Biomedical subjects

Eric Kalkhoven

Publications and source records attributed to Eric Kalkhoven.

8 recordsLinked to original sources

PubMed2006

Human Ccr4-Not complex is a ligand-dependent repressor of nuclear receptor-mediated transcription.

The Ccr4-Not complex is a highly conserved regulator of mRNA metabolism. The transcription regulatory function of this complex in higher eukaryotes, however, is largely unexplored. Here we report that CNOT1, the large human subunit, represses the ligand-dependent transcriptional activation function of oestrogen receptor (ER) alpha. Promoter recruitment assays indicate that CNOT1 contains an intrinsic ability to mediate transcriptional repression. Furthermore, CNOT1 can interact with the ligand-binding domain of ERalpha in a hormone-dependent fashion and is recruited with other Ccr4-Not subunits to endogenous oestrogen-regulated promoters dependent on the presence of ligand. In addition, siRNA-mediated depletion of endogenous CNOT1 or other Ccr4-Not subunits in breast cancer cells results in deregulation of ERalpha target genes. Finally, CNOT1 interacts in a ligand-dependent manner with RXR and represses transcription mediated by several RXR heterodimers. These findings define a function for the human Ccr4-Not complex as a transcriptional repressor of nuclear receptor signalling that is relevant for the understanding of molecular pathways involved in cancer.

Cell Cycle Proteins↗

PubMed2006

MOZ-TIF2 alters cofactor recruitment and histone modification at the RARbeta2 promoter: differential effects of MOZ fusion proteins on CBP- and MOZ-dependent activators.

MOZ-TIF2 and MOZ-CBP are leukemogenic fusion proteins associated with therapy-induced acute myeloid leukemia. These proteins are thought to subvert normal gene expression in differentiating hematopoietic progenitor cells. We have previously shown that MOZ-TIF2 inhibits transcription by CREB-binding protein (CBP)/p300-dependent activators such as nuclear receptors and p53. Here we have shown that MOZ-TIF2 associates with the RARbeta2 promoter in vivo, resulting in altered recruitment of CBP/p300, aberrant histone modification, and down-regulation of the RARbeta2 gene. In contrast, MOZ-TIF2 up-regulated transcription mediated by the MOZ/MYST3-dependent activator AML1/RUNX1. Both wild type MOZ and MOZ-TIF2 were found to colocalize with AML1, and MOZ-TIF2 was recruited to an AML1 target promoter. A MOZ-CBP fusion protein showed similar functions to MOZ-TIF2 in that it inhibited retinoic acid receptor-mediated transcription but enhanced AML1 reporter activation. Although it contains almost the entire CBP sequence, MOZ-CBP does not appear to associate with PML bodies. In summary, our results indicate that leukemogenic MOZ fusion proteins have differential effects on the activities of CBP-dependent and MOZ-dependent activators because of their ability to alter cofactor recruitment and chromatin modification at target promoters.

Animals↗

PubMed2005

MOZ-TIF2 inhibits transcription by nuclear receptors and p53 by impairment of CBP function.

Chromosomal rearrangements associated with acute myeloid leukemia (AML) include fusions of the genes encoding the acetyltransferase MOZ or MORF with genes encoding the nuclear receptor coactivator TIF2, p300, or CBP. Here we show that MOZ-TIF2 acts as a dominant inhibitor of the transcriptional activities of CBP-dependent activators such as nuclear receptors and p53. The dominant negative property of MOZ-TIF2 requires the CBP-binding domain (activation domain 1 [AD1]), and coimmunoprecipitation and fluorescent resonance energy transfer experiments show that MOZ-TIF2 interacts with CBP directly in vivo. The CBP-binding domain is also required for the ability of MOZ-TIF2 to extend the proliferative potential of murine bone marrow lineage-negative cells in vitro. We show that MOZ-TIF2 displays an aberrant nuclear distribution and that cells expressing this protein have reduced levels of cellular CBP, leading to depletion of CBP from PML bodies. In summary, our results indicate that disruption of the normal function of CBP and CBP-dependent activators is an important feature of MOZ-TIF2 action in AML.

Acetyltransferases↗

PubMed2004

CBP and p300: HATs for different occasions.

The transcriptional coactivators CREB binding protein (CBP) and p300 are key regulators of RNA polymerase II-mediated transcription. Genetic alterations in the genes encoding these regulatory proteins and their functional inactivation have been linked to human disease. Findings in patients, knockout mice and cell-based studies indicate that the ability of these multidomain proteins to acetylate histones and other proteins is critical for many biological processes. Furthermore, despite their high degree of homology, accumulating evidence indicates that CBP and p300 are not completely redundant but also have unique roles in vivo. Recent studies suggest that these functional differences could be due to differential association with other proteins or differences in substrate specificity between these acetyltransferases. Inactivation of the acetyltransferase function of either CBP or p300 in various experimental systems will no doubt teach us more about the specific biological roles of these proteins. Given the wide range of human diseases in which CBP and/or p300 have been implicated, understanding the mechanisms that regulate their activity in vivo could help to develop novel approaches for the development of therapeutic strategies.

Acetyltransferases↗

PubMed2003

Loss of CBP acetyltransferase activity by PHD finger mutations in Rubinstein-Taybi syndrome.

Disruption of one copy of the human CREB binding protein (CBP or CREBBP) gene leads to the Rubinstein-Taybi syndrome (RTS), a developmental disorder characterized by retarded growth and mental functions, broad thumbs, broad big toes and typical facial abnormalities. The CREB binding protein (CBP) is an essential transcriptional coactivator for many different transcription factors. CBP has the intrinsic ability to acetylate histones and other proteins, which is regarded as an important step in transcription activation. In vitro studies have shown that this enzymatic activity critically depends on the integrity of a plant homeodomain (PHD)-type zinc finger in the HAT domain of CBP. We therefore investigated whether PHD finger mutations are present in RTS patients. Mutational analysis of 39 patients revealed eight novel heterozygous mutations in the HAT domain of CBP, one of which alters a conserved PHD finger amino acid (E1278K), while a second mutation deletes exon 22, which encodes the central region of the PHD finger. Functional analysis of these RTS-associated PHD finger mutants showed that they lacked in vitro acetyltransferase activity towards histones and CBP itself and displayed reduced coactivator function for the transcription factor CREB. Importantly, in EBV-transformed lymphoblastoid cells from the exon 22 deletion patient we found approximately 50% less endogenous CBP HAT activity. These findings therefore underscore the functional importance of the PHD finger in vivo and imply that reduction of CBP HAT activity, as exemplified here by disruption of the PHD finger, is sufficient to cause RTS.

Acetyltransferases↗

PubMed2003

Cascade of distinct histone modifications during collagenase gene activation.

Gene activation in eukaryotes requires chromatin remodeling, in part via histone modifications. To study the events at the promoter of a mitogen-inducible gene, we examined the induction of expression of the collagenase gene. It has been established that the collagenase gene can be activated by c-Jun and c-Fos and that the transcriptional coactivator p300 is involved in the activation. As expected, we found histone acetyltransferase activity at the collagenase promoter during activation. Interestingly, we also found histone methyltransferase and kinase activity. Strikingly, the first modification observed is methylation of histone H3 lysine 4, which correlates with the binding of the SET9 methyltransferase and the assembly of a complex consisting of c-Jun, c-Fos, TATA binding protein, and RNA polymerase II. The assembly of the preinitiation complex also shows an ordered binding of the acetyltransferase p300, the RSK2 kinase, and the SWI/SNF component Brg-1. Our results suggest that collagenase gene activation involves a dynamic recruitment of different factors and that in addition to acetylation, histone H3 lysine 4 di- and trimethylation and histone H3 serine 10 phosphorylation are important steps in the activation of this gene.

Acetyltransferases↗

PubMed2002

The PHD type zinc finger is an integral part of the CBP acetyltransferase domain.

Histone acetyltransferases (HATs) such as CBP and p300 are regarded as key regulators of RNA polymerase II-mediated transcription, but the critical structural features of their HAT modules remain ill defined. The HAT domains of CBP and p300 are characterized by the presence of a highly conserved putative plant homeodomain (PHD) (C4HC3) type zinc finger, which is part of the functionally uncharacterized cysteine-histidine-rich region 2 (CH2). Here we show that this region conforms to the PHD type zinc finger consensus and that it is essential for in vitro acetylation of core histones and the basal transcription factor TFIIE34 as well as for CBP autoacetylation. PHD finger mutations also reduced the transcriptional activity of the full-length CBP protein when tested on transfected reporter genes. Importantly, similar results were obtained on integrated reporters, which reflect a more natural chromatinized state. Taken together, our results indicate that the PHD finger forms an integral part of the enzymatic core of the HAT domain of CBP.

Acetylation↗

PubMed2002

Scaffold/matrix attachment region elements interact with a p300-scaffold attachment factor A complex and are bound by acetylated nucleosomes.

The transcriptional coactivator p300 regulates transcription by binding to proteins involved in transcription and by acetylating histones and other proteins. These transcriptional effects are mainly at promoter and enhancer elements. Regulation of transcription also occurs through scaffold/matrix attachment regions (S/MARs), the chromatin regions that bind the nuclear matrix. Here we show that p300 binds to the S/MAR binding protein scaffold attachment factor A (SAF-A), a major constituent of the nuclear matrix. Using chromatin immunoprecipitations, we established that both p300 and SAF-A bind to S/MAR elements in the transiently silent topoisomerase I gene prior to its activation at G(1) during cell cycle. This binding is accompanied by local acetylation of nucleosomes, suggesting that p300-SAF-A interactions at S/MAR elements of nontranscribed genes might poise these genes for transcription.

Acetylation↗