Overlapping functions of the pRb family in the regulation of rRNA synthesis.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

Regulation of RNA polymerase III transcription during cell cycle entry.

Increased rates of RNA polymerase (pol) III transcription constitute a central feature of the mitogenic response, but little is known about the mechanism(s) responsible. We demonstrate that the retinoblastoma protein RB plays a major role in suppressing pol III transcription in growth-arrested fibroblasts. RB knockout cells are compromised in their ability to down-regulate pol III following serum withdrawal. RB binds and represses the pol III-specific transcription factor TFIIIB during G(0) and early G(1), but this interaction decreases as cells approach S phase. Full induction of pol III coincides with mid- to late G(1) phase, when RB becomes phosphorylated by cyclin D- and E-dependent kinases. TFIIIB only associates with the underphosphorylated form of RB, and overexpression of cyclins D and E stimulates pol III transcription in vivo. The RB-related protein p130 also contributes to the repression of TFIIIB in growth-arrested fibroblasts. These observations provide insight into the mechanisms responsible for controlling pol III transcription during the switch between growth and quiescence.

Regulation of RNA polymerase I transcription in response to F9 embryonal carcinoma stem cell differentiation.

Dramatic changes in the patterns of transcription are a common feature of early development. We have used F9 embryonal carcinoma cells as a model system to study gene regulation during an early stage of murine embryogenesis. We find that transcription by RNA polymerase I decreases when F9 cells differentiate into parietal endoderm. The reduced rate of transcription is associated with a down-regulation of several components of the class I transcription apparatus. The most substantial change involves the essential factor SL1, which is a multisubunit complex that contains the TATA-binding protein and three TATA-binding protein-associated factors (TAFs). The abundance of two of these TAFs, TAFI48 and TAFI95, decreases during F9 cell differentiation. Developmental regulation of a specific class of genes may therefore be achieved through changes in the availability of TAFs.

Regulation of a TATA-binding protein-associated factor during cellular differentiation.

RNA polymerase III transcription is down-regulated when F9 embryonal carcinoma cells differentiate into parietal endoderm. This reflects a decrease in the activity of TFIIIB, a multisubunit complex that is required for all class III gene expression. Two essential components of TFIIIB are the TATA-binding protein (TBP) and an associated polypeptide called BRF that is specific to this complex. The abundance of both TBP and BRF decreases during F9 cell differentiation. Whereas the amount of TBP assembled into TFIIIB is down-regulated, this is not the case for all TBP-containing complexes. BRF levels show a more dramatic decline that appears sufficient to account for the overall change in transcriptional activity. Developmental regulation of a specific class of genes may therefore be achieved through changes in the availability of a TBP-associated factor.

Transcription by RNA polymerases I and III: a potential link between cell growth, protein synthesis and the retinoblastoma protein.

The rate of protein synthesis is a critical determinant of cellular growth. Abnormal activation of this process is a frequent feature of transformed and tumour cells. Several distinct components of the translation apparatus have been shown to be deregulated in response to malignant transformation. Indeed, overexpression of certain translation factors has been found to predispose cells to transformation or even initiate it. The latest twist to this story comes from the discovery that the retinoblastoma protein RB plays a major role in restricting the production of tRNA and rRNA. RB is an important tumour suppressor. Its ability to limit the synthesis of these principle determinants of biosynthetic capacity could provide a mechanism for restraining cell growth. The loss of this control may constitute a significant step towards tumour progression.

Altered intestinal macrophage phenotype in ovine paratuberculosis.

The expression of macrophage surface markers that are likely to be important in antigen presentation and cell interactions was examined in normal sheep and those with clinical paratuberculosis. Immunohistological studies demonstrated that intestinal macrophages in diseased sheep expressed MHC class II, LFA-1 and CR4 antigens weakly compared with normal tissues. Reverse transcriptase-polymerase chain reaction analysis of MHC class II mRNA in intestinal whole tissue samples showed no significant difference between control and diseased groups. A reduction in molecules such as MHC class II and LFA-1 on the surface of infected macrophages could have implications for survival of the intracellular mycobacteria and the persistence of infection.

Phenotypic characterisation of intestinal lymphocytes in ovine paratuberculosis by immunohistochemistry.

Characterisation of the T-cell subsets in intestinal lesions in sheep with paratuberculosis may contribute to our understanding of the pathogenesis of this disease. To determine the phenotype and distribution of lymphocytes in the normal sheep intestinal mucosa and in Mycobacterium avium subspecies paratuberculosis infected sheep, immunohistochemistry was performed on 12 normal sheep and 18 naturally infected, clinically diseased sheep of which 12 showed lepromatous and six tuberculoid forms of the disease. Immunoperoxidase staining was carried out on frozen sections of ileum using monoclonal antibodies against ovine CD4, CD8, and gamma delta T-cell receptor (TCR) markers. In all three sample groups, cells appeared to be non-randomly distributed throughout the lamina propria. Higher densities of lymphocytes were present in villus than in crypt areas. CD8+ cells were located principally around the epithelial basement membrane, whereas CD4+ cells were localised towards the central villus area of the lamina propria. Lymphocytes bearing the gamma delta T-cell receptor were more widely distributed, both in epithelial and lamina propria compartments. Ileum with tuberculoid lesions had higher densities of CD4 and gamma delta T-cell subsets while lepromatous lesions had lower densities of CD4 and CD8 cells compared with normal tissues. The median relative percentage of CD4+ cells was increased and that of CD8+ cells decreased in tuberculoid cases, with a corresponding increase in the CD4:CD8 ratio, while the relative percentage of gamma delta + cells was increased in lepromatous cases.

Increased intestinal TNF-alpha, IL-1 beta and IL-6 expression in ovine paratuberculosis.

Mycobacterium avium subspecies paratuberculosis is an intracellular parasite of intestinal macrophages and causes a chronic granulomatous enteritis in sheep and other ruminants (paratuberculosis or Johne's disease). Macrophages can be produced a variety of immunoregulatory cytokines that may influence mycobacterial killing and produce disordered inflammation within the gut. In this study, messenger RNA (mRNA) was extracted from intestinal tissue from control and multibacillary diseased sheep and profiles for the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), IL-6, transforming growth factor-beta1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF) were semi-quantified using reverse transcriptase polymerase chain reactions (RT-PCR). Infected intestinal tissues had significantly increased mRNA for TNF-alpha, IL-1beta and IL-6 but TGF-beta1 and GM-CSF mRNA levels were significantly different from controls. Supernatants from in vitro intestinal cultures were assayed for TNF-alpha activity using the PK(15)-1512 cytotoxicity bioassay and levels were significantly raised in diseased samples. TNF-alpha was not detected in any serum samples. Further analysis on intestinal tissues from sheep with the different, paucibacillary, form of the disease showed significant elevation of TNF-alpha mRNA but not other cytokines tested. Increased pro-inflammatory cytokine expression in the intestine coincident with a failed or misdirected immune response may contribute to the pathogenesis of paratuberculosis and the persistence of a chronic inflammatory state.