New insights for Drosophila GAGA factor in larvae

GAGA factor plays important roles during Drosophila embryogenesis and its maternal contribution is essential for early development. Here, the role of GAGA factor was studied in 3rd instar larvae using depletion and overexpression conditions in wing disc and transcriptome analysis. We found that genes changing expression were different to those previously described using GAGA mutants in embryos. No apparent phenotypes on GAGA depletion could usually be observed at larval stages in imaginal discs but a strong effect on salivary gland polytene chromosomes was observed. In the adult, GAGA depletion produced many defects like abnormal cell proliferation in the wing, impaired dorsal closure and resulted in homeotic transformation of abdominal segment A5. Unexpectedly, no effects on Ultrabithorax expression were observed. Short overexpression of GAGA factor in 3rd instar larvae also resulted in activation of a set of genes not previously described to be under GAGA regulation, and in lethality at pupa. Our results suggest a little contribution of GAGA factor on gene transcription in wing discs and a change of the genes regulated in comparison with embryo. GAGA factor activity thus correlates with the global changes in gene expression that take place at the embryo-to-larva and, later, at the larva-to-pupa transitions.This work was supported by grants of the Ministerio de Educación y Ciencia of the Spanish Government (BFU-2007-64395/BMC), the MICINN (CSD2006-49 and BFU2009-07111) and the Generalitat de Catalunya (SGR2009-1023). M.B. was supported by I3P CPG_06_0034 contract from CSIC, and grant BFU-2007-64395/BMC contract of the Spanish Government. D.P. was supported by an FPU fellowship from the Spanish GovernmentPeer Reviewe

General, negative feedback mechanism for regulation of Trithorax-like gene expression in vivo: new roles for GAGA factor in flies

Expression of every gene is first regulated at the transcriptional level. While some genes show acute and discrete periods of expression others show a rather steady expression level throughout development. An example of the latter is Trithorax-like (Trl) a member of the Trithorax group that encodes GAGA factor in Drosophila. Among other functions, GAGA factor has been described to stimulate transcription of several genes, including some homeotic genes. Here we show that GAGA factor is continuously down-regulating the expression of its own promoter using a negative feedback mechanism in vivo. Like its expression, repression by GAGA factor is ubiquitous, prevents its accumulation, and takes place throughout development. Experimental alteration of GAGA factor dosage results in several unexpected phenotypes, not related to alteration of homeotic gene expression, but rather to functions that take place later during development and affect different morphogenetic processes. The results suggest that GAGA factor is essential during development, even after homeotic gene expression is established, and indicate the existence of an upper limit for GAGA factor dosage that should not be exceeded

High mobility group protein 1 interacts specifically with the core domain of human TATA box-binding protein and interferes with transcription factor IIB within the pre-initiation complex

This work was supported by Grants CHRX-CT94-0482 and FMRXCT970109 from the European Union and Grants PB96-0812 and UE96-0021 from the Spanish Comisión Interministerial de Ciencia y Tecnologı́a and was carried out within the framework of the "Centre de Referència en Biotecnologia" of the Generalitat de Cataluny

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

t(10;12)(q24;q15): A new cytogenetic marker in hematological malignancies

Cytogenetic studies have played a crucial role in the discovery of genes involved in several diseases. In the field of oncohematology, cytogenetics is still necessary for the classification and prognosis of many diseases. Here we report a new recurrent chromosome translocation, t(10;12)(q24;q15), in two patients with different hematological malignancies: myelodysplastic syndrome with excess blasts (MDS-EB), and myelofibrosis (MF) secondary to essential thrombocythemia (ET). The chromosome alteration was observed as a sole karyotype change in the patient with MDS-EB, both at the initial diagnosis and following progression to MDS-EB2. A putative HMGA2-KLLN rearrangement by RNA-sequencing was detected in this patient. The patient with ET, had a normal karyotype at diagnosis and the t(10;12)(q24;q15) translocation emerged as a sole cytogenetic alteration after transformation, and when MF was evident. We reviewed the literature to determine whether this chromosome abnormality had previously been described in other hematological patients and found two cases: an aggressive T-cell lymphoblastic lymphoma (T-LBL) and a case of transformed chronic myeloproliferative syndrome (CMS), in both of which t(10;12)(q24;q15) was also the only karyotype change. The clinical evolution of all four cases suggested that t(10;12)(q24;q15) is associated with a poor outcome in oncohematological patients

Lysine 27 dimethylation of Drosophila linker histone dH1 contributes to heterochromatin organization independently of H3K9 methylation

Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3–9, HP1a or Su(var)4–20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.MICIN/AEI 10.13039/501100011033 [BFU2015-65082-P and PGC2018-094538-B-100]; ‘FEDER, una manera de hacer Europa’; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; this work was carried out within the framework of the ‘Centre de Referencia en Biotecnologia’ of ` the Generalitat de Catalunya. Funding for open access charge: MINECO [PGC2018-094538-B-100]. Conflict of interest statement. None declared

HMGB1 interacts with many apparently unrelated proteins by recognizing short amino acid sequences

The chromatin high mobility group protein 1 (HMGB1) is a very abundant and conserved protein that is structured into two HMG box domains plus a highly acidic C-terminal domain. From the ability to bind DNA nonspecifically and to interact with various proteins, several functions in DNA-related processes have been assigned to HMGB1. Nevertheless, its functional role remains the subject of controversy. Using a phage display approach we have shown that HMGB1 can recognize several peptide motifs. A computer search of the protein data bases found peptide homologies with proteins already known to interact with HMGB1, like p53, and have allowed us to identify new potential candidates. Among them, transcriptional activators like the heterogeneous nuclear ribonucleoprotein K (hnRNP K), repressors like methyl-CpG binding protein 2 (MeCP2), and co-repressors like the retinoblastoma susceptibility protein (pRb) and Groucho-related gene proteins 1 (Grg1) and 5 (Grg5) can be found. A detailed analysis of the interaction of Grgl with HMGB1 confirmed that the binding region contained the sequence homologous to one of the peptides identified. Our results have led us to propose that HMGB1 may play a central role in the stabilization and/or assembly of several multifunctional complexes through protein-protein interactions.This work was supported by a European Union Grant FMRX-CT97-0109 and Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT) of the Generalitat de Catalunya Grant SGR97-55. This work was carried out in the context of the Centre de Referència en Biotecnologia of the CIRIT of the Generalitat de CatalunyaPeer Reviewe

Histone H1 at heterochromatin prevents R-loop formation

Trabajo presentado en las VIII Jornadas de Cromatina y Epigenética, de la Societat Catalana de Biologia, celebradas en Barcelona (España) el día 16 de marzo de 2018

Triple-stranded DNA

Capítulo en: Fritz Eckstein; David M. J. Lilley (eds.). Nucleic Acids and Molecular Biology. Berlin: Springer, 1995, p.1-21. (Nucleic Acids and Molecular Biology ; 9)The molecular biology of DNA is determined to a great extent by the chemical properties of its constitutive polynucleotide chains and, in particular, by the stability of the different complexes to which they can give rise. Most frequently, DNA is found in the form of an antiparallel doublestranded association but the formation of DNA complexes containing either three or four strands has also been extensively reported in the literature. The formation of a triple-stranded nucleic acid was first reported in 1957 in the case of the RNA triplex U(A · U) (Felsenfeld et al. 1957) and it was followed by the demonstration that the RNA homopolymers polyI and polyA could also form the triple-stranded helixes I(I · I) and I(A · I) (Rich 1958a,b). In the following decade, formation of triple-stranded conformations was also demonstrated for a variety of RNA and DNA homopolymers, as well as for RNA-DNA hybrids. Recently, triple-stranded DNA has received renewed attention. Most of the recent interest in triple-stranded DNA came after the discovery that DNA triplexes could also be intramolecular. In the case of an intramolecular triplex, the third strand, which associates to the double-stranded DNA fragment, is donated by the same DNA molecule. Intramolecular triplexes are therefore a source of DNA structural polymorphism, which adds to the known capability of the DNA molecule to exist under structurally different double-stranded conformations. On the other hand, in the case of an intermolecular triplex, the third strand is donated by a different DNA or RNA molecule, normally a singlestranded oligomer. Intermolecular triplexes provide a means for the specific recognition of double-stranded DNA by single-stranded DNA or RNA molecules. Here, after a brief introductory summary about the general aspects underlying the formation of triple-stranded DNA, we will review the recent progress on the study of the structural and functional properties of intra- and intermolecular DNA triplexesPeer reviewe

Caracterització funcional del factor GAGA de Drosophila melanogaster

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