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The unfolded protein response (UPR) maintains protein homeostasis by governing the processing capacity of the endoplasmic reticulum (ER) to manage ER client loads; however, key regulators within the UPR remain to be identified. Activation of the UPR sensor PERK (EIFAK3/PEK) results in the phosphorylation of the α subunit of eIF2 (eIF2α-P), which represses translation initiation and reduces influx of newly synthesized proteins into the overloaded ER. As part of this adaptive response, eIF2α-P also induces a feedback mechanism through enhanced transcriptional and translational expression of Gadd34 (Ppp1r15A),which targets type 1 protein phosphatase for dephosphorylation of eIF2α-P to restore protein synthesis. Here we describe a novel mechanism by which Gadd34 expression is regulated through the activity of the zinc finger transcription factor NMP4 (ZNF384, CIZ). NMP4 functions to suppress bone anabolism, and we suggest that this occurs due to decreased protein synthesis of factors involved in bone formation through NMP4-mediated dampening of Gadd34 and c-Myc expression. Loss of Nmp4 resulted in an increase in c-Myc and Gadd34 expression that facilitated enhanced ribosome biogenesis and global protein synthesis. Importantly, protein synthesis was sustained during pharmacological induction of the UPR through a mechanism suggested to involve GADD34-mediated dephosphorylation of eIF2α-P. Sustained protein synthesis sensitized cells to pharmacological induction of the UPR, and the observed decrease in cell viability was restored upon inhibition of GADD34 activity. We conclude that NMP4 is a key regulator of ribosome biogenesis and the UPR, which together play a central role in determining cell viability during endoplasmic reticulum stress.

Nmp4/CIZ proteins (nuclear matrix protein 4/cas interacting zinc finger protein) contribute to gene regulation in bone, blood, and testis. In osteoblasts, they govern the magnitude of gene response to osteotropic factors like parathyroid hormone (PTH). Nmp4/CIZ is recurrently involved in acute leukemia and it has been implicated in spermatogenesis. However, these conserved proteins, derived from a single gene, are expressed in numerous tissues indicative of a more generalized housekeeping function in addition to their tissue-specific roles. To address how Nmp4/CIZ expression is governed, we characterized the 5' regulatory region of the mouse Nmp4 gene, located on chromosome 6. Two adjacent promoters P(1) [-2521 nucleotide (nt)/-597 nt] and P(2) (-2521 nt/+1 nt) initiate transcription of alternative first exons (U(1) and U(2)). Both promoters lack TATA and CCAAT boxes but contain initiator sites and CpG islands. Northern analysis revealed expression of both U(1) and U(2) in numerous adult tissues consistent with the constitutive and ubiquitous activity of a housekeeping gene. Sequence analysis identified numerous potential transcription factor-binding sites significant to osteogenesis, hematopoeisis, and gonadal development. The promoters are active in both osteoblast-like cells and in the M12 B-lymphocyte cell line. Low doses of PTH attenuated P(1)/P(2) activity in osteoblast-like cells. The Nmp4/CIZ promoters are autoregulated and deletion analysis identified regions that drive P(1) and P(2) basal activities as well as regions that contain positive and negative regulatory elements affecting transcription. The Nmp4/CIZ promoters comprise a genomic regulatory architecture that supports constitutive expression as well as cell- and tissue-specific regulation.

An organism ultimately reflects the coordinate expression of its genome. The misexpression of a gene can have catastrophic consequences for an organism, yet the mechanics of transcription is a local phenomenon within the cell nucleus. Chromosomal and nuclear position often dictate the activity of a specific gene. Transcription occurs in territories and in discrete localized foci within these territories. The proximity of a gene or trans-acting factor to heterochromatin can have profound functional significance. The organization of heterochromatin changes with cell development, thus conferring temporal changes on gene activity. The protein-protein interactions that engage the trans-acting factor also contribute to context-dependent transcription. Multi-protein assemblages known as enhanceosomes govern gene expression by local committee thus dictating regional transcription factor function. Local DNA architecture can prescribe enhancesome membership. The local bending of the double helix, typically mediated by architectural transcription factors, is often critical for stabilizing enhanceosomes formed from trans-acting proteins separated over small and large distances. The recognition element to which a transcription factor binds is of functional significance because DNA may act as an allosteric ligand influencing the conformation and thus the activity of the transactivation domain of the binding protein, as well as the recruitment of other proteins to the enhanceosome. Here, we review and attempt to integrate these local determinants of gene expression.

Bone cells undergo changes in cell structure during phenotypic development. Parathyroid hormone (PTH) induces a change in osteoblast shape, a determinant of collagen expression. We hypothesize that alterations in bone cell shape reflect and direct gene expression as governed, in part, by nuclear organization. In this study, we determined whether the expression of nuclear matrix proteins that mediate nuclear architecture, NuMA, topoisomerase II (topo II)-alpha, and -beta, were altered during osteoblast development and response to PTH in vivo. NuMA forms an interphase nuclear scaffold in some cells, the absence of which may accommodate alterations in nuclear organization necessary for specific functions. Topo II enzymes are expressed in bone cells; the alpha-isoform is specific to proliferating cells. We used immunohistochemistry and flow cytometry to determine whether NuMA is expressed in the primary spongiosa of the rat metaphyseal femur and whether expression of NuMA, topo II-alpha, and II-beta changes during osteoblast development or with PTH treatment. NuMA and topo II-beta were expressed in marrow cells, osteoblasts, osteocytes, and chondrocytes. These proteins were not detected in osteoclasts in vivo, but were observed in cultured cells. Bone marrow cells expressed topo II-alpha. All three proteins were expressed in cultures of rat osteoblast-like UMR-106 cells. PTH treatment downregulated the number of topo II-alpha-immunopositive cells, correlated with a decrease in S-phase cells, in both bone tissue and cell culture. We conclude that, in vivo, nuclear matrix composition is altered during bone cell development and that anabolic doses of PTH attenuate the proliferative capacity of osteogenic cells, in part, by targeting topo II-alpha expression.

Tooth eruption activates a localized resorption and formation of alveolar bone and these activities depend upon the adjacent parts, coronal and basal, respectively, of the dental follicle-enamel epithelium. In this study the nuclear matrix-intermediate filament (NM-IF) proteins of these tissues were isolated in order to continue investigations into the molecular mechanisms underlying eruption. Dental follicles were removed from the third and fourth premolar of dogs at 13, 16 and 20 weeks (pre-, early, and mid-to-late eruption of these teeth) and NM-IF proteins were extracted from the coronal and basal halves. Most of the NM-IF protein profiles of these coronal and basal parts on one-dimensional, sodium dodecyl sulphate-polyacrylamide gel electrophoresis were remarkably constant, indicating an essentially uniform cellular composition. However, differences between these tissues were observed and some of these changed during eruption. Based on recent observations that nuclear matrix changes reflect and may even mediate cell-specific changes in gene expression, these findings suggest that changes in nuclear matrix proteins may be related to the molecular basis for some aspects of differential gene expression in the coronal and basal regions of the dental follicle and account for the ability of these tissues to activate bone resorption and formation during tooth eruption.

Metamorphic competence in the opisthobranch gastropod Aplysia californica Cooper coincides with the appearance of four to six irregularly shaped red spots on the right side of the outer periviceral membrane beneath the larval shell along with a red line overlying the mantle margin. If this pigmented pattern is not manifest, the animal will not respond to the metamorphic substratum, a number of red seaweeds including Agardhiella subulata (C. Agardh) Kraft et Wayne and Laurencia paciflca Kylin. Preliminary observations indicate that an exogenous factor(s), present in summer seawater in Woods Hole, Massachusetts, but absent during the winter months, is required for the onset of metamorphic competence. This factor is rendered inactive by heating fresh summer seawater for short periods or is removed via ultrafiltration using 30–100-K membranes. Exudants and crude extracts from red seaweeds (Agardhiella sp. and Gracillaria sp.) trigger the competence transition and metamorphosis. We have yet to determine whether the competence and metamorphic inducers are the same compound. Nevertheless, this factor does not appear to be required for normal larval growth and development preceding the competence transition. The possible ecological significance of our laboratory observations is briefly discussed.