Drosophila melanogaster linker histone dH1 is required for transposon silencing and to preserve genome integrity

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial LicenseHistone H1 is an intrinsic component of chromatin, whose important contribution to chromatin structure is well-established in vitro. Little is known, however, about its functional roles in vivo. Here, we have addressed this question in Drosophila, a model system offering many advantages since it contains a single dH1 variant. For this purpose, RNAi was used to efficiently deplete dH1 in flies. Expression-profiling showsthatdH1depletion affects expression of a relatively small number of genes in a regional manner. Furthermore, depletion up-regulates inactive genes, preferentially those located in heterochromatin, while active euchromatic genes are down-regulated, suggesting that the contribution of dH1 to transcription regulation is mainly structural, organizing chromatin for proper gene-expression regulation. Up-regulated genes are remarkably enriched in transposons. In particular, R1/R2 retrotransposons, which specifically integrate in the rDNA locus, are strongly up-regulated. Actually, depletion increases expression of transposon-inserted rDNA copies, resulting in synthesis of aberrant rRNAs and enlarged nucleolus. Concomitantly, dH1-depleted cells accumulate extra-chromosomal rDNA, show increased γH2Av content, stop proliferation and activate apoptosis, indicating that depletion causes genome instability and affects proliferation. Finally, the contributions to maintenance of genome integrity and cell proliferation appear conserved in human hH1s, as their expression rescues proliferation of dH1-depleted cells. © The Author(s) 2012. Published by Oxford University Press.MICINN (CSD2006-49 and BFU2009-07111); CSIC (200420E583 and 201120E001); Generalitat de Catalunya (SGR2009-1023); IRB fellowship (to O.V.). This work was carried out within the framework of the ‘Centre de Referència en Biotecnologia’ of the ‘Generalitat de Catalunya’. Funding for open access charge: MICINN.Peer Reviewe

Molecular mechanisms of gene activation and gene expression mediated by CCAAT/enhancer binding proteins

Der Transkriptionsfaktor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) koordiniert Proliferationshemmung und Differenzierung von myeloiden VorlŠuferzellen und Adipozyten. C/EBPa ist ein transkriptioneller Aktivator von abstammungspezifischen Genen und blockiert den Zellzyklus durch Repression von proliferationsfšrdernden E2F Zielgenen. Die hier gezeigten Daten zeigen, dass auch umgekehrt E2F die transkriptionelle und differenzierungsfšrdernde AktivitŠt von C/EBPa entgegenwirkt. Somit besitzen E2F-C/EBPa eine zentrale Schalterfunktion zwischen Proliferation und Differenzierung. Der Repressionsmechanismus durch E2F ist in mehreren Aspekten neuartig: Zum erstenmal wurde gezeigt, dass E2F einen anderen Transkriptionsfaktor reprimieren kann. E2F reprimiert die transkriptionelle AktivitŠt von C/EBPa ohne Bindung an cis-regulatorischen Elemente, sondern durch direkte Protein-Protein Interaktionen, die die Bindung von C/EBPa an DNA verhindern. Diese Form der transkriptionellen Repression geschieht unabhŠngig von "Pocket-Proteinen''". Patienten mit Akuter Myeloiden LeukŠmie (AML) weisen hŠufig eine gestšrte DNA Bindung von C/EBPa auf, welche ursachlich fŸr granulozitŠren Funktionsstšrungen sein kšnnte. Daher wŠre es wichtig zu analysieren ob E2F die DNA Bindung von C/EBPa in AML Patienten beeintrŠchtigt und ob auf E2F gerichtete Therapien granulozitŠre Reifung wiederherstellen. C/EBPa blockiert Zellproliferation durch vielseitigen Mechanismen. Hier wurde gezeigt, dass C/EBPa mit UBF1, dem Co-Aktivator der RNA Polymerase I, an chromosomalen Foci positioniert wird. Eine €hnlichkeit zu anderen fokalen Strukturen suggeriert, dass C/EBPa die Transkription von Polymerase I regulierten rRNA Gene reprimieren und somit ribosomale Biogenese beeintrŠchtigen kšnnte. Die Assoziation zwischen C/EBPa und UBF1 wird durch die Histon-Methyltransferase SUV39H1 stimuliert. Demnach kšnnte die antiproliferative Funktion von C/EBPa nicht nur auf der Regulierung von RNA Pol II-abhŠngiger Transkription, sondern auch auf der Repression von RNA Pol I regulierter rRNA Synthese basieren.The transcription factor CCAAT/Enhancer-Binding Protein alpha (C/EBPa) coordinates proliferation arrest and differentiation of myeloid progenitors and adipocytes. C/EBPa acts as a transcriptional activator of lineage specific genes and blocks the cell cycle by repressing transcription of E2F-regulated genes. Data presented here suggest that also inversely E2F interferes with the transcriptional activity of C/EBPa, counteracting C/EBPa-mediated differentiation processes. Thus, E2F-C/EBPa are part of a switch mechanism between proliferation and differentiation. The mechanism by which E2F suppresses C/EBPa-mediated transactivation is novel in several aspects. E2F acts as a co-repressor of another transcription factor, C/EBPa, without binding to cis-regulatory elements, but by direct protein-protein interactions that abolish the binding of C/EBPa to DNA. This mechanism of transcriptional repression occurs independent of pocket proteins. Disturbed DNA binding of C/EBPa is often observed in AML patients suggesting a causative role in granulocytic disorders. Thus, it would be of main interest to analyze whether E2F mediates disruption of C/EBPa''s DNA-binding in AML patients and whether therapies directed against E2F could restore granulocytic maturation. Despite the extensive knowledge of mechanisms involved in the inhibitory function of C/EBPa, it has not been addressed whether C/EBPa may impinge on cell proliferation by affecting the ribosomal biogenesis of a cell. This work demonstrates an association of C/EBPa to the RNA Pol I transcription factor UBF1, both proteins retained in large chromosomal foci. Similarities to other focal structures associated to UBF1, suggest that C/EBPa may repress transcription of Pol I-transcribed rRNA genes, and thus affect ribosomal biogenesis. The enrichment of C/EBPa at sites of UBF1 is induced by the histone methyltransferase SUV39H1. Thus, C/EBPa may not only control lineage commitment and cell proliferation by regulating genes transcribed by RNA Pol II, but also may act as a repressor of RNA Pol I mediated rRNA synthesis

Repression of Transcriptional Activity of C/EBPα by E2F-Dimerization Partner Complexes▿†

The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates proliferation arrest and the differentiation of myeloid progenitors, adipocytes, hepatocytes, keratinocytes, and cells of the lung and placenta. C/EBPα transactivates lineage-specific differentiation genes and inhibits proliferation by repressing E2F-regulated genes. The myeloproliferative C/EBPα BRM2 mutant serves as a paradigm for recurrent human C-terminal bZIP C/EBPα mutations that are involved in acute myeloid leukemogenesis. BRM2 fails to repress E2F and to induce adipogenesis and granulopoiesis. The data presented here show that, independently of pocket proteins, C/EBPα interacts with the dimerization partner (DP) of E2F and that C/EBPα-E2F/DP interaction prevents both binding of C/EBPα to its cognate sites on DNA and transactivation of C/EBP target genes. The BRM2 mutant, in addition, exhibits enhanced interaction with E2F-DP and reduced affinity toward DNA and yet retains transactivation potential and differentiation competence that becomes exposed when E2F/DP levels are low. Our data suggest a tripartite balance between C/EBPα, E2F/DP, and pocket proteins in the control of proliferation, differentiation, and tumorigenesis

A novel and essential mechanism determining specificity and activity of protein phosphatase 2A (PP2A) in vivo

Protein phosphatase 2A (PP2A) is an essential intracellular serine/threonine phosphatase containing a catalytic subunit that possesses the potential to dephosphorylate promiscuously tyrosine-phosphorylated substrates in vitro. How PP2A acquires its intracellular specificity and activity for serine/threonine-phosphorylated substrates is unknown. Here we report a novel and phylogenetically conserved mechanism to generate active phospho-serine/threonine-specific PP2A in vivo. Phosphotyrosyl phosphatase activator (PTPA), a protein of so far unknown intracellular function, is required for the biogenesis of active and specific PP2A. Deletion of the yeast PTPA homologs generated a PP2A catalytic subunit with a conformation different from the wild-type enzyme, as indicated by its altered substrate specificity, reduced protein stability, and metal dependence. Complementation and RNA-interference experiments showed that PTPA fulfills an essential function conserved from yeast to man

C/EBPβΔuORF mice—a genetic model for uORF-mediated translational control in mammals

Upstream ORFs (uORFs) are translational control elements found predominantly in transcripts of key regulatory genes. No mammalian genetic model exists to experimentally validate the physiological relevance of uORF-regulated translation initiation. We report that mice deficient for the CCAAT/enhancer-binding protein β (C/EBPβ) uORF initiation codon fail to initiate translation of the autoantagonistic LIP (liver inhibitory protein) C/EBPβ isoform. C/EBPβΔuORF mice show hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. These data and the widespread prevalence of uORFs in mammalian transcriptomes suggest a comprehensive role of uORF-regulated translation in (patho)physiology

Role for Histone Deacetylase 1 in Human Tumor Cell Proliferation▿

Posttranslational modifications of core histones are central to the regulation of gene expression. Histone deacetylases (HDACs) repress transcription by deacetylating histones, and class I HDACs have a crucial role in mouse, Xenopus laevis, zebra fish, and Caenorhabditis elegans development. The role of individual class I HDACs in tumor cell proliferation was investigated using RNA interference-mediated protein knockdown. We show here that in the absence of HDAC1 cells can arrest either at the G1 phase of the cell cycle or at the G2/M transition, resulting in the loss of mitotic cells, cell growth inhibition, and an increase in the percentage of apoptotic cells. On the contrary, HDAC2 knockdown showed no effect on cell proliferation unless we concurrently knocked down HDAC1. Using gene expression profiling analysis, we found that inactivation of HDAC1 affected the transcription of specific target genes involved in proliferation and apoptosis. Furthermore, HDAC2 downregulation did not cause significant changes compared to control cells, while inactivation of HDAC1, HDAC1 plus HDAC2, or HDAC3 resulted in more distinct clusters. Loss of these HDACs might impair cell cycle progression by affecting not only the transcription of specific target genes but also other biological processes. Our data support the idea that a drug targeting specific HDACs could be highly beneficial in the treatment of cancer