Id helix-loop-helix proteins inhibit nucleoprotein complex formation by the TCF ETS-domain transcription factors.
The Id subfamily of helix-loop-helix (HLH) proteins plays a fundamental role in the regulation of cellular proliferation and differentiation. Id proteins are thought to inhibit differentiation mainly through interaction with other HLH proteins and by blocking their DNA-binding activity. Members of the ternary complex factor (TCF) subfamily of ETS-domain proteins have key functions in regulating immediate-early gene expression in response to mitogenic stimulation. TCFs form DNA-bound complexes with the serum response factor (SRF) and are direct targets of MAP kinase (MAPK) signal transduction cascades. In this study we demonstrate functional interactions between Id proteins and TCFs. Ids bind to the ETS DNA-binding domain and disrupt the formation of DNA-bound complexes between TCFs and SRF on the c-fos serum response element (SRE). Inhibition occurs by disrupting protein-DNA interactions with the TCF component of this complex. In vivo, the Id proteins cause down-regulation of the transcriptional activity mediated by the TCFs and thereby block MAPK signalling to SREs. Therefore, our results demonstrate a novel facet of Id function in the coordination of mitogenic signalling and cell cycle entry. (+info)
Identification of a novel transcriptional activity of mammalian Id proteins.
The Id proteins are a family of related mammalian helix-loop-helix (HLH) proteins which can interact with other HLH proteins but lack a basic region and are thus not thought to bind to DNA. Instead, they are hypothesized to act as dominant negative regulators of DNA-binding basic HLH (bHLH) proteins, by forming inactive heterodimers with these proteins. All four Id family proteins possess related HLH dimerization domains and can interact with similar bHLH proteins, although with differing affinities. The functions of the largely unrelated N- and C-terminal regions of the proteins are unknown. In this study, we have identified a novel transcriptional activity of the mammalian Id proteins. We show that when fused to the heterologous GAL4 DNA-binding domain, all four of the mammalian Id proteins can activate GAL4-dependent transcription. The HLH domain is necessary for the transactivation activity observed, suggesting that interaction with a cellular HLH protein is required. Co-transfection with exogenous Class A bHLH proteins (E-proteins) greatly potentiates the transactivation, which is abolished upon co-transfection with Class B bHLH proteins. These results are consistent with the idea that the Id proteins have a transcriptional activity when present in a DNA-binding complex. (+info)
Id genes are direct targets of bone morphogenetic protein induction in embryonic stem cells.
Bone morphogenetic proteins (BMPs) are morphogenetic signaling molecules essential for embryonic patterning. To obtain molecular insight into the influence of BMPs on morphogenesis, we searched for new genes directly activated by BMP signaling. In vitro cultured mouse embryonic stem (ES) cells were used, cultivated in chemically defined growth medium (CDM). CDM-cultured ES cells responded very selectively to stimulation by various mesoderm inducers (BMP2/4, activin A, and basic fibroblast growth factor). BMP2/4 rapidly induced transcript levels of the homeobox genes Msx-1 and Msx-2 and the proto-oncogene JunB, whereas c-jun transcripts displayed delayed albeit prolonged increase. Using differential display cDNA cloning, six direct BMP target genes were identified. These include Id3, which showed strong mRNA induction, and the moderately induced Cyr61, DEK, and eIF4AII genes, as well as a gene encoding a GC-binding protein. Besides Id3, also the Id1 and Id2 genes were activated by BMP4 in both ES cells and a range of different cell lines. Id genes encode negative regulators of basic helix-loop-helix transcription factors. In vivo we observed local ectopic expression of Id3 and Msx-2 mRNAs in Ft/+ embryos at overlapping regions of ectopic Bmp4 misexpression. We therefore propose that the Msx and Id genes are direct target genes of embryonic BMP4 signaling in vivo. (+info)
Immortalization of primary human keratinocytes by the helix-loop-helix protein, Id-1.
Basic helix-loop-helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix-loop-helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein. (+info)
Id-1 and Id-2 are overexpressed in pancreatic cancer and in dysplastic lesions in chronic pancreatitis.
Id proteins antagonize basic helix-loop-helix proteins, inhibit differentiation, and enhance cell proliferation. In this study we compared the expression of Id-1, Id-2, and Id-3 in the normal pancreas, in pancreatic cancer, and in chronic pancreatitis (CP). Northern blot analysis demonstrated that all three Id mRNA species were expressed at high levels in pancreatic cancer samples by comparison with normal or CP samples. Pancreatic cancer cell lines frequently coexpressed all three Ids, exhibiting a good correlation between Id mRNA and protein levels, as determined by immunoblotting with highly specific anti-Id antibodies. Immunohistochemistry using these antibodies demonstrated the presence of faint Id-1 and Id-2 immunostaining in pancreatic ductal cells in the normal pancreas, whereas Id-3 immunoreactivity ranged from weak to strong. In the cancer tissues, many of the cancer cells exhibited abundant Id-1, Id-2, and Id-3 immunoreactivity. Scoring on the basis of percentage of positive cells and intensity of immunostaining indicated that Id-1 and Id-2 were increased significantly in the cancer cells by comparison with the respective controls. Mild to moderate Id immunoreactivity was also seen in the ductal cells in the CP-like areas adjacent to these cells and in the ductal cells of small and interlobular ducts in CP. In contrast, in dysplastic and atypical papillary ducts in CP, Id-1 and Id-2 immunoreactivity was as significantly elevated as in the cancer cells. These findings suggest that increased Id expression may be associated with enhanced proliferative potential of pancreatic cancer cells and of proliferating or dysplastic ductal cells in CP. (+info)
The adenovirus E1A domains required for induction of DNA rereplication in G2/M arrested cells coincide with those required for apoptosis.
Induction of apoptosis by adenovirus E1A in rodent cells is stimulated by wild type (wt) p53 but completely suppressed by mutated p53. The suppression is overcome by coexpression with Id proteins (Ids). The cells expressing E1A and Ids undergo apoptosis after accumulation in S phase, suggesting that S phase events are perturbed by E1A and Ids. The E1A domains required for induction of apoptosis, analysed by transfection with expression vectors for E1A, Ids and their mutants, followed by flow cytometry, reside in N-terminal (positions 17 - 38), CR1 and CR2 regions. Interaction of E1A with Ids requires the N-terminal and CR1 regions. The cyclin D1 promoter activity in S phase was reduced severely by E1A and this reduction is caused through CR1 and CR2 regions required for interaction with pRB. Analysis of DNA synthesis in G2/M arrested cells indicated that E1A is capable of inducing >4 N cells and this E1A-mediated DNA rereplication is enhanced by coexpression with Id-1H. The E1A domains required for induction of DNA rereplication coincide with those required for apoptosis. (+info)
Massive apoptosis of thymocytes in T-cell-deficient Id1 transgenic mice.
Id1 is an inhibitor of a group of basic helix-loop-helix transcription factors, collectively called E proteins, which includes E12, E47, E2-2, and HEB. We have generated transgenic mice in which Id1 is specifically expressed in T cells. The total number of thymocytes in these mice is less than 4% of that in wild-type mice. The majority of the transgenic thymocytes are CD4 and CD8 double negative and bear the cell surface markers of multipotent progenitor cells. A small number of thymocytes, however, differentiate into CD4 or CD8 single-positive T cells, which also display different characteristics from their wild-type counterparts. More importantly, apoptotic cells constitute about 50% of the total thymocytes. These apoptotic thymocytes have rearranged their T-cell receptor genes, suggesting that they are differentiating T cells. This finding has raised the possibility that the T-cell deficiency in Id1 transgenic mice is the result of a massive apoptosis of differentiating T cells triggered by Id1 expression as opposed to a developmental block at the earliest progenitor stage. The progenitor cells accumulated in the transgenic mice might have survived because they are not susceptible to the apoptotic signals. Despite the massive cell death of the thymocytes at young ages, Id1 transgenic mice frequently develop T-cell lymphoma later in their life span, and lymphomagenesis appears to occur at different stages of T-cell development. Taken together, our data suggest that E proteins, being the targets of Id1, are essential regulators for normal T-cell differentiation and tumor suppression. (+info)
Functional antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) trans-factors for the regulation of fatty acid synthase promoter in adipocytes.
We show that Id (inhibitor of DNA binding) 2 and Id3, dominant negative members of the helix-loop-helix (HLH) family, interact with the adipocyte determination and differentiation factor 1 (ADD1)/sterol regulatory element-binding protein (SREBP) 1c, a transcription factor of the basic HLH-leucine zipper family that controls the expression of several key genes of adipose metabolism. Gel mobility-shift assays performed with in vitro-translated ADD1, Id2 or Id3 proteins and a fatty acid synthase (FAS) promoter oligonucleotide showed evidence for a marked inhibition of the formation of DNA-ADD1 complexes by Id2 or Id3 proteins. Co-immunoprecipitation studies using in vitro-translated proteins demonstrated further the physical interaction of Id and ADD1/SREBP-1c proteins in the absence of DNA. Using the FAS gene as a model of an ADD1-regulated promoter in transiently transfected isolated rat adipocytes or mature 3T3-L1 adipocytes, a potent inhibition of the activity of the FAS-chloramphenicol acetyltransferase reporter gene was observed by overexpression of Id2 or Id3. Reciprocally, co-transfection of Id3 antisense and ADD1 expression vectors in preadipocytes potentiated the ADD1/SREBP-1c effect on the FAS promoter activity. Finally, in the non adipogenic NIH-3T3 cell line, most of the ADD1-mediated trans-activation of the FAS promoter was counteracted by co-transfection of Id2 or Id3 expression vectors. Previous studies have indicated Id gene expression to be down-regulated during adipogenesis [Moldes, Lasnier, Feve, Pairault and Djian (1997) Mol. Cell. Biol. 17, 1796-1804]. We here demonstrated that there was a dramatic rise of Id2 and Id3 mRNA levels when 3T3-L1 adipocytes or isolated rat fat cells were exposed to lipolytic and anti-lipogenic agents, forskolin and isoproterenol. Taken together, our data show that Id products are functionally involved in modulating ADD1/SREBP-1c transcriptional activity, and thus lipogenesis in adipocytes. (+info)