Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinitypurified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks. multidimensional protein identification technology ͉ proteomics ͉ spectrum counting ͉ mass spectrometry S ince its discovery in yeast (1, 2) and subsequent isolation and initial characterizations in human cells (3), the transcriptional coactivator complex Mediator has been the subject of numerous studies to characterize both its composition and function. Researchers have used a combination of immunoprecipitation (3-14), ion exchange/size-exclusion chromatography (8, 10, 15-19), glycerol gradient (6, 7, 12, 13, 19, 20), SDS/PAGE/ silver stain/Western blot analysis (6,7,9,10,15,18,21), MS (9,16,19,21,22), and ChIP (21,23) to determine the composition of Mediator complexes. Varying in size and subunit composition, the larger complexes (thyroid hormone receptor-associated protein complex, SRB/Med-containing cofactor complex, negative regulator of activated transcription, vitamin D receptor interacting protein complex, and activator recruited cofactor) ranged from 1 to 2 MDa and were composed of Ϸ30 subunits (3, 9-16, 21, 22, 24), whereas the smaller complexes (cofactor required for Sp1, positive cofactor 2, and positive cofactor 4) ranged from Ϸ500 to 700 kDa and were composed of Ϸ9-17 subunits (5-7, 17-20, 25, 26). The main shared difference between large and small complexes appears to be the presence or absence of the kinase module. Furthermore, different complexes possessed different levels of basal transcription, which could be accounted for by several possibilities, including purification method (high ionic strength may have washed away necessary proteins), the adding back of RNA polymerase (pol) II and basal transcription factors, and testing only for activated transcription.Although these studies form the foundation for our present understanding of Mediator, the relationship between different forms of Mediator was unclear and the composition of each form was unclear. The first attempt at standardizing Mediator complex...

The Maf-family transcription factor Nrl is a key regulator of photoreceptor differentiation in mammals. Ablation of the Nrl gene in mice leads to functional cones at the expense of rods. We show that a 2.5-kb Nrl promoter segment directs the expression of enhanced GFP specifically to rod photoreceptors and the pineal gland of transgenic mice. GFP is detected shortly after terminal cell division, corresponding to the timing of rod genesis revealed by birthdating studies. In Nrl ؊/؊ retinas, the GFP؉ photoreceptors express S-opsin, consistent with the transformation of rod precursors into cones. We report the gene profiles of freshly isolated flow-sorted GFP؉ photoreceptors from wild-type and Nrl ؊/؊ retinas at five distinct developmental stages. Our results provide a framework for establishing gene regulatory networks that lead to mature functional photoreceptors from postmitotic precursors. Differentially expressed rod and cone genes are excellent candidates for retinopathies.gene profiling ͉ gene regulation ͉ neuronal differentiation ͉ retina ͉ transcription factor E volution of higher-order sensory and behavioral functions in mammals is accompanied by increasingly complex regulation of gene expression (1). As much as 10% of the human genome is presumably dedicated to the control of transcription. Exquisitely timed expression of cell-type-specific genes, together with spatial and quantitative precision, depends on the interaction between transcriptional control machinery and extracellular signals (2, 3). Neuronal heterogeneity and functional diversity result from combinatorial and cooperative actions of regulatory proteins that form complicated yet precise transcriptional networks to generate unique gene expression profiles. A key transcription factor, combined with its cognate regulatory cis-sequence codes, specifies a particular node in the gene regulatory networks that guide differentiation and development (4).The retina offers an ideal paradigm for investigating regulatory networks underlying neuronal differentiation. The genesis of six types of neurons and Müller glia in the vertebrate retina proceeds in a predictable sequence during development (5). Subsets of multipotent retinal neuroepithelial progenitors exit the cell cycle at specific time points and acquire a particular cell fate under the influence of intrinsic genetic program and extrinsic factors (5-7). Pioneering studies using thymidine labeling and retroviral vectors established the order and birthdates of neurons in developing retina (5,(8)(9)(10)). The current model of retinal differentiation proposes that a heterogeneous pool of progenitors passes through states of competence, where it can generate a distinct subset of neurons (5). One can predict that, at the molecular level, this competence is acquired by combinatorial action of specific transcriptional regulatory proteins. Genetic ablation studies of transcription factors involved in early murine eye specification are consistent with combinatorial regulation (11-13).Rod and cone photorecep...

The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4–containing nucleosomes is a subject of debate. ChIP-chip experiments and high resolution quantitative PCR confirm that there is a single Cse4 nucleosome at each centromere, and additional regions of the genome contain Cse4 nucleosomes at low levels. Using unbiased biochemical, cell biological, and genetic approaches we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the non-histone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating Scm3 is not essential for the formation of functional centromeric chromatin. Additionally, octameric Cse4 nucleosomes can be reconstituted in vitro. The Cse4-Cse4 interaction domain appears to be essential and interaction occurs in vivo in the centromeric nucleosome. Taken together, our experimental evidence supports the model that the Cse4-nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.

It is widely postulated that tissue aging could be, at least partially, caused by reduction of stem cell number, activity, or both. However, the mechanisms of controlling stem cell aging remain largely a mystery. Here, we use Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that age-dependent decline in the functions of stem cells and their niche contributes to overall stem cell aging. BMP signaling activity from the niche significantly decreases with age, and increasing BMP signaling can prolong GSC life span and promote their proliferation. In addition, the age-dependent E-cadherin decline in the stem cell-niche junction also contributes to stem cell aging. Finally, overexpression of SOD, an enzyme that helps eliminate free oxygen species, in either GSCs or their niche alone can prolong GSC life span and increase GSC proliferation. Therefore, this study demonstrates that stem cell aging is controlled extrinsically and intrinsically in the Drosophila ovary.