Small Number Of Transcription Factors Research Articles

Interference with Sin3 function induces epigenetic reprogramming and differentiation in breast cancer cells

Sin3A/B is a master transcriptional scaffold and corepressor that plays an essential role in the regulation of gene transcription and maintenance of chromatin structure, and its inappropriate recruitment has been associated with aberrant gene silencing in cancer. Sin3A/B are highly related, large, multidomian proteins that interact with a wide variety of transcription factors and corepressor components, and we examined whether disruption of the function of a specific domain could lead to epigenetic reprogramming and derepression of specific subsets of genes. To this end, we selected the Sin3A/B-paired amphipathic alpha-helices (PAH2) domain based on its established role in mediating the effects of a relatively small number of transcription factors containing a PAH2-binding motif known as the Sin3 interaction domain (SID). Here, we show that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction of a SID decoy that interferes with PAH2 binding to SID-containing partner proteins reverted the silencing of genes involved in cell growth and differentiation. In particular, the SID decoy led to epigenetic reprogramming and reexpression of the important breast cancer-associated silenced genes encoding E-cadherin, estrogen receptor alpha, and retinoic acid receptor beta and impaired tumor growth in vivo. Interestingly, the SID decoy was effective in the triple-negative M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) breast cancer cell line, restoring sensitivity to 17beta-estradiol, tamoxifen, and retinoids. Therefore, the development of small molecules that can block interactions between PAH2 and SID-containing proteins offers a targeted epigenetic approach for treating this type of breast cancer that may also have wider therapeutic implications.

Abstract 572: Interference with Sin3 PAH-2 domain function induces epigenetic reprogramming, differentiation and growth inhibition in breast cancer cells

Abstract Our recent work in understanding silenced retinoic acid response genes in breast cancer led us to explore the role of transcription repressor complexes in gene silencing in breast cancer. To this end we constructed a set of tagged vectors that contain a specific MAD1 motif called SID (mSin3A interaction domain), which binds with high affinity to block the function of the Sin3. Sin3A/B serve as multisubunit co-repressor scaffold protein that regulate gene transcription by recruiting histone deacetylase and histone demethylase activities to sequence-specific transcriptional repressors which are aberrant in breast cancer. The PAH2 domain of Sin3A/B binds with high affinity to a small number of transcription factors, and offers a more specific epigenetic target which contributes to the development of breast cancer. PAH-2 domain a specific component of a transcriptional repressor complex that plays an important role in modulating a small number of transcription factors containing the Sin3 PAH-2 interaction domain (SID). Here we demonstrated that in both human and mouse breast cancer cells, the targeted disruption of Sin3 function by introduction with their partners by the expression of SID transcript or peptide decoy interfered with PAH2 binding to SID-containing partner proteins as measured by co-immunoprecipitation and mammalian two-hybrid assays, reverteds the silencing of several genes involved in cell growth and differentiation. We observed that the SID decoy induced clear signs of differentiation in both human and mouse breast cancer cellsIn particular, the which include theSID decoys led to acinar morphogenesis in 3D cultures, increased adherence to collagen type-IV and laminin, reduced invasive phenotype and impaired tumor growth in vivo (>75%). This was associated with epigenetic reprogramming characterized by a marked increase in H3K4 2/3 methylation and a modest increase in H3 acetylation in the promoter region, promoter DNA demethylation and re-expression of the important breast cancer-associated silenced genes encoding E-cadherin, and, estrogen receptor α (ERα) and retinoic acid receptor β (RARβ)and impairment in tumor growth in vivo. There was increased expression of E-cadherin, CRBP1 and p27 known RAR response genes. The re-expression of ERα and RARβ in the “triple negative” MDA-MB-231 breast cancer cell line is functional since there was significant growth inhibition by tamoxifen after stimulation with 17b-estradiol and RAR activation by atRA and AM580. Therefore, the development of small molecules that mimic the 13 amino acid SID peptide and block interactions between PAH2 and SID-containing proteins This offers a new novel approach for treating this type of breast cancer and may also provide wider therapeutic implicationstriple negative breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 572.

Coordination of Lipid Metabolism in Membrane Biogenesis

Bilayer synthesis during membrane biogenesis involves the concerted assembly of multiple lipid species, requiring coordination of the level of lipid synthesis, uptake, turnover, and subcellular distribution. In this review, we discuss some of the salient conclusions regarding the coordination of lipid synthesis that have emerged from work in mammalian and yeast cells. The principal instruments of global control are a small number of transcription factors that target a wide range of genes encoding enzymes that operate in a given metabolic pathway. Critical in mammalian cells are sterol regulatory element binding proteins (SREBPs) that stimulate expression of genes for the uptake and synthesis of cholesterol and fatty acids. From work with Saccharomyces cerevisiae, much has been learned about glycerophospholipid and ergosterol regulation through Ino2p/Ino4p and Upc2p transcription factors, respectively. Lipid supply is fine-tuned through a multitude of negative feedback circuits initiated by both end products and intermediates of lipid synthesis pathways. Moreover, there is evidence that the diversity of membrane lipids is maintained through cross-regulatory effects, whereby classes of lipids activate the activity of enzymes operating in another metabolic branch.

Adult mice generated from induced pluripotent stem cells

Recent landmark experiments have shown that transient overexpression of a small number of transcription factors can reprogram differentiated cells into induced pluripotent stem (iPS) cells that resemble embryonic stem (ES) cells. These iPS cells hold great promise for medicine because they have the potential to generate patient-specific cell types for cell replacement therapy and produce in vitro models of disease, without requiring embryonic tissues or oocytes. Although current iPS cell lines resemble ES cells, they have not passed the most stringent test of pluripotency by generating full-term or adult mice in tetraploid complementation assays, raising questions as to whether they are sufficiently potent to generate all of the cell types in an organism. Whether this difference between iPS and ES cells reflects intrinsic limitations of direct reprogramming is not known. Here we report fertile adult mice derived entirely from iPS cells that we generated by inducible genetic reprogramming of mouse embryonic fibroblasts. Producing adult mice derived entirely from a reprogrammed fibroblast shows that all features of a differentiated cell can be restored to an embryonic level of pluripotency without exposure to unknown ooplasmic factors. Comparing these fully pluripotent iPS cell lines to less developmentally potent lines may reveal molecular markers of different pluripotent states. Furthermore, mice derived entirely from iPS cells will provide a new resource to assess the functional and genomic stability of cells and tissues derived from iPS cells, which is important to validate their utility in cell replacement therapy and research applications.

Structural Mechanism of Signal Transduction between the RNA-binding Domain and the Phosphotransferase System Regulation Domain of the LicT Antiterminator

LicT belongs to a family of bacterial transcriptional antiterminators, which control the expression of sugar-metabolizing operons in response to phosphorylations by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Previous studies of LicT have revealed the structural basis of RNA recognition by the dimeric N-terminal co-antiterminator (CAT) domain on the one hand and the conformational changes undergone by the duplicated regulation domain (PRD1 and PRD2) upon activation on the other hand. To investigate the mechanism of signal transduction between the effector and regulation modules, we have undertaken the characterization of a fragment, including the CAT and PRD1 domains and the linker in-between. Comparative experiments, including RNA binding assays, NMR spectroscopy, limited proteolysis, analytical ultracentrifugation, and circular dichroism, were conducted on native CAT-PRD1 and on a constitutively active CAT-PRD1 mutant carrying a D99N substitution in PRD1. We show that in the native state, CAT-PRD1 behaves as a rather unstable RNA-binding deficient dimer, in which the CAT dimer interface is significantly altered and the linker region is folded as a trypsin-resistant helix. In the activated mutant form, the CAT-PRD1 linker becomes protease-sensitive, and the helix content decreases, and the CAT module adopts the same dimeric conformation as in isolated CAT, thereby restoring the affinity for RNA. From these results, we propose that a helix-to-coil transition in the linker acts as the structural relay triggered by the regulatory domain for remodeling the effector dimer interface. In essence, the structural mechanism modulating the LicT RNA antitermination activity is thus similar to that controlling the DNA binding activity of dimeric transcriptional regulators.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

Reconstructing a Network of Stress-Response Regulators via Dynamic System Modeling of Gene Regulation

Unicellular organisms such as yeasts have evolved mechanisms to respond to environmental stresses by rapidly reorganizing the gene expression program. Although many stress-response genes in yeast have been discovered by DNA microarrays, the stress-response transcription factors (TFs) that regulate these stress-response genes remain to be investigated. In this study, we use a dynamic system model of gene regulation to describe the mechanism of how TFs may control a gene's expression. Then, based on the dynamic system model, we develop the Stress Regulator Identification Algorithm (SRIA) to identify stress-response TFs for six kinds of stresses. We identified some general stress-response TFs that respond to various stresses and some specific stress-response TFs that respond to one specific stress. The biological significance of our findings is validated by the literature. We found that a small number of TFs is probably sufficient to control a wide variety of expression patterns in yeast under different stresses. Two implications can be inferred from this observation. First, the response mechanisms to different stresses may have a bow-tie structure. Second, there may be regulatory cross-talks among different stress responses. In conclusion, this study proposes a network of stress-response regulators and the details of their actions.

Universal patterns of purifying selection at noncoding positions in bacteria

To investigate the dependence of the number of regulatory sites per intergenic region on genome size, we developed a new method for detecting purifying selection at noncoding positions in clades of related bacterial genomes. We comprehensively quantified evidence of purifying selection at noncoding positions across bacteria and found several striking universal patterns. Consistent with selection acting at transcriptional regulatory elements near the transcription start, we find a universal positional profile of selection with respect to gene starts and ends, showing most evidence of selection immediately upstream and least immediately downstream from genes. A further set of universal features indicates that selection for translation initiation efficiency is the major determinant of the sequence composition around translation start in all clades. In addition to a peak in selection at ribosomal binding sites, the region immediately around translation start shows a universal pattern of high adenine frequency, significant selection at silent positions, and avoidance of RNA secondary structure. Surprisingly, although the number of transcription factors (TF) increases quadratically with genome size, we present several lines of evidence that small and large genomes have the same average number of regulatory sites per intergenic region. By comparing the sequence diversity of the most and least conserved DNA words in intergenic regions across clades we provide evidence that the structure of transcription regulatory networks changes dramatically with genome size: Small genomes have a small number of TFs with a large number of target sites, whereas large genomes have a large number of TFs with a small number of target sites each.

Factor analysis for gene regulatory networks and transcription factor activity profiles.

BackgroundMost existing algorithms for the inference of the structure of gene regulatory networks from gene expression data assume that the activity levels of transcription factors (TFs) are proportional to their mRNA levels. This assumption is invalid for most biological systems. However, one might be able to reconstruct unobserved activity profiles of TFs from the expression profiles of target genes. A simple model is a two-layer network with unobserved TF variables in the first layer and observed gene expression variables in the second layer. TFs are connected to regulated genes by weighted edges. The weights, known as factor loadings, indicate the strength and direction of regulation. Of particular interest are methods that produce sparse networks, networks with few edges, since it is known that most genes are regulated by only a small number of TFs, and most TFs regulate only a small number of genes.ResultsIn this paper, we explore the performance of five factor analysis algorithms, Bayesian as well as classical, on problems with biological context using both simulated and real data. Factor analysis (FA) models are used in order to describe a larger number of observed variables by a smaller number of unobserved variables, the factors, whereby all correlation between observed variables is explained by common factors. Bayesian FA methods allow one to infer sparse networks by enforcing sparsity through priors. In contrast, in the classical FA, matrix rotation methods are used to enforce sparsity and thus to increase the interpretability of the inferred factor loadings matrix. However, we also show that Bayesian FA models that do not impose sparsity through the priors can still be used for the reconstruction of a gene regulatory network if applied in conjunction with matrix rotation methods. Finally, we show the added advantage of merging the information derived from all algorithms in order to obtain a combined result.ConclusionMost of the algorithms tested are successful in reconstructing the connectivity structure as well as the TF profiles. Moreover, we demonstrate that if the underlying network is sparse it is still possible to reconstruct hidden activity profiles of TFs to some degree without prior connectivity information.

Identifying Stress Transcription Factors Using Gene Expression and TF-Gene Association Data

Unicellular organisms such as yeasts have evolved to survive environmental stresses by rapidly reorganizing the genomic expression program to meet the challenges of harsh environments. The complex adaptation mechanisms to stress remain to be elucidated. In this study, we developed Stress Transcription Factor Identification Algorithm (STFIA), which integrates gene expression and TF-gene association data to identify the stress transcription factors (TFs) of six kinds of stresses. We identified some general stress TFs that are in response to various stresses, and some specific stress TFs that are in response to one specific stress. The biological significance of our findings is validated by the literature. We found that a small number of TFs may be sufficient to control a wide variety of expression patterns in yeast under different stresses. Two implications can be inferred from this observation. First, the adaptation mechanisms to different stresses may have a bow-tie structure. Second, there may exist extensive regulatory cross-talk among different stress responses. In conclusion, this study proposes a network of the regulators of stress responses and their mechanism of action.

STAT3 as a central mediator of neoplastic cellular transformation

Much of the focus in understanding the molecular pathogenesis of tumors has centered on kinases that are activated in cancer. However, cancers driven by a diversity of activated kinases may have very similar pathological and clinical properties. This likely relates to the fact that the biological characteristics of a tumor are driven by the pattern of gene expression in that tumor, and that a wide spectrum of activating events at the cell surface and in the cytoplasm converge on a relatively small number of transcription factors that regulate the expression of key target genes. One transcription factor that has been found to be activated inappropriately in a wide range of human cancers is STAT3. STAT3 target genes are involved in fundamental events of tumor development including proliferation, survival, self-renewal, invasion, and angiogenesis. Furthermore, there is strong evidence that STAT3 is critical for these processes, in that inhibition of STAT3 by a variety of means can exert an anti-cancer effect. Since normal cells are relatively tolerant of interruption in STAT3 signaling, these findings suggest that STAT3 may also be an excellent target for the molecular therapy of cancer.

Comprehensive Analysis of Combinatorial Regulation using the Transcriptional Regulatory Network of Yeast

Studies on various model systems have shown that a relatively small number of transcription factors can set up strikingly complex spatial and temporal patterns of gene expression. This is achieved mainly by means of combinatorial or differential gene regulation, i.e. regulation of a gene by two or more transcription factors simultaneously or under different conditions. While a number of specific molecular details of the mechanisms of combinatorial regulation have emerged, our understanding of the general principles of combinatorial regulation on a genomic scale is still limited. In this work, we approach this problem by using the largest assembled transcriptional regulatory network for yeast. A specific network transformation procedure was used to obtain the co-regulatory network describing the set of all significant associations among transcription factors in regulating common target genes. Analysis of the global properties of the co-regulatory network suggested the presence of two classes of regulatory hubs: (i) those that make many co-regulatory associations, thus serving as integrators of disparate cellular processes; and (ii) those that make few co-regulatory associations, and thereby specifically regulate one or a few major cellular processes. Investigation of the local structure of the co-regulatory network revealed a significantly higher than expected modular organization, which might have emerged as a result of selection by functional constraints. These constraints probably emerge from the need for extensive modular backup and the requirement to integrate transcriptional inputs of multiple distinct functional systems. We then explored the transcriptional control of three major regulatory systems (ubiquitin signaling, protein kinase and transcriptional regulation systems) to understand specific aspects of their upstream control. As a result, we observed that ubiquitin E3 ligases are regulated primarily by unique transcription factors, whereas E1 and E2 enzymes share common transcription factors to a much greater extent. This suggested that the deployment of E3s unique to specific functional contexts may be mediated significantly at the transcriptional level. Likewise, we were able to uncover evidence for much higher upstream transcription control of transcription factors themselves, in comparison to components of other regulatory systems. We believe that the results presented here might provide a framework for testing the role of co-regulatory associations in eukaryotic transcriptional control.

Chipping away at the Embryonic Stem Cell Network

Critical transcription factors, notably OCT4, SOX2, and NANOG, are necessary to maintain self-renewal and pluripotency, two properties characteristic of embryonic stem (ES) cells. By analyzing the genome-wide localization of these factors at promoter regions in human ES cells, Boyer et al. (2005) demonstrate frequent promoter co-occupancy at numerous target genes. As they discuss in this issue of Cell, their findings indicate the presence of a complex network of autoregulatory and feedforward loops in human ES cells.

Identifying regulatory networks by combinatorial analysis of promoter elements.

Several computational methods based on microarray data are currently used to study genome-wide transcriptional regulation. Few studies, however, address the combinatorial nature of transcription, a well-established phenomenon in eukaryotes. Here we describe a new approach using microarray data to uncover novel functional motif combinations in the promoters of Saccharomyces cerevisiae. In addition to identifying novel motif combinations that affect expression patterns during the cell cycle, sporulation and various stress responses, we observed regulatory cross-talk among several of these processes. We have also generated motif-association maps that provide a global view of transcription networks. The maps are highly connected, suggesting that a small number of transcription factors are responsible for a complex set of expression patterns in diverse conditions. This approach may be useful for modeling transcriptional regulatory networks in more complex eukaryotes.

Studies on Neisseria gonorrhoeae cultured in liquid medium.

Experiments were carried out to determine the minimum amount of inoculum of gonococci that is needed to produce growth in ANM liquid medium. This was found to be less than had previously been thought. Minimum inocula were used to study penicillin sensitivity in liquid medium. This work showed that growth occurred at higher concentrations of penicillin with larger inocula. Evidence was also obtained which suggested that in some circumstances penicillin acts bacteriostatically rather than bacteriocidally. It was also shown that growth of gonococci can be inhibited in liquid medium by the presence of hyperimmune serum, the inhibition being more marked in the presence of fresh complement. This work is continuing in the hope that it may provide a basis for a much-needed serological test for gonorrhoea. This would be especially valuable in chronic assymptomatic infections.