p33ING1b regulates acetylation of p53 in oral squamous cell carcinoma via SIR2.

BACKGROUND: Oral squamous cell carcinoma (OSCC), a form of head and neck squamous cell carcinoma (HNSCC) has a poor 5-year survival rate. OSCC patients are often treated with cisplatin but resistance to chemotherapy is often observed. This makes it important identification of alternative therapeutic targets which will result in more favorable outcome in OSCC patients. The plant homeodomain (PHD)-containing protein Inhibitor of Growth family of tumor suppressor proteins (p33ING1b) has been indicated as a tumor suppressor in different cancers including OSCC. This protein has been shown to function by modulating transcriptional activity of p53; however, the exact mechanism(s) are not well defined. METHODS: Expression of total and acetylated p53 and p33ING1b protein was determined in OSCC cell lines YD-9, YD-8, and YD-38 by immunoblot analysis. Effect of modulation of p33ING1b protein expression on acetylation of p53 and cell proliferation was determined by immunoblot and MTT assay. Effect of modulation of p33ING1b protein expression on transactivation of p53 was assessed by heterologous promoter-based reporter and chromatin immunoprecipitation. Effect of modulation of expression of p33ING1b on SIR2 mRNA and protein was determined by quantitative real-time PCR and immunoblot analyses. Impact of modulation of p33ING1b alone or in combination with SIR2 on chemosensitivity of YD-9 and YD-8 cells to cisplatin was determined in time and dose-dependent cell proliferation assays. RESULTS: Here, using a panel of OSCC cell lines with wild type or mutant p53, we show that p33ING1b expression is correlated to acetylation of p53 at lysine 382 residue. Increased acetylation of p53 following overexpression of p33ING1b was associated with increased expression of the pro-apoptotic proteins BAX, p21, and cleaved-Caspase 3, and decreased cell proliferation. Reporter assays with p21 and BAX promoters showed that p33ING1b expression levels directly correlated to promoter activity of these 2 genes. Chromatin immunoprecipitation assay showed that transcriptional regulation of p21 and BAX by acetylated p53 is dependent on expression level of p33ING1b. Differential acetylation of p53 following modulation of p33ING1b expression was indirect. Expression of p33ING1b was found to be inversely correlated to the NAD-dependent deacetylase silent information regulator 2 (SIR2). SIR2 was transcriptionally regulated by p33ING1b. Relative expression of p33ING1b was found to dictate chemosensitivity of OSCC cell lines to cisplatin treatment. Concomitant overexpression of p33ING1b and knockdown of SIR2 had a synergistic effect on chemosensitivity of OSCC cell lines to cisplatin, compared to either overexpression of p33ING1b or knockdown of SIR2 alone. CONCLUSIONS: The results from the current study thus elucidate that p33ING1b regulates p53 acetylation irrespective of p53 mutation and subsequent transactivation by transcriptional regulation of SIR2 expression. The results also indicate that p33ING1b and SIR2 are potentially attractive therapeutic targets.

Nuclear entrapment of p33ING1b by inhibition of exportin-1: A trigger of apoptosis in head and neck squamous cell cancer.

The effect of deregulation of nuclear export mediated by exportin-1, with consequent cellular mislocalization of p33ING1b, a member of the tumor suppressor gene family, has not been previously investigated in head and neck squamous cell cancer (HNSCC). We evaluated the effect of reversing cytoplasmic p33ING1b localization through inhibition of exportin-1 by leptomycin B (LMB) and the effect of nuclear entrapment of p33ING1b on molecular alterations in primary and metastatic HNSCC lines. The expression and location of exportin-1 and p33ING1b were analyzed by a quantitative real­time reverse transcription polymerase chain reaction PCR (qRT-PCR), a Western blot, and immunostaining. Cell proliferation and migration assays were conducted to determine the effect of exportin-1 inhibition on the cell lines. Exportin-1 was overexpressed in metastatic HNSCC, whereas p33ING1b was poorly expressed. Exportin-1 inhibition induced nuclear entrapment and upregulation of p33ING1b, extensive apoptosis, and growth arrest. It also suppressed cell migration. Cytoplasmic p33ING1b-mediated regulation of cell growth and nuclear entrapment of p33ING1b via inhibition of exportin-1 may be a key mechanism for inducing HNSCC apoptosis.

ING1 inhibits Twist1 expression to block EMT and is antagonized by the HDAC inhibitor vorinostat.

ING1 is a chromatin targeting subunit of the Sin3a histone deacetylase (HDAC) complex that alters chromatin structure to subsequently regulate gene expression. We find that ING1 knockdown increases expression of Twist1, Zeb 1&2, Snai1, Bmi1 and TSHZ1 drivers of EMT, promoting EMT and cell motility. ING1 expression had the opposite effect, promoting epithelial cell morphology and inhibiting basal and TGF-ß-induced motility in 3D organoid cultures. ING1 binds the Twist1 promoter and Twist1 was largely responsible for the ability of ING1 to reduce cell migration. Consistent with ING1 inhibiting Twist1 expression in vivo, an inverse relationship between ING1 and Twist1 levels was seen in breast cancer samples from The Cancer Genome Atlas (TCGA). The HDAC inhibitor vorinostat is approved for treatment of multiple myeloma and cutaneous T cell lymphoma and is in clinical trials for solid tumours as adjuvant therapy. One molecular target of vorinostat is INhibitor of Growth 2 (ING2), that together with ING1 serve as targeting subunits of the Sin3a HDAC complex. Treatment with sublethal (LD25-LD50) levels of vorinostat promoted breast cancer cell migration several-fold, which increased further upon ING1 knockout. These observations indicate that correct targeting of the Sin3a HDAC complex, and HDAC activity in general decreases luminal and basal breast cancer cell motility, suggesting that use of HDAC inhibitors as adjuvant therapies in breast cancers that are prone to metastasize may not be optimal and requires further investigation.

Inhibitor of Growth Factors Regulate Cellular Senescence.

The Inhibitor of Growth (ING) proteins are a group of tumor suppressors with five conserved genes. A common motif of ING factors is the conserved plant homeodomain (PHD), with which they bind to chromatin as readers of the histone mark trimethylated histone H3 (H3K4me3). These genes often produce several protein products through alternative splicing events. Interestingly, ING1 and ING2 participate in the establishment of the repressive mSIN3a-HDAC complexes, whereas ING3, ING4, and ING5 are associated with the activating HAT protein complexes. In addition to the modulation of chromatin's structure, they regulate cell cycle transition, cellular senescence, repair of DNA damage, apoptosis, and angiogenic pathways. They also have fundamental effects on regulating cellular senescence in cancer cells. In the current review, we explain their role in cellular senescence based on the evidence obtained from cell line and animal studies, particularly in the context of cancer.

CG7379 and ING1 suppress cancer cell invasion by maintaining cell-cell junction integrity.

Approximately 90% of cancer-related deaths can be attributed to a tumour's ability to spread. We have identified CG7379, the fly orthologue of human ING1, as a potent invasion suppressor. ING1 is a type II tumour suppressor with well-established roles in the transcriptional regulation of genes that control cell proliferation, response to DNA damage, oncogene-induced senescence and apoptosis. Recent work suggests a possible role for ING1 in cancer cell invasion and metastasis, but the molecular mechanism underlying this observation is lacking. Our results show that reduced expression of CG7379 promotes invasion in vivo in Drosophila, reduces the junctional localization of several adherens and septate junction components, and severely disrupts cell-cell junction architecture. Similarly, ING1 knockdown significantly enhances invasion in vitro and disrupts E-cadherin distribution at cell-cell junctions. A transcriptome analysis reveals that loss of ING1 affects the expression of several junctional and cytoskeletal modulators, confirming ING1 as an invasion suppressor and a key regulator of cell-cell junction integrity.

Altered endocytosis in cellular senescence.

Cellular senescence occurs in response to diverse stresses (e.g., telomere shortening, DNA damage, oxidative stress, oncogene activation). A growing body of evidence indicates that alterations in multiple components of endocytic pathways contribute to cellular senescence. Clathrin-mediated endocytosis (CME) and caveolae-mediated endocytosis (CavME) represent major types of endocytosis that are implicated in senescence. More recent research has also identified a chromatin modifier and tumor suppressor that contributes to the induction of senescence via altered endocytosis. Here, molecular regulators of aberrant endocytosis-induced senescence are reviewed and discussed in the context of their capacity to serve as senescence-inducing stressors or modifiers.

Antithetic hTERT Regulation by Androgens in Prostate Cancer Cells: hTERT Inhibition Is Mediated by the ING1 and ING2 Tumor Suppressors.

The human telomerase is a key factor during tumorigenesis in prostate cancer (PCa). The androgen receptor (AR) is a key drug target controlling PCa growth and regulates hTERT expression, but is described to either inhibit or to activate. Here, we reveal that androgens repress and activate hTERT expression in a concentration-dependent manner. Physiological low androgen levels activate, while, notably, supraphysiological androgen levels (SAL), used in bipolar androgen therapy (BAT), repress hTERT expression. We confirmed the SAL-mediated gene repression of hTERT in PCa cell lines, native human PCa samples derived from patients treated ex vivo, as well as in cancer spheroids derived from androgen-dependent or castration resistant PCa (CRPC) cells. Interestingly, chromatin immuno-precipitation (ChIP) combined with functional assays revealed a positive (pARE) and a negative androgen response element (nARE). The nARE was narrowed down to 63 bp in the hTERT core promoter region. AR and tumor suppressors, inhibitor of growth 1 and 2 (ING1 and ING2, respectively), are androgen-dependently recruited. Mechanistically, knockdown indicates that ING1 and ING2 mediate AR-regulated transrepression. Thus, our data suggest an oppositional, biphasic function of AR to control the hTERT expression, while the inhibition of hTERT by androgens is mediated by the AR co-repressors ING1 and ING2.

mRNA and protein of p33ING1 in normal and cancer tissues.

BACKGROUND: Inhibitor growth protein 1 (ING1) is a tumor suppressor, and its down-regulation is involved in the progression and aggressive phenotypes of human malignancies through its interactions with the H3K4me3 and p53. METHODS: We collected datasets to analyze the relationship between ING1b mRNA expression and accumulative survival rate, and carried out immunohistochemistry analyses to determine the expression profiles of the p33ING1 protein on the mouse, normal human, and human cancer tissue microarrays. RESULTS: Compared with normal tissues, the ING1b mRNA was highly expressed in various types of cancer tissues, including, colorectal, lung, and breast cancers, and was positively correlated with the overall survival rate of gastric cancer patients. In mouse tissues, the subcellular location of p33ING1 was frequently nuclear; however, it was occasionally cytoplasmic or nucleocytoplasmic. There was a positive detection in the neuron body, a part of glial cells, the glandular epithelium of the stomach, intestines, breast, hepatocytes, heart, skeletal muscle cells, the bronchial and alveolar epithelium, and nephric tubules. In human tissues, the p33ING1 protein, apart from its cytoplasmic distribution, was distributed in the nuclei of the tongue, esophagus, stomach, intestine, lung, trachea, skin, appendix, cervix, endometrium, ovary, and breast. p33ING1 immunoreactivity was strongly detected in the stomach, trachea, skin, cervix, and breast, while it was weak in the other tissues. The positive rate of p33ING1 was 41.0% in the tested cancer entities (489/1,194). In general, p33ING1 expression was restricted to only the cytoplasm for all cancers, whereas it was found in the nucleus of renal clear cells, ovarian and colorectal cancers. Among them, p33ING1 was expressed in more than half of squamous cell carcinomas derived from the esophagus and cervix, while it was rarely expressed in hepatocellular (21.0%) and renal clear cell carcinoma (19.4%). CONCLUSIONS: The findings suggest that p33ING1 might be participated in the repair and regeneration of organs or tissues the repair and regeneration of organs or tissue, and the carcinogenesis of the highly proliferative epithelium.

The ING1a model of rapid cell senescence.

Replicative capacity of normal human cells decreases as telomeric sequence is lost at each division. It is believed that when a subset of chromosomes reach a critically short length, an ATM-initiated and p53-mediated transcriptional response inhibits cell growth, promoting cell senescence. In addition to loss of telomeric sequence, senescence can be induced by other stresses including ionizing radiation, oxidative damage, chemical crosslinkers like the chemotherapeutic agent cisplatin, as well as overactivation of oncogenes and tumor suppressors. Our group found that the expression of an isoform of the INhibitor of Growth 1 gene called ING1a increases approximately 10-fold as fibroblasts approach senescence and that forced expression rapidly induces a senescent phenotype in primary diploid fibroblasts, epithelial and endothelial cells that resembles replicative senescence by most physical and biochemical measures. ING1a induces these changes through strongly inhibiting endocytosis to block mitogen signaling by inducing the expression of intersectin 2, a key scaffolding protein of the endosomal pathway. This, in turn increases the expression of Rb and of p57Kip2 and p16INK4a that serve to maintain Rb is an active, growth inhibitory state. The ING1a model is currently being used to better understand the mechanism(s) responsible for activating Rb to enforce the senescent state.

Exploiting ING2 Epigenetic Modulation as a Therapeutic Opportunity for Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) has been the leading cause of cancer-related death worldwide, over the last few decades. Survival remains extremely poor in the metastatic setting and, consequently, innovative therapeutic strategies are urgently needed. Inhibitor of Growth Gene 2 (ING2) is a core component of the mSin3A/Histone deacetylases complex (HDAC), which controls the chromatin acetylation status and modulates gene transcription. This gene has been characterized as a tumor suppressor gene and its status in cancer has been scarcely explored. In this review, we focused on ING2 and other mSin3A/HDAC member statuses in NSCLC. Taking advantage of existing public databases and known pharmacological properties of HDAC inhibitors, finally, we proposed a therapeutic model based on an ING2 biomarker-guided strategy.

Down-regulation of miR-500 and miR-628 suppress non-small cell lung cancer proliferation, migration and invasion by targeting ING1.

BACKGROUND: MicroRNAs (miRNAs) have been consistently demonstrated to be involved in non-small cell lung cancer (NSCLC) as either tumor oncogenes or tumor suppressors. However, the detailed role of miR-500 and miR-628 in NSCLC remain poorly understood. METHODS: The expressions of miR-500 and miR-628 in NSCLC tissues and cell lines were measured by quantitative real-time PCR (qRT-PCR). Cells migration, invasion, proliferation, adhesion and apoptosis abilities were test to analyze the biological functions of miR-500 and miR-628 in NSCLC. A bioinformatic analysis was conducted to predict the target genes regulated by miR-500 and miR-628 using TargetScan (http://www.targetscan.org/mamm/). Luciferase reporter assay was employed to validate the direct targeting of ING1 by miR-500 and miR-628. RESULTS: In this study, miR-500 and miR-628 were up-regulated with NSCLC tissues. Furthermore, inhibition of miR-500 and miR-628 significantly suppressed NSCLC cells proliferation, migration, invasion and adhesion, and induced NSCLC cells apoptosis. Additionally, the result showed that ING1 functioned as the direct target for miR-500 and miR-628, which was a core tumor suppressor in regulating NSCLC progression. Over-expression of ING1 could dramatically inhibit NSCLC cells proliferation, migration and invasion, and promote cells apoptosis. CONCLUSION: These results brought new insights into the oncogenic role of miR-500 and miR-628 in NSCLC, indicating that miR-500 and miR-628 might be the novel biomarkers for the diagnosis and prognosis of NSCLC.

A Functional Role for the Epigenetic Regulator ING1 in Activity-induced Gene Expression in Primary Cortical Neurons.

Epigenetic regulation of activity-induced gene expression involves multiple levels of molecular interaction, including histone and DNA modifications, as well as mechanisms of DNA repair. Here we demonstrate that the genome-wide deposition of inhibitor of growth family member 1 (ING1), which is a central epigenetic regulatory protein, is dynamically regulated in response to activity in primary cortical neurons. ING1 knockdown leads to decreased expression of genes related to synaptic plasticity, including the regulatory subunit of calcineurin, Ppp3r1. In addition, ING1 binding at a site upstream of the transcription start site (TSS) of Ppp3r1 depends on yet another group of neuroepigenetic regulatory proteins, the Piwi-like family, which are also involved in DNA repair. These findings provide new insight into a novel mode of activity-induced gene expression, which involves the interaction between different epigenetic regulatory mechanisms traditionally associated with gene repression and DNA repair.

The tumor suppressor ING1b is a novel corepressor for the androgen receptor and induces cellular senescence in prostate cancer cells.

The androgen receptor (AR) signaling is critical for prostate cancer (PCa) progression to the castration-resistant stage with poor clinical outcome. Altered function of AR-interacting factors may contribute to castration-resistant PCa (CRPCa). Inhibitor of growth 1 (ING1) is a tumor suppressor that regulates various cellular processes including cell proliferation. Interestingly, ING1 expression is upregulated in senescent primary human prostate cells; however, its role in AR signaling in PCa was unknown. Using a proteomic approach by surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) combined with immunological techniques, we provide here evidence that ING1b interacts in vivo with the AR. The interaction was confirmed by co-immunoprecipitation, in vitro GST-pull-down, and quantitative intracellular colocalization analyses. Functionally, ING1b inhibits AR-responsive promoters and endogenous key AR target genes in the human PCa LNCaP cells. Conversely, ING1b knockout (KO) mouse embryonic fibroblasts (MEFs) exhibit enhanced AR activity, suggesting that the interaction with ING1b represses the AR-mediated transcription. Also, data suggest that ING1b expression is downregulated in CRPCa cells compared with androgen-dependent LNCaP cells. Interestingly, its ectopic expression induces cellular senescence and reduces cell migration in both androgen-dependent and CRPCa cells. Intriguingly, ING1b can also inhibit androgen-induced growth in LNCaP cells in a similar manner as AR antagonists. Moreover, ING1b upregulates different cell cycle inhibitors including p27(KIP1), which is a novel target for ING1b. Taken together, our findings reveal a novel corepressor function of ING1b on various AR functions, thereby inhibiting PCa cell growth.

A novel crosstalk between the tumor suppressors ING1 and ING2 regulates androgen receptor signaling.

The androgen receptor (AR) is a transcriptional factor that has a pivotal role in the development of normal and also cancerous prostate. Therefore, analyzing AR signaling is essential to understand cancerogensis and proliferation of prostate cancer (PCa). Inhibitor of growth 1 (ING1) and ING2 are tumor suppressors with reduced expression in many cancer types. There are also indications of misregulation of ING1 and ING2 in PCa. However, the roles of ING1 and ING2 in PCa and AR signaling are poorly understood. Here, we show that surprisingly the ING1b knockdown (KD) represses AR-mediated transactivation on AR key target genes in the human LNCaP PCa cells. This is associated with growth reduction of LNCaP cells by ING1 KD. In line with this, using Ing1 knockout (KO) mice, we provide further evidence that ING1 deficiency downregulates prostate-specific AR target genes in vivo. Further analyses suggest that KD of ING1b results in induction of both cellular senescence and the cell cycle inhibitor p16 INK4a . The unexpected finding that the ING1 KD results in growth inhibition was further analyzed and can be explained by a compensatory mechanism through enhanced levels of ING2 protein in ING1-deficient condition. Accordingly, the data suggest that ING2 interacts with AR and hampers the AR transcriptional activation, causes growth arrest, and induces cellular senescence. The data further suggest that ING2 upregulates p16 INK4a , which is a novel target for ING2. Taken together, our data suggest that ING2 is a novel corepressor for AR. ING2 levels are increased upon downregulation of ING1 expression indicating a compensatory mechanism and suggests a novel crosstalk between ING1 and ING2 tumor suppressors to inhibit AR signaling and induce cellular senescence in PCa cells. KEY MESSAGE: • The tumor suppressors ING1 and 2 are dysregulated in human prostate cancer. • ING1 deficiency reduces AR-mediated gene expression in vitro and in vivo. • ING2, like ING1, inhibits AR-mediated transactivation and prostate cancer cell growth. • ING1 regulates ING2. • ING1 and ING2 crosstalk with each other to inhibit AR signaling in prostate cancer.

Ectopic expression of p33ING1b suppresses proliferation and induces apoptosis in colonic adenocarcinoma cells.

Inhibitor of growth 1b (ING1b) is considered to be a class II tumor suppressor gene. Although decreased expression of p33ING1b has previously been reported in colorectal cancer (CRC), its role in CRC has remained to be elucidated. The present study was designed to assess the function of p33ING1b in CRC and to further evaluate its underlying mechanisms of action. Western blot analysis confirmed that ING1b gene expression was significantly decreased in CRC tissues compared with that of adjacent non-tumorous colorectal tissues. Furthermore, recombinant adenovirus-mediated ectopic expression of p33ING1b resulted in growth inhibition, G1-phase cell cycle arrest and apoptosis in the SW480, HT29 and LoVo colorectal adenocarcinoma cell lines. The results suggested that the downregulation of ING1b contributes to colorectal carcinogenesis and that ectopic expression of ING1b may be a potentially useful therapeutic approach for CRC.

Aging with ING: a comparative study of different forms of stress induced premature senescence.

Cell senescence contributes to organismal aging and is induced by telomere erosion and an ensuing DNA damage signal as cells reach the end of their replicative lifespan in vitro or in vivo. Stresses induced by oncogene or tumor suppressor hyperactivation, oxidative stress, ionizing radiation and other DNA damaging agents result in forms of stress induced premature senescence (SIPS) that show similarities to replicative senescence. Since replicative senescence and SIPS occur over many days and many population doublings of the mass cultures of primary cells used to study senescence, the sequence of events that occur downstream of senescence signaling can be challenging to define. Here we compare a new model of ING1a-induced senescence with several other forms of senescence. The ING1a epigenetic regulator synchronously induces senescence in mass cultures several-fold faster than all other agents, taking 24 and 36 hours to activate the Rb/ p16INK4a, but not the p53 tumor suppressor axis to efficiently induce senescence. ING1a induces expression of intersectin 2, a scaffold protein necessary for endocytosis, altering the stoichiometry of endocytosis proteins, subsequently blocking growth factor uptake leading to activation of Rb signaling to block cell growth. ING1a acts as a novel link in the activation of the Rb pathway that can impose senescence in the absence of activating p53-mediated DNA damage signaling, and should prove useful in defining the molecular events contributing to Rb-induced senescence.

The ING tumor suppressor genes: status in human tumors.

ING genes (ING1-5) were identified has tumor suppressor genes. ING proteins are characterized as Type II TSGs since they are involved in the control of cell proliferation, apoptosis and senescence. They may also function as Type I TSGs since they are also involved in DNA replication and repair. Most studies have reported that they are frequently lost in human tumors and epigenetic mechanisms or misregulation of their transcription may be involved. Recently, studies have described that this loss may be caused by microRNA inhibition. Here, we summarize the current knowledge on ING functions, their involvement in tumor suppression and, in order to give a full assessment of the current knowledge, we review all the studies that have examined ING status in human cancers.

p33ING1b methylation in fecal DNA as a molecular screening tool for colorectal cancer and precancerous lesions.

The present study aimed to investigate the feasibility of detecting p33 inhibitor of growth 1b (p33ING1b) gene methylation in fecal DNA as a screening method for colorectal carcinoma (CRC) and precancerous lesions. The methylation of p33ING1b was analyzed in fecal samples from 61 patients with CRCs, 27 patients with precancerous lesions (advanced adenoma) and 20 normal individuals by nested methylation-specific polymerase chain reaction (nMSP) and fecal occult blood test. Methylated p33ING1b was detected in 73.77% of CRC patients and 62.96% of adenoma patients. By contrast, only 5% of normal individuals had methylated p33ING1b. These results indicated 73.77% sensitivity for detecting CRC, 62.96% sensitivity for detecting precancerous lesions and 95% specificity of the assay for detecting CRCs and precancerous lesions. The detection of p33ING1b methylation status by incubation of DNA contained in agarose beads for bisulfite modification, followed by nMSP, is a promising non-invasive screening method for CRCs and precancerous lesions.

Mammalian DNA demethylation: multiple faces and upstream regulation.

DNA cytosine methylation is a reversible epigenetic mark regulating gene expression. Aberrant methylation profiles are concomitant with developmental defects and cancer. Numerous studies in the past decade have identified enzymes and pathways responsible for active DNA demethylation both on a genome-wide as well as gene-specific scale. Recent findings have strengthened the idea that 5-methylcytosine oxidation catalyzed by members of the ten-eleven translocation (Tet1-3) oxygenases in conjunction with replication-coupled dilution of the conversion products causes the majority of genome-wide erasure of methylation marks during early development. In contrast, short and long patch DNA excision repair seems to be implicated mainly in gene-specific demethylation. Growth arrest and DNA damage-inducible protein 45 a (Gadd45a) regulates gene-specific demethylation within regulatory sequences of limited lengths raising the question of how such site specificity is achieved. A new study identified the protein inhibitor of growth 1 (Ing1) as a reader of the active chromatin mark histone H3 lysine 4 trimethylation (H3K4me3). Ing1 binds and directs Gadd45a to target sites, thus linking the histone code with DNA demethylation.