Oxidative Stress Is Associated with Overgrowth in Drosophila l(3)mbt Mutant Imaginal Discs

The loss-of-function conditions for an l(3)malignant brain tumour (l(3)mbt) in larvae reared at 29 degrees C results in malignant brain tumours and hyperplastic imaginal discs. Unlike the former that have been extensively characterised, little is known about the latter. Here we report the results of a study of the hyperplastic l(3)mbt mutant wing imaginal discs. We identify the l(3)mbt wing disc tumour transcriptome and find it to include genes involved in reactive oxygen species (ROS) metabolism. Furthermore, we show the presence of oxidative stress in l(3)mbt hyperplastic discs, even in apoptosis-blocked conditions, but not in l(3)mbt brain tumours. We also find that chemically blocking oxidative stress in l(3)mbt wing discs reduces the incidence of wing disc overgrowths. Our results reveal the involvement of oxidative stress in l(3)mbt wing discs hyperplastic growth

The tumour suppressor brain tumour (Brat) regulates linker histone dBigH1 expression in the Drosophila female germline and the early embryo

Linker histones H1 are essential chromatin components that exist as multiple developmentally regulated variants. In metazoans, specific H1s are expressed during germline development in a tightly regulated manner. However, the mechanisms governing their stage-dependent expression are poorly understood. Here, we address this question in Drosophila, which encodes for a single germline-specific dBigH1 linker histone. We show that during female germline lineage differentiation, dBigH1 is expressed in germ stem cells and cystoblasts, becomes silenced during transit-amplifying (TA) cystocytes divisions to resume expression after proliferation stops and differentiation starts, when it progressively accumulates in the oocyte. We find that dBigH1 silencing during TA divisions is post-transcriptional and depends on the tumour suppressor Brain tumour (Brat), an essential RNA-binding protein that regulates mRNA translation and stability. Like other oocyte-specific variants, dBigH1 is maternally expressed during early embryogenesis until it is replaced by somatic dH1 at the maternal-to-zygotic transition (MZT). Brat also mediates dBigH1 silencing at MZT. Finally, we discuss the situation in testes, where Brat is not expressed, but dBigH1 is translationally silenced too

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

Estudio funcional de la variante embrionaria de la histona H1 de Drosophila

[spa] En los metazoos existen múltiples variantes de histona H1, siendo algunas de ellas específicas de la línea germinal y la embriogénesis temprana. Las variantes embrionarias están presentes mientras el genoma del zigoto está transcripcionalmente inactivo y posteriormente son reemplazadas por las variantes de H1 somáticas. En el caso de Drosophila, la reducción de los niveles de la variante embrionaria, dBigH1, tiene como resultado la activación prematura del genoma zigótico, lo que sugiere una posible implicación de dBigH1 en la represión del genoma. Durante el desarrollo de esta tesis doctoral hemos utilizado células S2 de Drosophila, que no expresan dBigH1, para caracterizar los efectos diferenciales de dBigH1 sobre la transcripción en comparación con la variante somática, dH1. Hemos visto que cuando expresamos dBigH1 en células S2 se incorpora a la cromatina uniformemente y, además, reemplaza la dH1. La expresión de dBigH1 en estas células, además, regula negativamente la transcripción. Esta regulación negativa se debe a que dBigH1 interfiere en la unión de la RNA polimerasa II y en la acetilación de las histonas. Por otro lado, en este trabajo también hemos estudiado la función de los diferentes dominios de dBigH1 y hemos encontrado que el dominio C-terminal es necesario para su unión a la cromatina. En cambio, la región N-terminal contiene una región rica en residuos ácidos, llamada dominio ED, que es el responsable del reemplazo de dH1 y la represión de la transcripción ya que cuando expresamos una forma truncada de la proteína que no contiene el dominio ED, dBigH1 no interfiere en la unión de la RNA polimerasa II y la acetilación de las histonas. Además, dBigH1 está presente en la línea germinal femenina. dBigH1 se expresa en las células madre germinales y en sus hijas, los cistoblastos. Posteriormente desaparece en la región de divisiones mitóticas y se vuelve a expresar en las cámaras ováricas. A medida que se desarrollan las cámaras ováricas, la expresión de dBigH1 queda restringida al oocito. Además, en la línea germinal femenina encontramos dH1, que coexiste con dBigH1 en las células madre germinales, cistoblastos y, durante los primeros estadios del desarrollo de las cámaras ováricas, el oocito. En este trabajo hemos visto que las células que contienen ambas variantes son activas transcripcionalmente. Sin embargo, cuando el genoma del oocito está totalmente silenciado únicamente encontramos dBigH1, a excepción de los estadios 8-11, en los que dBigH1 permite la reanudación de la actividad transcripcional. En este trabajo también hemos estudiado la regulación de la expresión de dBigH1 en la línea germinal femenina y hemos encontrado que está regulada postranscripcionalmente mediante señales localizadas en la región 3’UTR del mRNA. Finalmente, hemos encontrado que uno de los responsables de la regulación de dBigH1 es Brat, probablemente de manera directa a través de la unión al 3’UTR del mRNA de dBigH1.[eng] Linker histones H1 are one of the main components of chromatin. Histone H1 is a highly heterogeneous family of proteins and several variants have been described in metazoa. Some of them are specifically expressed in the germline and are retained in the early embryo. However, in Drosophila, only one somatic H1, dH1, and one embryonic and germline-specific variant, dBigH1, have been described. dBigH1 is present during early embriogenesis, when the zygotic genome is silenced and is replaced at cellularization by dH1. The reduction of dBigH1 levels results in a premature activation of the zygotic genome, suggesting a role of dBigH1 in transcriptional silencing. We report here that ectopic expression of dBigH1 in Drosophila S2 cells results in dBigH1 binding across chromatin and replacement of dH1. This binding and replacement result in down-regulation of gene expression by altering RNApol II binding and histone acetylation at promoters. We also show that the effects of dBigH1 depend on the highly acidic ED-domain of the N-terminal tail, since a truncated form lacking this domain does not replace dH1 or affect transcription. We also show that dBiH1 is present in the female germline. dBigH1 is expressed in the germ stem cells and cystoblasts, disappears in the mitotic divisions region and is expressed again in cysts cells that are surrounded by the follicular cells to form an egg chamber. As the egg chambers develop, dBigH1 expression is restricted to the oocyte. In the germline dBigH1 and dH1 coexist in germ stem cells, cystoblasts and early oocytes. However, dBigH1 is the only variant present in the oocyte when is completely silenced. We demonstrate also that in cyst cells, dBigH1 expression is postranscriptionally regulated through signals present in the mRNA 3’UTR region. Finally, we show that Brat participates in the down-regulation of dBigH1 expression, probably through a direct mechanism

The germline linker histone dBigH1 and the translational regulator bam form a repressor loop essential for male germ stem cell differentiation

Drosophila spermatogenesis constitutes a paradigmatic system to study maintenance, proliferation, and differentiation of adult stem cell lineages. Each Drosophila testis contains 6–12 germ stem cells (GSCs) that divide asymmetrically to produce gonialblast cells that undergo four transit-amplifying (TA) spermatogonial divisions before entering spermatocyte differentiation. Mechanisms governing these crucial transitions are not fully understood. Here, we report the essential role of the germline linker histone dBigH1 during early spermatogenesis. Our results suggest that dBigH1 is a general silencing factor that represses Bam, a key regulator of spermatogonia proliferation that is silenced in spermatocytes. Reciprocally, Bam represses dBigH1 during TA divisions. This double-repressor mechanism switches dBigH1/Bam expression from off/on in spermatogonia to on/off in spermatocytes, regulating progression into spermatocyte differentiation. dBigH1 is also required for GSC maintenance and differentiation. These results show the critical importance of germline H1s for male GSC lineage differentiation, unveiling a regulatory interaction that couples transcriptional and translational repression.This work was supported by grants from the MINECO (BFU2012-30724 and BFU2015-65082P), the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204), and the European Community FEDER program. This work was carried out within the framework of the ‘‘Centre de Refere` ncia en Biotecnologia’’ of the ‘‘Generalitat de Catalunya.’’ S.P.-M. and P.C.-C. acknowledge receipt of FPI fellowships from the MINECO.Peer reviewe

Chromatin organization and function in Drosophila

Eukaryotic genomes are packaged into high-order chromatin structures organized in discrete territories inside the cell nucleus, which is surrounded by the nuclear envelope acting as a barrier. This chromatin organization is complex and dynamic and, thus, determining the spatial and temporal distribution and folding of chromosomes within the nucleus is critical for understanding the role of chromatin topology in genome function. Primarily focusing on the regulation of gene expression, we review here how the genome of Drosophila melanogaster is organized into the cell nucleus, from small scale histone-DNA interactions to chromosome and lamina interactions in the nuclear space

The embryonic linker histone dBigH1 alters the functional state of active chromatin

Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. Here we directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. We show that dBigH1 binds across chromatin, replaces somatic dH1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription.Funding: MINECO [BFU2015-65082-P, PGC2018-094538-B-100]; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; European Community FEDER program (to F.A.); MEC ‘Centro de Excelencia Severo Ochoa 2013–2017’ [SEV-2012-0208]; MINECO [SAF2016-75006-P to M.B.]; ‘Centre de Referència en Biotecnologia’ of the Generalitat de Catalunya. Funding for open access charge: MINEC

Histone H1: Lessons from Drosophila

Eukaryotic genomes are structured in the formof chromatin with the help of a set of five small basic proteins, the histones. Four of them are highly conserved through evolution, form the basic unit of the chromatin, the nucleosome, and have been intensively studied and are well characterized. The fifth histone, histone H1, adds to this basic structure through its interaction at the entry/exit site of DNA in the nucleosomeandmakes an essential contribution to the higher order folding of the chromatin fiber. Histone H1 is the less conserved histone and the less known of them. Though for long time considered as a general repressor of gene expression, recent studies in Drosophila have rejected this view and have contributed to uncover important functions on genome stability and development. Here we present some of the most recent data obtained in the Drosophila model system and discuss how the lessons learnt in these studies compare and could be applied to all other eukaryotes. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.Work in the authors' laboratory is supported by grants from MINECO (BFU2012-30724) and the Generalitat de Catalunya (SGR2014-204)Peer Reviewe