Protein S-glutathionylation stimulate adipogenesis by stabilizing C/EBPß in 3T3L1 cells.

Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) ß. Protein S-glutathionylation decreased the interaction of C/EBPß and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier E3 ligase that facilitates C/EBPß degradation. Experiments with truncated mutant C/EBPß demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPß. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPß. The C201S, C296S double-mutant C/EBPß prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPß, stabilizing and increasing C/EBPß protein levels.

Regulation of senescence and the SASP by the transcription factor C/EBPß.

Oncogene-induced senescence (OIS) serves as an important barrier to tumor progression in cells that have acquired activating mutations in RAS and other oncogenes. Senescent cells also produce a secretome known as the senescence-associated secretory phenotype (SASP) that includes pro-inflammatory cytokines and chemokines. SASP factors reinforce and propagate the senescence program and identify senescent cells to the immune system for clearance. The OIS program is executed by several transcriptional effectors that include p53, RB, NF-κB and C/EBPß. In this review, we summarize the critical role of C/EBPß in regulating OIS and the SASP. Post-translational modifications induced by oncogenic RAS signaling control C/EBPß activity and dimerization, and these alterations switch C/EBPß to a pro-senescence form during OIS. In addition, C/EBPß is regulated by a unique 3'UTR-mediated mechanism that restrains its activity in tumor cells to facilitate senescence bypass and suppression of the SASP.

An interaction between MKL1, BRG1, and C/EBPß mediates palmitate induced CRP transcription in hepatocytes.

Non-alcoholic steatohepatitis (NASH) is one of the most predominant disorders in metabolic syndrome. Induction of pro-inflammatory mediators in hepatocytes exposed to free fatty acids represents a hallmark event during NASH pathogenesis. C-reactive protein (CRP) is a prototypical pro-inflammatory mediator. In the present study, we investigated the mechanism by which megakaryocytic leukemia 1 (MKL1) mediates palmitate (PA) induced CRP transcription in hepatocytes. We report that over-expression of MKL1, but not MKL2, activated the CRP promoter whereas depletion or inhibition of MKL1 repressed the CRP promoter. MKL1 potentiated the induction of the CRP promoter activity by PA treatment. Importantly, MKL1 knockdown by siRNA or pharmaceutical inhibition by CCG-1423 attenuated the induction of endogenous CRP expression in hepatocytes. Similarly, primary hepatocytes isolated from wild type (WT) mice produced more CRP than those isolated from MKL1 deficient (KO) mice when stimulated with PA. Mechanistically, the sequence-specific transcription factor CCAAT-enhancer-binding protein (C/EBPß) interacted with MKL1 and recruited MKL1 to activate CRP transcription. Reciprocally, MKL1 modulated C/EBPß activity by recruiting the chromatin remodeling protein BRG1 to the CRP promoter to alter histone modifications. In conclusion, our data delineate a novel epigenetic mechanism underlying augmented hepatic inflammation during NASH pathogenesis.

Molecular cloning, characterisation, and expression patterns of pigeon CCAAT/enhancer binding protein-α and -ß genes.

1. CCAAT/enhancer binding proteins (C/EBPs), as a family of transcription factors, consists of six functionally and structurally related proteins which share a conserved basic leucine zipper (bZIP) DNA-binding domain. The aim of this study was to clone the full-length coding sequences (CDS) of C/EBP-α and -ß genes, and determine the abundance of these two genes in various tissues of white king pigeon (C. livia). 2. The complete cDNA sequences of C/EBP-α and -ß genes were cloned from pigeons by using PCR combined with rapid amplification of cDNA ends (RACE). The sequences were bioinformatically analysed, and the tissue distribution determined by quantitative real-time RT-PCR (qRT-PCR). 3. The results showed that the full-length cDNA sequences of pigeon C/EBP-α and -ß genes were 2,807bp and 1,778bp, respectively. The open reading frames of C/EBP-α (978 bp) and -ß (987bp) encoded 325 amino acids and 328 amino acids, respectively. The pigeon C/EBP-α and C/EBP-ß proteins were predicted to have a conserved basic leucine zipper (bZIP) domain, which is a common structure feature of the C/EBP family. Multiple sequence alignments indicated that pigeon C/EBP-α and -ß shared more than 90% amino-acid identity with their corresponding homologues in other avian species. Phylogenetic analysis revealed that these two proteins were highly conserved across different species and evolutionary processes. QRT-PCR results indicated that the pigeon C/EBP-α and -ß mRNA transcripts were expressed in all investigated organs. The mRNA expression levels of pigeon C/EBP-α in descending order, were in spleen, heart, liver, lung, kidney and muscle. The pigeon C/EBP-ß gene had the most abundant expression in lung, followed by the kidney, with minimal expression detected in muscle. 4. This study investigated the full-length cDNA sequences, genetic characteristics and tissue distribution of pigeon C/EBP-α and -ß genes and found that they may have functions in various tissues of pigeon. This provides a foundation for further study for regulatory mechanisms of these two genes in birds.

p300 Mediates Muscle Wasting in Lewis Lung Carcinoma.

C/EBPß is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBPß in the cancer milieu are poorly defined. Here, we report cancer-induced muscle wasting requires the transcriptional cofactor p300, which is critical for the activation of C/EBPß. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBPß in myotubes, particularly at its Lys39 residue. Overexpression of C/EBPß with mutated Lys39 impaired Lewis lung carcinoma (LLC)-induced activation of the C/EBPß-dependent catabolic response, which included upregulation of E3 ligases UBR2 and atrogin1/MAFbx, increased LC3-II, and loss of muscle proteins both in myotubes and mouse muscle. Silencing p300 in myotubes or overexpressing a dominant negative p300 mutant lacking acetyltransferase activity in mouse muscle attenuated LLC tumor-induced muscle catabolism. Administration of pharmacologic p300 inhibitor C646, but not PCAF/GCN5 inhibitor CPTH6, spared LLC tumor-bearing mice from muscle wasting. Furthermore, mice with muscle-specific p300 knockout were resistant to LLC tumor-induced muscle wasting. These data suggest that p300 is a key mediator of LLC tumor-induced muscle wasting whose acetyltransferase activity may be targeted for therapeutic benefit in this disease. SIGNIFICANCE: These findings demonstrate that tumor-induced muscle wasting in mice is abrogated by knockout, mutation of Lys39 or Asp1399, and pharmacologic inhibition of p300.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1331/F1.large.jpg.

Structural basis for effects of CpA modifications on C/EBPß binding of DNA.

CCAAT/enhancer binding proteins (C/EBPs) regulate gene expression in a variety of cells/tissues/organs, during a range of developmental stages, under both physiological and pathological conditions. C/EBP-related transcription factors have a consensus binding specificity of 5'-TTG-CG-CAA-3', with a central CpG/CpG and two outer CpA/TpG dinucleotides. Methylation of the CpG and CpA sites generates a DNA element with every pyrimidine having a methyl group in the 5-carbon position (thymine or 5-methylcytosine (5mC)). To understand the effects of both CpG and CpA modification on a centrally-important transcription factor, we show that C/EBPß binds the methylated 8-bp element with modestly-increased (2.4-fold) binding affinity relative to the unmodified cognate sequence, while cytosine hydroxymethylation (particularly at the CpA sites) substantially decreased binding affinity (36-fold). The structure of C/EBPß DNA binding domain in complex with methylated DNA revealed that the methyl groups of the 5mCpA/TpG make van der Waals contacts with Val285 in C/EBPß. Arg289 recognizes the central 5mCpG by forming a methyl-Arg-G triad, and its conformation is constrained by Val285 and the 5mCpG methyl group. We substituted Val285 with Ala (V285A) in an Ala-Val dipeptide, to mimic the conserved Ala-Ala in many members of the basic leucine-zipper family of transcription factors, important in gene regulation, cell proliferation and oncogenesis. The V285A variant demonstrated a 90-fold binding preference for methylated DNA (particularly 5mCpA methylation) over the unmodified sequence. The smaller side chain of Ala285 permits Arg289 to adopt two alternative conformations, to interact in a similar fashion with either the central 5mCpG or the TpG of the opposite strand. Significantly, the best-studied cis-regulatory elements in RNA polymerase II promoters and enhancers have variable sequences corresponding to the central CpG or reduced to a single G:C base pair, but retain a conserved outer CpA sequence. Our analyses suggest an important modification-dependent CpA recognition by basic leucine-zipper transcription factors.

A combined computational and experimental approach reveals the structure of a C/EBPß-Spi1 interaction required for IL1B gene transcription.

We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1ß, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPß) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPß leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPß DBDs, along with the C/EBPß C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPß-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPß's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPß to cognate DNA and in transcription of the C/EBPß-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.

Withaferin A, a natural compound with anti-tumor activity, is a potent inhibitor of transcription factor C/EBPß.

Recent work has shown that deregulation of the transcription factor Myb contributes to the development of leukemia and several other human cancers, making Myb and its cooperation partners attractive targets for drug development. By employing a myeloid Myb-reporter cell line we have identified Withaferin A (WFA), a natural compound that exhibits anti-tumor activities, as an inhibitor of Myb-dependent transcription. Analysis of the inhibitory mechanism of WFA showed that WFA is a significantly more potent inhibitor of C/EBPß, a transcription factor cooperating with Myb in myeloid cells, than of Myb itself. We show that WFA covalently modifies specific cysteine residues of C/EBPß, resulting in the disruption of the interaction of C/EBPß with the co-activator p300. Our work identifies C/EBPß as a novel direct target of WFA and highlights the role of p300 as a crucial co-activator of C/EBPß. The finding that WFA is a potent inhibitor of C/EBPß suggests that inhibition of C/EBPß might contribute to the biological activities of WFA.

Identification of a 5-Methylcytosine Site that may Regulate C/EBPß Binding and Determine Tissue-Specific Expression of the BPI Gene in Piglets.

Bactericidal/permeability-increasing protein (BPI) plays an important role in innate immune defense in mammals. A previous study showed that BPI gene expression correlates to gram-negative bacteria resistance. However, this gene showed tissue-specific expression in piglets and strongly expressed only in the digestive tract. To investigate the mechanisms governing the tissue-specificity, bisulfite sequencing PCR and next generation sequencing were used for high accuracy methylation quantitation of CpG islands of BPI gene upstream in 11 different tissues from weaned Yorkshire piglets. Additionally, qPCR was used to examine mRNA levels of BPI gene as well as transcription factor. We additionally analyzed transcriptional regulation by studying key 5-methylcytosine sites and transcription factors. Results showed that BPI mRNA levels significantly correlated with the overall methylation as well as methylation at mC-15 which was non-CpG site, no significant correlation could be found between the BPI and transcription factor mRNA levels, EMSA test showed that C/EBPß could interact with BPI wild-type promoter DNA, but not methylated DNA. So we confirmed that methylation of mC-15 residue could inhibit the ability of C/EBPß binding to the BPI promoter and affect the expression, and this mechanism probably plays a role in the tissue specificity of BPI gene expression in weaned piglets.

C/EBPß (CEBPB) protein binding to the C/EBP|CRE DNA 8-mer TTGC|GTCA is inhibited by 5hmC and enhanced by 5mC, 5fC, and 5caC in the CG dinucleotide.

During mammalian development, some methylated cytosines (5mC) in CG dinucleotides are iteratively oxidized by TET dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). The effect of these cytosine oxidative products on the sequence-specific DNA binding of transcription factors is being actively investigated. Here, we used the electrophoretic mobility shift assay (EMSA) to examine C/EBPα and C/EBPß homodimers binding to all 25 chemical forms of a CG dinucleotide for two DNA sequences: the canonical C/EBP 8-mer TTGC|GCAA and the chimeric C/EBP|CRE 8-mer TTGC|GTCA. 5hmC in the CG dinucleotide in the C/EBP|CRE motif 8-mer TGAC|GCAA inhibits binding of C/EBPß but not C/EBPα. Binding was increased by 5mC, 5fC and 5caC. Circular dichroism monitored thermal denaturations for C/EBPß bound to the C/EBP|CRE motif confirmed the EMSA. The structural differences between C/EBPα and C/EBPß that may account for the difference in binding 5hmC in the 8-mer TGAC|GCAA are explored.

The inflammation-related gene S100A12 is positively regulated by C/EBPß and AP-1 in pigs.

S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS) or porcine circovirus type 2 (PCV2). In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBPß) and activator protein-1 (AP-1) genes were up-regulated in PK-15 (ATCC, CCL-33) cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBPß or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA) to confirm that C/EBPß and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBPß and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBPß and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBPß and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.

The transcription factor CCAAT enhancer-binding protein ß protects rat cerebellar granule neurons from apoptosis through its transcription-activating isoforms.

CCAAT enhancer-binding protein ß is a transcription factor that is involved in many brain processes, although its role in neuronal survival/death remains unclear. By using primary cultures of rat cerebellar granule neurons, we have shown here that CCAAT enhancer-binding protein ß is present as all of its isoforms: the transcriptional activators liver activator proteins 1 and 2, and the transcriptional inhibitor liver inhibitory protein. We have also shown that liver activator protein 1 undergoes post-translational modifications, such as phosphorylation and sumoylation. These isoforms have different subcellular localizations, liver activator protein 2 being found in the cytosolic fraction only, liver inhibitory protein in the nucleus only, and liver activator protein 1 in both fractions. Through neuronal apoptosis induction by shifting mature cerebellar granule neurons to low-potassium medium, we have demonstrated that nuclear liver activator protein 1 expression decreases and its phosphorylation disappears, whereas liver inhibitory protein levels increase in the nuclear fraction, suggesting a pro-survival role for liver activator protein transcriptional activation and a pro-apoptotic role for liver inhibitory protein transcriptional inhibition. To confirm this, we transfected cerebellar granule neurons with plasmids expressing liver activator protein 1, liver activator protein 2, or liver inhibitory protein respectively, and observed that both liver activator proteins, which increase CCAAT-dependent transcription, but not liver inhibitory protein, counteracted apoptosis, thus demonstrating the pro-survival role of liver activator proteins. These data significantly improve our current understanding of the role of CCAAT enhancer-binding protein ß in neuronal survival/apoptosis.

Protein inhibitor of activated STAT 1 (PIAS1) is identified as the SUMO E3 ligase of CCAAT/enhancer-binding protein ß (C/EBPß) during adipogenesis.

It is well recognized that PIAS1, a SUMO (small ubiquitin-like modifier) E3 ligase, modulates such cellular processes as cell proliferation, DNA damage responses, and inflammation responses. Recent studies have shown that PIAS1 also plays a part in cell differentiation. However, the role of PIAS1 in adipocyte differentiation remains unknown. CCAAT/enhancer-binding protein ß (C/EBPß), a major regulator of adipogenesis, is a target of SUMOylation, but the E3 ligase responsible for the SUMOylation of C/EBPß has not been identified. The present study showed that PIAS1 functions as a SUMO E3 ligase of C/EBPß to regulate adipogenesis. PIAS1 expression was significantly and transiently induced on day 4 of 3T3-L1 adipocyte differentiation, when C/EBPß began to decline. PIAS1 was found to interact with C/EBPß through the SAP (scaffold attachment factor A/B/acinus/PIAS) domain and SUMOylate it, leading to increased ubiquitination and degradation of C/EBPß. C/EBPß became more stable when PIAS1 was silenced by RNA interference (RNAi). Moreover, adipogenesis was inhibited by overexpression of wild-type PIAS1 and promoted by knockdown of PIAS1. The mutational study indicated that the catalytic activity of SUMO E3 ligase was required for PIAS1 to restrain adipogenesis. Importantly, the inhibitory effect of PIAS1 overexpression on adipogenesis was rescued by overexpressed C/EBPß. Thus, PIAS1 could play a dynamic role in adipogenesis by promoting the SUMOylation of C/EBPß.

Interaction and cooperation of the CCAAT-box enhancer-binding protein ß (C/EBPß) with the homeodomain-interacting protein kinase 2 (Hipk2).

CCAAT box/enhancer-binding protein ß (C/EBPß) is a bZip transcription factor that plays crucial roles in important cellular processes such as differentiation and proliferation of specific cell types. Previously, we showed that C/EBPß cooperates with the coactivator p300 through a novel mechanism that involves the C/EBPß-induced phosphorylation of multiple sites in the carboxyl-terminal domain of p300 by protein kinase Hipk2. We have now examined the interaction and cooperation of C/EBPß, p300, and Hipk2 in more detail. We show that Hipk2 and C/EBPß are direct physical binding partners whose interaction is mediated by sequences located in the amino-terminal and central domains of Hipk2 and the amino-terminal part of C/EBPß. In addition to phosphorylating p300 recruited to C/EBPß, Hipk2 also phosphorylates C/EBPß at sites that have previously been shown to plays key roles in the regulation of C/EBPß activity. Silencing of Hipk2 expression disrupts adipocyte differentiation of 3T3-L1 cells, a physiological C/EBPß-dependent differentiation process indicating that the cooperation of C/EBPß and Hipk2 is functionally relevant. Finally, we demonstrate that C/EBPα, a related C/EBP family member whose amino-terminal sequences differ significantly from that of C/EBPß, is unable to interact and cooperate with Hipk2. Instead, our data suggest that C/EBPα cooperates with the protein kinase Jnk to induce phosphorylation of p300. Overall, our data identify Hipk2 as a novel regulator of C/EBPß and implicate different protein kinases in the cooperation of p300 with C/EBPß and C/EBPα.

Lymphoid to myeloid cell trans-differentiation is determined by C/EBPß structure and post-translational modifications.

The transcription factor C/EBPß controls differentiation, proliferation, and functionality of many cell types, including innate immune cells. A detailed molecular understanding of how C/EBPß directs alternative cell fates remains largely elusive. A multitude of signal-dependent post-translational modifications (PTMs) differentially affect the protean C/EBPß functions. In this study we apply an assay that converts primary mouse B lymphoid progenitors into myeloid cells in order to answer the question how C/EBPß regulates (trans-) differentiation and determines myeloid cell fate. We found that structural alterations and various C/EBPß PTMs determine the outcome of trans-differentiation of lymphoid into myeloid cells, including different types of monocytes/macrophages, dendritic cells, and granulocytes. The ability of C/EBPß to recruit chromatin remodeling complexes is required for the granulocytic trans-differentiation outcome. These novel findings reveal that PTMs and structural plasticity of C/EBPß are adaptable modular properties that integrate and rewire epigenetic functions to direct differentiation to diverse innate immune system cells, which are crucial for the organism survival.

CG methylated microarrays identify a novel methylated sequence bound by the CEBPB|ATF4 heterodimer that is active in vivo.

To evaluate the effect of CG methylation on DNA binding of sequence-specific B-ZIP transcription factors (TFs) in a high-throughput manner, we enzymatically methylated the cytosine in the CG dinucleotide on protein binding microarrays. Two Agilent DNA array designs were used. One contained 40,000 features using de Bruijn sequences where each 8-mer occurs 32 times in various positions in the DNA sequence. The second contained 180,000 features with each CG containing 8-mer occurring three times. The first design was better for identification of binding motifs, while the second was better for quantification. Using this novel technology, we show that CG methylation enhanced binding for CEBPA and CEBPB and inhibited binding for CREB, ATF4, JUN, JUND, CEBPD, and CEBPG. The CEBPB|ATF4 heterodimer bound a novel motif CGAT|GCAA 10-fold better when methylated. The electrophoretic mobility shift assay (EMSA) confirmed these results. CEBPB ChIP-seq data using primary female mouse dermal fibroblasts with 50× methylome coverage for each strand indicate that the methylated sequences well-bound on the arrays are also bound in vivo. CEBPB bound 39% of the methylated canonical 10-mers ATTGC|GCAAT in the mouse genome. After ATF4 protein induction by thapsigargin which results in ER stress, CEBPB binds methylated CGAT|GCAA in vivo, recapitulating what was observed on the arrays. This methodology can be used to identify new methylated DNA sequences preferentially bound by TFs, which may be functional in vivo.

Regulation of C/EBPß and resulting functions in cells of the monocytic lineage.

Monocyte/macrophages play an important role in orchestrating the immune response. The present review refers to C/EBPß, which is a key transcription factor regulating monocytic gene expression. Following a general introduction to C/EBPß, this article focuses on activators and regulators of the C/EBPß system in monocytic cells, including differentiating agents, cytokines, and bacterial products as well as associated signaling pathways. Furthermore, C/EBPß target genes in monocytic cells are summarized and resulting functions are described, including regulation of proliferation and differentiation as well as orchestration of processes of mainly the innate immune response. In addition, a variety of disease stages are described in which a dysregulation of the C/EBPß system may be involved. A detailed knowledge of the C/EBPß system in monocytic cells may help to further understand the difference between inflammatory and malignant proliferation as well as additional regulatory facets of innate immunity.

Human CCAAT/enhancer-binding protein ß interacts with chromatin remodeling complexes of the imitation switch subfamily.

Transcription factor C/EBPß is involved in several cellular processes, such as proliferation, differentiation, and energy metabolism. This factor exerts its activity through recruitment of different proteins or protein complexes, including the ATP-dependent chromatin remodeling complex SWI/SNF. The C/EBPß protein is found as three major isoforms, C/EBPß1, -2, and -3. They are generated by translation at alternative AUG initiation codons of a unique mRNA, C/EBPß1 being the full-length isoform. It has been found that C/EBPß1 participates in terminal differentiation processes. Conversely, C/EBPß2 and -3 promote cell proliferation and are involved in malignant progression in a number of tissues. The mechanisms by which C/EBPß2 and -3 promote cell proliferation and tumor progression are not fully understood. In this work, we sought to identify proteins interacting with hC/EBPß using a proteomics approach. We found that all three isoforms interact with hSNF2H and hACF, components of ACF and CHRAC chromatin remodeling complexes, which belong to the imitation switch subfamily. Additional protein-protein interaction studies confirmed this finding and also showed that hC/EBPß directly interacts with hACF1. By overexpressing hC/EBPß, hSNF2H, and hACF1 in HepG2 cells and analyzing variations in expression of cyclin D1 and other C/EBPß target genes, we observed a functional interaction between C/EBPß and SNF2H/ACF1, characterized mainly by suppression of C/EBPß transactivation activity in the presence of SNF2H and ACF1. Consistent with these findings, induction of differentiation of HepG2 cells by 1% DMSO was accompanied by a reduction in the level of cyclin D1 expression and the appearance of hC/EBPß, hSNF2H, and hACF1 on the promoter region of this gene.

Regulatory evolution through divergence of a phosphoswitch in the transcription factor CEBPB.

There is an emerging consensus that gene regulation evolves through changes in cis-regulatory elements and transcription factors. Although it is clear how nucleotide substitutions in cis-regulatory elements affect gene expression, it is not clear how amino-acid substitutions in transcription factors influence gene regulation. Here we show that amino-acid changes in the transcription factor CCAAT/enhancer binding protein-ß (CEBPB, also known as C/EBP-ß) in the stem-lineage of placental mammals changed the way it responds to cyclic AMP/protein kinase A (cAMP/PKA) signalling. By functionally analysing resurrected ancestral proteins, we identify three amino-acid substitutions in an internal regulatory domain of CEBPB that are responsible for the novel function. These amino-acid substitutions reorganize the location of key phosphorylation sites, introducing a new site and removing two ancestral sites, reversing the response of CEBPB to GSK-3ß-mediated phosphorylation from repression to activation. We conclude that changing the response of transcription factors to signalling pathways can be an important mechanism of gene regulatory evolution.

C/EBPß-Thr217 phosphorylation signaling contributes to the development of lung injury and fibrosis in mice.

BACKGROUND: Although C/EBPß(ko) mice are refractory to Bleomycin-induced lung fibrosis the molecular mechanisms remain unknown. Here we show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-ß on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice. METHODOLOGY/PRINCIPAL FINDINGS: Mice expressing the non-phosphorylatable C/EBPß-Ala217 transgene had a marked reduction in lung injury on day-13 after Bleomycin exposure, compared to C/EBPß(wt) mice, judging by the decrease of CD68(+) activated monocytes/macrophages, bone marrow-derived CD45(+) cells and lung cytokines as well as by the normal surfactant protein-C expression by lung pneumocytes. On day-21 after Bleomycin treatment, C/EBPß(wt) mice but not mice expressing the dominant negative C/EBPß-Ala217 transgene developed severe lung fibrosis as determined by quantitative collagen assays. All mice were of identical genetic background and back-crossed to the parental wild-type inbreed FVB mice for at least ten generations. Treatment of C/EBPß(wt) mice with a cell permeant, C/EBPß peptide that inhibits phosphorylation of C/EBPß on Thr217 (40 µg instilled intracheally on day-2 and day-6 after the single Bleomycin dose) also blocked the progression of lung injury and fibrosis induced by Bleomycin. Phosphorylation of human C/EBPß on Thr266 (human homologue phosphoacceptor) was induced in collagen-activated human lung fibroblasts in culture as well as in activated lung fibroblasts in situ in lungs of patients with severe lung fibrosis but not in control lungs, suggesting that this signaling pathway may be also relevant in human lung injury and fibrosis. CONCLUSIONS/SIGNIFICANCE: These data suggest that the RSK-C/EBPß phosphorylation pathway may contribute to the development of lung injury and fibrosis.