studies of the biological factors that mediate epididymal sperm maturation.

Regional differences along the epididymis are essential for the establishment of the luminal environment required for sperm maturation. In the current study, 19 morphologically distinct segments of the rat epididymis were identified by microdissection. Total RNA was isolated from each segment and subjected to microarray analysis. Segmental analysis of epididymal gene expression identified more than 16,000 expressed qualifiers, whereas profiling of RNA from whole rat epididymis identified approximately 12,000 expressed qualifiers. Screening a panel of normal rat tissues identified both epididymal-selective and epididymal-specific transcripts. In addition, more than 3500 qualifiers were shown to be present and differentially upregulated or downregulated by more than fourfold between any two segments. The present study complements our previous segment-dependent analysis of gene expression in the mouse epididymis and allows for comparative analyses between datasets. A total of 492 genes was shown to be present on both the MOE430 (mouse) and RAE230_2 (rat) microarrays, expressed in the epididymis of both species, and differentially expressed by more than fourfold in between segments in each species. Moreover, in-depth quantitative RT-PCR analysis of 36 members of the beta defensin gene family showed highly conserved patterns of expression along the lengths of the mouse and rat epididymides. These analyses elucidate global gene expression patterns along the length of the rat epididymis and provide a novel evaluation of conserved and nonconserved gene expression patterns in the epididymides of the two species. Furthermore, these data provide a powerful resource for the research community for future studies of biological factors that mediate sperm maturation and storage.

TREM-1 (triggering receptor expressed on myeloid cells-1) is an orphan immunoreceptor expressed on monocytes, macrophages, and neutrophils. TREM-1 associates with and signals via the adapter protein DAP12/TYROBP, which contains an ITAM. TREM-1 activation by receptor cross-linking has been shown to be proinflammatory and to amplify some cellular responses to TLR ligands such as bacterial LPS. To investigate the cellular consequences of TREM-1 activation, we have characterized global gene expression changes in human monocytes in response to TREM-1 cross-linking in comparison to and combined with LPS. Both TREM-1 activation and LPS up-regulate chemokines, cytokines, matrix metalloproteases, and PTGS/COX2, consistent with a core inflammatory response. However, other immunomodulatory factors are selectively induced, including SPP1 and CSF1 (i.e., M-CSF) by TREM-1 activation and IL-23 and CSF3 (i.e., G-CSF) by LPS. Additionally, cross-talk between TREM-1 activation and LPS occurs on multiple levels. Although synergy in GM-CSF protein production is reflected in commensurate mRNA abundance, comparable synergy in IL-1β protein production is not. TREM-1 activation also attenuates the induction of some LPS target genes, including those that encode IL-12 cytokine family subunits. Where tested, positive TREM-1 outputs are greatly reduced by the PI3K inhibitor wortmannin, whereas this attenuation is largely PI3K independent. These experiments provide a detailed analysis of the cellular consequences of TREM-1 activation and highlight the complexity in signal integration between ITAM- and TLR-mediated signaling.

Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes, and spermatids suggests the existence of precise programs of gene expression in these cells and in their neighboring somatic Sertoli cells. The objective of this study was to identify the genes that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, and XIII-XIV of the cycle were isolated by microdissection, whereas Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes, and round spermatids were purified from enzymatically dispersed testes. Microarray analysis by using Rat Genome 230 2.0 arrays identified 16,971 probe sets that recognized testicular transcripts, and 398 of these were identified as testis-specific. Expression of 1,286 probe sets were found to differ at least 4-fold between two cell types and also across the stages of the cycle. Pathway and annotated cluster analyses of those probe sets predicted that entire biological pathways and processes are regulated cyclically in specific cells. Important among these are the cell cycle, DNA repair, and embryonic neuron development. Taken together, these data indicate that stage-regulated gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells.array analysis ͉ spermatogenesis ͉ seminiferous tubules ͉ stages of the cycle of the seminiferous epithellum ͉ contraception I n mammals, spermatogenesis encompasses a series of precisely timed cellular events that take place in a highly organized tissue, the seminiferous epithelium. Within each cross-section of this tissue, spermatogonia, spermatocytes, and spermatids are in intimate physical association with somatic Sertoli cells, and, together, these cells progress synchronously through the stages of the cycle of the seminiferous epithelium (1, 2). Four cycles are required for spermatogonia, and their progeny to complete spermatogenesis. The synchrony of germ cell development has a significant effect on the structures and functions of both the germ cells and their associated somatic Sertoli cells, suggesting the existence of precise and coordinated cyclic programs of gene expression. However, because investigators have evaluated the stage-dependent changes in expression of only a few genes, whether or not cyclic gene expression is a rare or prevalent characteristic of these cells is unknown (3-7). It is also not known whether genes encoding components of important biological pathways and processes are coordinately regulated in a cyclic manner. However, such genes may represent new targets for the development of male contraceptives.Given the coordinated nature of spermatogenesis, we hypothesized that the expression of large numbers of genes differ substantially, both between specific cell types within the seminiferous epithelium and within a given cell type as it progresses through the stages of...

ABSTRACT:Mesenchymal stems cells have a demonstrated ability to differentiate into muscle, bone, and fat. Determining whether these same cells have the ability to differentiate into tendon-like fibroblasts has been hampered by the lack of specific tendon cell marker genes. In order to identify molecular markers of mature tendon, expression profiling was used to identify genes expressed in adult rat and human tendon tissue compared to other musculoskeletal tissues. Using this technique, approximately 1,600 transcripts appeared to be selectively expressed in rat tendon tissue and approximately 300 transcripts appeared to be selectively expressed in human tendon tissue, with 20 genes selectively expressed in both human and rat tendon tissue. Of these common tendon-selective genes, thrombospondin-4 (THBS4) and tenomodulin (TNMD) were found to have the highest tendon-selective expression compared to other tissues examined. Interestingly, expression of these tendon-selective genes, which are present in primary tendon fibroblasts, is lost when these cells are placed in two-dimensional culture systems. In conclusion, this study has defined a set of tendon-selective genes present in both adult rat and human tendons. Identification of tendon-selective genes provides potential molecular tools to facilitate a better understanding of tendon development and tendon repair. ß

Author contributions. C.S., P.L.C and S.Z. contributed equally to this work. M. Cella designed, performed and interpreted experiments. R.G. and S.Z. analyzed scRNA-seq data and wrote methods for scRNA-seq analysis. C.S. generated Aiolos-and T-bet-transduced MNK3 cells. M.L.R. and V.P. analyzed the microarray data and RNA-seq data. K.Z. and M.N.A. provided bioinformatic support. J.K.B., K.Y. and V.C. helped in flow cytometry data presentation and analysis. C.F. and R.F. generated libraries for scRNA-seq. J.S. provided critical advice for Cytof analysis. W.G., L.-L.L. and M.B. provided critical insights to the study. S.G., R.A.F. and L.S. provided key reagents. P.L.C. performed cut and run experiment and interpreted data under supervision of E.M.O. S.A.J. and M. Colonna supervised the study. M. Cella, S.A.J. and M. Colonna wrote the manuscript and all the authors contributed editing and suggestions.