Serine-arginine-rich (SR) proteins play a key role in alternative pre-mRNA splicing in eukaryotes. We recently showed that a large SR protein called Son has unique repeat motifs that are essential for maintaining the subnuclear organization of pre-mRNA processing factors in nuclear speckles. Motif analysis of Son highlights putative RNA interaction domains that suggest a direct role for Son in pre-mRNA splicing. Here, we used in situ approaches to show that Son localizes to a reporter minigene transcription site, and that RNAi-mediated Son depletion causes exon skipping on reporter transcripts at this transcription site. A genome-wide exon microarray analysis was performed to identify human transcription and splicing targets of Son. Our data show that Son-regulated splicing encompasses all known types of alternative splicing, the most common being alternative splicing of cassette exons. We confirmed that knockdown of Son leads to exon skipping in pre-mRNAs for chromatin-modifying enzymes, including ADA, HDAC6 and SetD8. This study reports a comprehensive view of human transcription and splicing targets for Son in fundamental cellular pathways such as integrin-mediated cell adhesion, cell cycle regulation, cholesterol biosynthesis, apoptosis and epigenetic regulation of gene expression.

The ectoparasitic mite, Sarcoptes scabiei that burrows in the epidermis of mammalian skin has a long co-evolution with its hosts. Phenotypic studies show that the mites have the ability to modulate cytokine secretion and expression of cell adhesion molecules in cells of the skin and other cells of the innate and adaptive immune systems that may assist the mites to survive in the skin. The purpose of this study was to identify genes in keratinocytes and fibroblasts in human skin equivalents (HSEs) that changed expression in response to the burrowing of live scabies mites. Overall, of the more than 25,800 genes measured, 189 genes were up-regulated >2-fold in response to scabies mite burrowing while 152 genes were down-regulated to the same degree. HSEs differentially expressed large numbers of genes that were related to host protective responses including those involved in immune response, defense response, cytokine activity, taxis, response to other organisms, and cell adhesion. Genes for the expression of interleukin-1α (IL-1α) precursor, IL-1β, granulocyte/macrophage-colony stimulating factor (GM-CSF) precursor, and G-CSF precursor were up-regulated 2.8- to 7.4-fold, paralleling cytokine secretion profiles. A large number of genes involved in epithelium development and keratinization were also differentially expressed in response to live scabies mites. Thus, these skin cells are directly responding as expected in an inflammatory response to products of the mites and the disruption of the skin’s protective barrier caused by burrowing. This suggests that in vivo the interplay among these skin cells and other cell types, including Langerhans cells, dendritic cells, lymphocytes and endothelial cells, is responsible for depressing the host’s protective response allowing these mites to survive in the skin.

Functional inactivation of the retinoblastoma tumor suppressor gene product (RB) is a common event in human cancers. Classically, RB functions to constrain cellular proliferation, and loss of RB is proposed to facilitate the hyperplastic proliferation associated with tumorigenesis. To understand the repertoire of regulatory processes governed by RB, two models of RB loss were utilized to perform microarray analysis. In murine embryonic fibroblasts harboring germline loss of RB, there was a striking deregulation of gene expression, wherein distinct biological pathways were altered. Specifically, genes involved in cell cycle control and classically associated with E2F-dependent gene regulation were upregulated via RB loss. In contrast, a program of gene expression associated with immune function and response to pathogens was significantly downregulated with the loss of RB. To determine the specific influence of RB loss during a defined period and without the possibility of developmental compensation as occurs in embryonic fibroblasts, a second system was employed wherein Rb was acutely knocked out in adult fibroblasts. This model confirmed the distinct regulation of cell cycle and immune modulatory genes through RB loss. Analyses of ciselements supported the hypothesis that the majority of those genes upregulated with RB loss are regulated via the E2F family of transcription factors. In contrast, those genes whose expression was reduced with the loss of RB harbored different promoter elements. Consistent with these analyses, we found that disruption of E2F-binding function of RB was associated with the upregulation of gene expression. In contrast, cells harboring an RB mutant protein (RB-750F) that retains E2F-binding activity, but is specifically deficient in the association with LXCXE-containing proteins, failed to upregulate these same target genes. However, downregulation of genes involved in immune function was readily observed with disruption of the LXCXE-binding function of RB.Thus, these studies demonstrate that RB plays a significant role in both the positive and negative regulations of transcriptional programs and indicate that loss of RB has distinct biological effects related to both cell cycle control and immune function.

BackgroundMDM4, also called MDMX or HDMX in humans, is an important negative regulator of the p53 tumor suppressor. MDM4 is overexpressed in about 17% of all cancers and more frequently in some types, such as colon cancer or retinoblastoma. MDM4 is known to be post-translationally regulated by MDM2-mediated ubiquitination to decrease its protein levels in response to genotoxic stress, resulting in accumulation and activation of p53. At the transcriptional level, MDM4 gene regulation has been less clearly understood. We have reported that DNA damage triggers loss of MDM4 mRNA and a concurrent increase in p53 activity. These experiments attempt to determine a mechanism for down-regulation of MDM4 mRNA.Methodology/Principal FindingsHere we report that MDM4 mRNA is a target of hsa-mir-34a (miR-34a). MDM4 mRNA contains a lengthy 3′ untranslated region; however, we find that it is a miR-34a site within the open reading frame (ORF) of exon 11 that is responsible for the repression. Overexpression of miR-34a, but not a mutant miR-34a, is sufficient to decrease MDM4 mRNA levels to an extent identical to those of known miR-34a target genes. Likewise, MDM4 protein levels are decreased by miR-34a overexpression. Inhibition of endogenous miR-34a increased expression of miR-34a target genes and MDM4. A portion of MDM4 exon 11 containing this 8mer-A1 miR-34a site fused to a luciferase reporter gene is sufficient to confer responsiveness, being inhibited by additional expression of exogenous mir-34a and activated by inhibition of miR-34a.Conclusions/SignificanceThese data establish a mechanism for the observed DNA damage-induced negative regulation of MDM4 and potentially provide a novel means to manipulate MDM4 expression without introducing DNA damage.

The retinoblastoma tumor suppressor, RB, is a negative regulator of the cell cycle that is inactivated in the majority of human tumors. Cell cycle inhibition elicited by RB has been attributed to the attenuation of CDK2 activity. Although ectopic cyclins partially overcome RB-mediated S-phase arrest at the replication fork, DNA replication remains inhibited and cells fail to progress to G 2 phase. These data suggest that RB regulates an additional execution point in S phase. We observed that constitutively active RB attenuates the expression of specific dNTP synthetic enzymes: dihydrofolate reductase, ribonucleotide reductase (RNR) subunits R1/R2, and thymidylate synthase (TS). Activation of endogenous RB and related proteins by p16ink4a yielded similar effects on enzyme expression. Conversely, targeted disruption of RB resulted in increased metabolic protein levels (dihydrofolate reductase, TS, RNR-R2) and conferred resistance to the effect of TS or RNR inhibitors that diminish available dNTPs. Analysis of dNTP pools during RB-mediated cell cycle arrest revealed significant depletion, concurrent with the loss of TS and RNR protein. Importantly, the effect of active RB on cell cycle position and available dNTPs was comparable to that observed with specific antimetabolites. Together, these results show that RB-mediated transcriptional repression attenuates available dNTP pools to control Sphase progression. Thus, RB employs both canonical cyclin-dependent kinase/cyclin regulation and metabolic regulation as a means to limit proliferation, underscoring its potency in tumor suppression.The retinoblastoma tumor suppressor (RB) 1 functions as a negative regulator of cell cycle transitions (1-5). Due to its frequent inactivation in tumors (Ͼ60%), it is highly relevant to determine how RB functions to inhibit cellular proliferation and to elucidate its interaction with chemotherapeutic drugs.Biochemically, RB functions as a transcriptional co-repressor that mediates the inhibition of cell cycle progression (1-5). RB interacts with multiple cellular proteins, including the E2F family of transcriptional regulators (6). In addition to binding E2F, RB also interacts with histone deacetylase (HDAC) and SWI/SNF chromatin remodeling proteins to establish a repressor complex on the promoters of E2F-regulated genes (3,7,8). This activity of RB is critical for cell cycle inhibition. In G 0 and early G 1 , RB is hypophosphorylated and forms transcriptional repressor complexes to inhibit cell cycle progression. However, in response to mitogenic signaling, cyclin-dependent kinase (CDK)/cyclin complexes phosphorylate RB (9). Phosphorylation disrupts the association of RB with its interacting proteins, thereby alleviating transcriptional repression of E2F-regulated genes and facilitating cell cycle progression (1-5).Targets of E2F are known to encompass a variety of proteins involved in cell cycle progression (6,10,11). Consistent with the role of RB as a repressor of E2F, in disparate settings the expression/activity of cyclin E, cy...

Purpose: The transforming growth factor-h (TGF-h) signaling pathway has been frequently implicated in breast cancer. An intronic variant (Int7G24A) of TGF-h receptor type I (TGFBR1) is associated with kidney and bladder cancers in our recent study.We hypothesize that this germline variant may be involved in development and progression of breast cancer. Experimental Design: Case-control studies were designed from archived paraffin-embedded tissue specimens from the same geographic area with a homogenous ethnic population.We analyzed 223 patients (25 with preinvasive tumors and 198 with invasive and metastatic breast cancers) and 153 noncancer controls.The Int7G24A was identified by PCR-RFLP. Another germline deletion (TGFBR1*6A) and somatic mutations in theTGFBR1 were also analyzed by PCR and single-strand conformational polymorphism. Results: The Int7G24A allele was evident in 32% of patients with preinvasive neoplasms and 48% of patients with invasive breast cancers compared with 26% controls (P = 0.00008). In addition, 11 (5.6%) homozygous Int7G24A carriers were found in patients with invasive breast cancers, whereas only 3 (2 %) homozygous carriers were found in the control group. The TGFBR1*6A allele was not significantly associated with breast cancer patients and only one somatic mutation was found in 71breast cancers. Conclusion: These data suggest that the germline Int7G24A variant may represent a risk factor for invasive breast cancer and a marker for breast cancer progression. A separate study with alarger sample size is warranted to validate the association of the Int7G24A with human breast cancer.

The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the "chronic" RB-mediated arrest state and the "acute" arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation that is targeted at high frequency in human cancer (5,30,51,61,72,73). While RB has principally been considered as a regulator of G 1 phase, the importance of RB in governing DNA synthesis has become increasingly clear (14,33,35,41,52,58,60). Currently, there are two mechanisms through which RB has been postulated to inhibit replication. In the first, DNA synthesis may be influenced by the direct interaction of RB with components of the replication process. For example, recent data have suggested a direct role for RB/E2F in regulating origin function during Drosophila chorion gene amplification (10). Furthermore, under certain conditions RB has been localized to sites of DNA replication, although this remains controversial (16, 32). Additionally, RB has been demonstrated to directly interact with the DNA replication factors MCM7, DNA polymerase ␣, and replication factor C (RFC) p140 subunit (24,55,64,67). The importance of these interactions during cellular DNA replication has yet to be determined. However, these studies collectively suggest that RB could function in cis to directly inhibit DNA replication.In the second model, RB-mediated S-phase inhibition may be attributed to the active repression of requisite DNA replication factor expression. RB is known to bin...

Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNAdamaged HCT116 colon cancer cells and in isogenic p53 À/À cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of fulllength hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress.