Promoters Of Specific Target Genes Research Articles

Threonine Phosphorylation of IκBζ Mediates Inhibition of Selective Proinflammatory Target Genes

Transcription factors of the NF-κB family play a crucial role for immune responses by activating the expression of chemokines, cytokines, and antimicrobial peptides involved in pathogen clearance. IκBζ, an atypical nuclear IκB protein and selective coactivator of particular NF-κB target genes, has recently been identified as an essential regulator for skin immunity. This study discovered that IκBζ is strongly induced in keratinocytes that sense the fungal glucan zymosan A. Additionally, IκBζ is essential for the optimal expression of proinflammatory genes, such as IL6, CXCL5, IL1B, or S100A9. Moreover, this study found that IκBζ was not solely regulated on the transcriptional level but also by phosphorylation events. This study identified several IκBζ phosphorylation sites, including a conserved cluster of threonine residues located in the N-terminus of the protein, which can be phosphorylated by MAPKs. Surprisingly, IκBζ phosphorylation at this threonine cluster promoted the recruitment of histone deacetylase 1 to specific target gene promoters and, thus, negatively controlled transcription. Taken together, this study proposes a model of how an antifungal response translates to the expression of proinflammatory cytokines and highlights an additional layer of complexity in the regulation of the NF-κB responses in keratinocytes.

Host proteome linked to HPV E7-mediated specific gene hypermethylation in cancer pathways

BackgroundHuman papillomavirus (HPV) infection causes around 90% of cervical cancer cases, and cervical cancer is a leading cause of female mortality worldwide. HPV-derived oncoprotein E7 participates in cervical carcinogenesis by inducing aberrant host DNA methylation. However, the targeting specificity of E7 methylation of host genes is not fully understood but is important in the down-regulation of crucial proteins of the hallmark cancer pathways. In this study, we aim to link E7-driven aberrations in the host proteome to corresponding gene promoter hypermethylation events in the hope of providing novel therapeutic targets and biomarkers to indicate the progression of cervical cancer.MethodsHEK293 cells were transfected with pcDNA3.1-E7 plasmid and empty vector and subjected to mass spectrometry-based proteomic analysis. Down-regulated proteins (where relative abundance was determined significant by paired T-test) relevant to cancer pathways were selected as gene candidates for mRNA transcript abundance measurement by qPCR and expression compared with that in SiHa cells (HPV type 16 positive). Methylation Specific PCR was used to determine promoter hypermethylation in genes downregulated in both SiHa and transfected HEK293 cell lines. The FunRich and STRING databases were used for identification of potential regulatory transcription factors and the proteins interacting with transcription factor gene candidates, respectively.ResultsApproximately 400 proteins totally were identified in proteomics analysis. The transcripts of six genes involved in the host immune response and cell proliferation (PTMS, C1QBP, BCAP31, CDKN2A, ZMYM6 and HIST1H1D) were down-regulated, corresponding to proteomic results. Methylation assays showed four gene promoters (PTMS, C1QBP, BCAP31 and CDKN2A) were hypermethylated with 61, 55.5, 70 and 78% increased methylation, respectively. Those four genes can be regulated by the GA-binding protein alpha chain, specificity protein 1 and ETS-like protein-1 transcription factors, as identified from FunRich database predictions.ConclusionsHPV E7 altered the HEK293 proteome, particularly with respect to proteins involved in cell proliferation and host immunity. Down-regulation of these proteins appears to be partly mediated via host DNA methylation. E7 possibly complexes with the transcription factors of its targeting genes and DNMT1, allowing methylation of specific target gene promoters.

Apoptosis in human myelodysplastic syndrome CD34+ cells is modulated by the upregulation of TLRs and histone H4 acetylation via a β-arrestin 1 dependent mechanism

Excessive apoptosis of hematopoietic precursors in the bone marrow underlies the ineffective hematopoiesis characteristic of myelodysplastic syndrome (MDS). Toll-like receptor (TLR) signaling is abnormally activated in MDS and may be involved in excessive programmed cell death in the pathogenesis of MDS. TLRs expression and global histone H3/H4 acetylation were analyzed in bone marrow (BM) CD34+ cells from 20 lower-risk and 20 higher-risk MDS patients and 10 healthy controls. Apoptosis of BM CD34+ cells was examined by flow cytometry, and its correlation to histone acetylation and the expression of TLR2 and β-arrestin1 (β-arr1), measured by enzyme-linkedimmunosorbent assay and qRT-PCR, was assessed. TLR1, TLR2 and TLR6 expression and H4 acetylation levels were higher in lower-risk MDS patients than in higher-risk MDS patients or controls, and TLR2 expression and H4 acetylation levels were positively correlated with an increased rate of apoptosis. Lower-risk MDS was associated with increased β-arr1 expression and histone acetyltransferase p300 activity. In in vitro-cultured primary normal and lower-risk MDS CD34+ cells, TLR2 activation-induced apoptosis was mediated by the upregulation of β-arr1 leading to the recruitment of p300 and increased histone H4 acetylation. The nuclear accumulation of βarr1 following TLR2 activation promote H4 acetylation at specific target gene promoters and may thus affect transcription of target genes in BM CD34+ cells. The mechanisms underlying the deregulation of TLR2 and increased apoptosis in MDS may involve the β-arr1 mediated recruitment of p300 leading to increased levels of histone H4 acetylation.

Specific phosphorylation of histone demethylase KDM3A determines target gene expression in response to heat shock.

Author SummaryHistone methylation regulates gene expression and can have drastic consequences for health if the process is defective. Histone lysine demethylases (KDMs) counteract the activity of methyl-transferases and remove methyl group(s) from histones. KDM3A is a H3K9me2/1 demethylase that performs diverse functions via the regulation of its target genes, which are involved in spermatogenesis, metabolism, and cell differentiation. However, the mechanisms underlying KDM3A regulation of specific genes at specific times are largely unknown. Here we found that a physiological stress—elevated temperature—induces KDM3A phosphorylation in human cells via the MSK1 kinase. This phosphorylated form of KDM3A directly interacts with the transcription factor Stat1, which enables Stat1 to recruit KDM3A to Stat1-binding sequences at the promoters of specific target genes. KDM3A then acts to demethylate H3K9me2/1 at these targets, thereby causing specific gene expression in response to the thermal stress. We conclude that heat shock can affect the expression of many genes in human cells via a novel activation mechanism that is centered around the phosphorylation of KDM3A.

Minireview: Challenges and opportunities in development of PPAR agonists.

The clinical impact of the fibrate and thiazolidinedione drugs on dyslipidemia and diabetes is driven mainly through activation of two transcription factors, peroxisome proliferator-activated receptors (PPAR)-α and PPAR-γ. However, substantial differences exist in the therapeutic and side-effect profiles of specific drugs. This has been attributed primarily to the complexity of drug-target complexes that involve many coregulatory proteins in the context of specific target gene promoters. Recent data have revealed that some PPAR ligands interact with other non-PPAR targets. Here we review concepts used to develop new agents that preferentially modulate transcriptional complex assembly, target more than one PPAR receptor simultaneously, or act as partial agonists. We highlight newly described on-target mechanisms of PPAR regulation including phosphorylation and nongenomic regulation. We briefly describe the recently discovered non-PPAR protein targets of thiazolidinediones, mitoNEET, and mTOT. Finally, we summarize the contributions of on- and off-target actions to select therapeutic and side effects of PPAR ligands including insulin sensitivity, cardiovascular actions, inflammation, and carcinogenicity.

Peroxisome Proliferator-activated Receptor γ Agonists Induce Cell Cycle Arrest through Transcriptional Regulation of Krüppel-like Factor 4 (KLF4)

Peroxisome proliferator-activated receptor γ (PPARγ), a subgroup of ligand-activated nuclear receptors, plays critical roles in cell cycle regulation, differentiation, apoptosis, and invasion. PPARγ is involved in tumorigenesis and is a potent target for cancer therapy. PPARγ transactivation of KLF4 has been demonstrated in various studies; however, how PPARγ regulates KLF4 expression is not clear. In this study, we reveal that PPARγ regulates the expression of KLF4 by binding directly to the PPAR response element (PPRE) within the KLF4 promoter. The PPRE resides at -1657 to -1669 bp upstream of the KLF4 ATG codon, which is essential for the transactivation of troglitazone-induced KLF4 expression. Furthermore, we found that stable silencing of KLF4 obviously suppressed the G(1)/S arrest and anti-proliferation effects induced by PPARγ ligands. Taken together, our data indicate that up-regulation of KLF4 upon PPARγ activation is mediated through the PPRE in the KLF4 promoter, thus providing further insights into the PPARγ signal transduction pathway as well as a novel cancer therapeutic strategy.

Characterization of a new cancer-associated mutant of p53 with a missense mutation (K351N) in the tetramerization domain

Inactivation of the tumour suppressor p53 is central to carcinogenesis and acquisition of resistance to drug-induced apoptosis. The majority of alterations are missense mutations and occur within the DNA-binding domain. However, little is known about the point mutations in the tetramerization domain (TD). Here we investigated the properties of a new p53 mutant (Lys 351 to Asn) in the TD identified in a cisplatin-resistant ovarian carcinoma cell line (A2780 CIS). We found that K351N substitution significantly reduces the thermodynamic stability of p53 tetramers without affecting the overall half-life of the protein. Moreover, p53 K351N has a reduced ability to bind DNA and to trans-activate its specific target gene promoters, such as bax. Data obtained from the analysis of p53 subcellular localization revealed that K351N mutation inhibits the nuclear export of p53 and accumulation in the cytoplasm induced by cisplatin treatment. These results identify p53 K351N as a new cancer associated mutant with reduced tumour suppressor activity and altered functions in response to apoptotic stimuli.

Regulation of Dendritic Development by Neuron-Specific Chromatin Remodeling Complexes

The diversity of dendritic patterns is one of the fundamental characteristics of neurons and is in part regulated by transcriptional programs initiated by electrical activity. We show that dendritic outgrowth requires a family of combinatorially assembled, neuron-specific chromatin remodeling complexes (nBAF complexes) distinguished by the actin-related protein BAF53b and based on the Brg/Brm ATPases. nBAF complexes bind tightly to the Ca(2+)-responsive dendritic regulator CREST and directly regulate genes essential for dendritic outgrowth. BAF53b is not required for nBAF complex assembly or the interaction with CREST, yet is required for their recruitment to the promoters of specific target genes. The highly homologous BAF53a protein, which is a component of neural progenitor and nonneural BAF complexes, cannot replace BAF53b's role in dendritic development. Remarkably, we find that this functional specificity is conferred by the actin fold subdomain 2 of BAF53b. These studies suggest that the genes encoding the individual subunits of BAF complexes function like letters in a ten-letter word to produce biologically specific meanings (in this case dendritic outgrowth) by combinatorial assembly of their products.

Long-distance combinatorial linkage between methylation and acetylation on histone H3 N termini.

Individual posttranslational modifications (PTMs) on histones have well established roles in certain biological processes, notably transcriptional programming. Recent genomewide studies describe patterns of covalent modifications, such as H3 methylation and acetylation at promoters of specific target genes, or "bivalent domains," in stem cells, suggestive of a possible combinatorial interplay between PTMs on the same histone. However, detection of long-range PTM associations is often problematic in antibody-based or traditional mass spectrometric-based analyses. Here, histone H3 from a ciliate model was analyzed as an enriched source of transcriptionally active chromatin. Using a recently developed mass spectrometric approach, combinatorial modification states on single, long N-terminal H3 fragments (residues 1-50) were determined. The entire modification status of intact N termini was obtained and indicated correlations between K4 methylation and H3 acetylation. In addition, K4 and K27 methylation were identified concurrently on one H3 species. This methodology is applicable to other histones and larger polypeptides and will likely be a valuable tool in understanding the roles of combinatorial patterns of PTMs.

Gfi1b alters histone methylation at target gene promoters and sites of γ-satellite containing heterochromatin

Gfi1b is a 37 kDa nuclear protein with six C2H2 zinc-finger domains that can silence transcription upon binding to specific target gene promoters. Here we show by using a chromatin immunoprecipitation and cloning protocol that Gfi1b also binds to gamma-satellite sequences that mainly occur in pericentric heterochromatin. Immuno-FISH experiments demonstrated that Gfi1b is localized at foci of pericentric heterochromatin identified by DAPI staining. Elevated levels of Gfi1b correlated with increased histone H3 lysine 9 dimethylation at sites neighboring gamma-satellite sequences but also at Gfi1b target gene promoters. In Gfi1b-deficient cells, however, a decrease of histone H3 lysine 9 trimethylation and a loss of heterochromatic structures was observed. Strikingly, we found that Gfi1b binds to both SUV39H1 and G9A histone methyl transferases, which provides a direct link between histone methylation and Gfi1b at heterochromatic and euchromatic sites. We propose that Gfi1b functions in heterochromatin formation and silencing of euchromatic transcription by recruiting histone methyl transferases to either gamma-satellite sequences or specific target gene promoters.

Regulation of coactivator complex assembly and function by protein arginine methylation and demethylimination

Nuclear receptors activate transcription by recruiting multiple coactivators to the promoters of specific target genes. The functional synergy of the p160 coactivators [steroid receptor coactivator-1, glucocorticoid receptor interacting protein (GRIP1), or the activator for thyroid hormone and retinoid receptors], the histone acetyltransferases cAMP response element binding protein binding protein (CBP) and p300 and the histone methyltransferase coactivator-associated arginine methyltransferase (CARM1) depends on the methyltransferase activity of CARM1. CARM1 methylates histone H3 and other factors including the N-terminal region of p300. Here, we report that CARM1 also methylates Arg-2142 within the C-terminal GRIP1 binding domain (GBD) of p300. In the GBD, both Arg-2088 and Arg-2142 are important for binding GRIP1. Methylation of Arg-2142 inhibits the bimolecular interaction of GRIP1 to p300 in vitro and in vivo. This methylation mark of p300 GBD is removed by peptidyl deiminase 4, thereby enhancing the p300-GRIP1 interaction. These methylation and demethylimination events also alter the conformation and activity of the coactivator complex and regulate estrogen receptor-mediated transcription, and they thus represent unique mechanisms for regulating coactivator complex assembly, conformation, and function.

Integration of anteroposterior and dorsoventral regulation of Phox2b transcription in cranial motoneuron progenitors by homeodomain proteins.

Little is known about the molecular mechanisms that integrate anteroposterior (AP) and dorsoventral (DV) positional information in neural progenitors that specify distinct neuronal types within the vertebrate neural tube. We have previously shown that in ventral rhombomere (r)4 of Hoxb1 and Hoxb2 mutant mouse embryos, Phox2b expression is not properly maintained in the visceral motoneuron progenitor domain (pMNv), resulting in a switch to serotonergic fate. Here, we show that Phox2b is a direct target of Hoxb1 and Hoxb2. We found a highly conserved Phox2b proximal enhancer that mediates rhombomere-restricted expression and contains separate Pbx-Hox (PH) and Prep/Meis (P/M) binding sites. We further show that both the PH and P/M sites are essential for Hox-Pbx-Prep ternary complex formation and regulation of the Phox2b enhancer activity in ventral r4. Moreover, the DV factor Nkx2.2 enhances Hox-mediated transactivation via a derepression mechanism. Finally, we show that induction of ectopic Phox2b-expressing visceral motoneurons in the chick hindbrain requires the combined activities of Hox and Nkx2 homeodomain proteins. This study takes an important first step to understand how activators and repressors, induced along the AP and DV axes in response to signaling pathways, interact to regulate specific target gene promoters, leading to neuronal fate specification in the appropriate developmental context.

Functional roles of STAT family proteins: lessons from knockout mice.

STAT (signal transducers and activators of transcription) proteins are activated in response to a large number of cytokines, growth factors, and hormones. Upon activation following the binding of ligands to their receptors, STAT proteins dimerize, translocate to the nucleus, and bind to the promoters of specific target genes. To date, seven mammalian members of the STAT family have been identified. Although some cytokines and growth factors can activate multiple STAT proteins, some STATs are activated with considerable specificity. The physiological role of each individual STAT protein is now being examined through the study of "knockout" mice, harboring a null allele for the particular gene. STAT1-deficient mice exhibit a selective signaling defect in response to interferons. STAT4 and STAT6 are essential for Thl-and Th2-responses, respectively. STAT5a-deficient mice exhibit defective mammary gland development. A study of STAT5b-deficient mice indicates that STAT5b mediates the sexually dimorphic effects of growth hormone in the liver. STAT5a and 5b also play different biological roles in the immune system. STAT3-deficient mice die during early embryogenesis, but the role of STAT3 in adult tissues can be assessed by utilizing the CreloxP recombination system to ablate the gene later in life. Analyses of tissue-specific STAT3-deficient mice indicate that STAT3 plays a crucial role in a variety of biological functions, including cell growth, suppression of apoptosis, and cell motility.