László Henn

Metazoan genomes typically encode several linker histone variants, often expressed in a tissue‐ or developmental stage‐specific manner. The Drosophila melanogaster genome contains only two linker histone variants: H1 is present in somatic cells, while BigH1 substitutes H1 in the germline and early embryos. In the early stages of embryogenesis, BigH1 is replaced by H1 in the chromatin of somatic cells, contributing to the initiation and maintenance of the zygotic gene expression program. Nevertheless, the molecular mechanism of this exchange and the possible functions of post‐translational modifications of BigH1 in this process remain elusive. Here, we identify phosphorylation as a key post‐translational regulator of BigH1 dynamics. Using proteomics and targeted mutagenesis of the endogenous BigH1 locus, we show that the loss of N‐terminal phosphorylation results in persistent retention of BigH1 in somatic nuclei throughout embryogenesis, indicating a failure in BigH1 turnover. In contrast, disruption of C‐terminal phosphorylation does not markedly affect BigH1 clearance but increases defects during early nuclear divisions and compromises embryonic development, particularly under suboptimal conditions. Together, these findings demonstrate that domain‐specific phosphorylation differentially regulates BigH1 function, coordinating its early embryonic role with its subsequent removal from the chromatin.

During early embryogenesis the zygotic genome remains transcriptionally silent and expression relies on maternally deposited products. Maternal deposition of histones is crucial to preserve chromatin integrity during early embryo development, when the number of nuclei exponentially increases in the absence of zygotic expression. In the Drosophila embryo, histones are maternally deposited as both proteins and mRNAs. Histone transcripts are the only nonpolyadenylated cellular mRNAs. They contain a highly conserved 3′UTR stem-loop structure, which is recognized by the Stem-Loop Binding Protein (SLBP) that, in conjunction with U7 snRNP, regulates their unique 3′-end processing. Here we report that, unexpectedly, maternal histone mRNAs are polyadenylated and have a truncated 3′ stem-loop. This noncanonical 3′-end processing of maternal histone mRNAs occurs at their synthesis during oogenesis and requires SLBP, but not U7 snRNP. We show that maternal histone transcripts are subjected to cytoplasmic poly(A) tail elongation by Wisp, which results in their stabilization and is a requisite for translation. We also show that maternal histone transcripts remain largely quiescent and that their translation is activated upon loss of the embryonic linker histone dBigH1, which impairs chromatin assembly and induces DNA damage. Here, we discuss possible models to integrate these observations.

In most animals, the start of embryogenesis requires specific histones. In Drosophila linker histone variant BigH1 is present in early embryos. To uncover the specific role of this alternative linker histone at early embryogenesis, we established fly lines in which domains of BigH1 have been replaced partially or completely with that of H1. Analysis of the resulting Drosophila lines revealed that at normal temperature somatic H1 can substitute the alternative linker histone, but at low temperature the globular and C-terminal domains of BigH1 are essential for embryogenesis. In the presence of BigH1 nucleosome stability increases and core histone incorporation into nucleosomes is more rapid, while nucleosome spacing is unchanged. Chromatin formation in the presence of BigH1 permits the fast-paced nuclear divisions of the early embryo. We propose a model which explains how this specific linker histone ensures the rapid nucleosome reassembly required during quick replication cycles at the start of embryogenesis.

DUTP pyrophosphatase (dUTPase) is a dNTP‐sanitizing enzyme that prevents the appearance of potentially harmful uracil bases in DNA by hydrolyzing cellular dUTP. This function of dUTPase is found to be essential in many organisms including Drosophila melanogaster. Previously, we showed that the expression pattern of dUTPase determines the extent of uracil accumulation in the genome of different tissues. We wished to find the regulatory mechanism that eventually leaves a set of tissues with a uracil‐free and intact genome. We found that the expression pattern established by the promoter of Drosophila dUTPase overlaps with mRNA and protein expression, excluding the involvement of other post‐transcriptional contributions. This promoter was found to be active in primordial tissues, such as in the imaginal discs of larvae, in the larval brain and in reproductive organs. In the case of brain and imaginal tissues, we observed that the promoter activity depends on a DNA replication‐related element motif, the docking site of DNA replication‐related element binding factor, which is known as a transcriptional activator of genes involved in replication and proliferation. These results suggest that dUTPase expression is fine‐tuned to meet the requirements of DNA synthesis in tissues where the maintenance of genome integrity is of high importance.

In Drosophila melanogaster, primordial germ cells are specified at the posterior pole of the very early embryo. This process is regulated by the posterior localized germ plasm that contains a large number of RNAs of maternal origin. Transcription in the primordial germ cells is actively down-regulated until germ cell fate is established. Bulk expression of the zygotic genes commences concomitantly with the degradation of the maternal transcripts. Thus, during embryogenesis, maternally provided and zygotically transcribed mRNAs determine germ cell development collectively. In an effort to identify novel genes involved in the regulation of germ cell behavior, we carried out a large-scale RNAi screen targeting both maternal and zygotic components of the embryonic germ line transcriptome. We identified 48 genes necessary for distinct stages in germ cell development. We found pebble and fascetto to be essential for germ cell migration and germ cell division, respectively. Our data uncover a previously unanticipated role of mei-P26 in maintenance of embryonic germ cell fate. We also performed systematic co-RNAi experiments, through which we found a low rate of functional redundancy among homologous gene pairs. As our data indicate a high degree of evolutionary conservation in genetic regulation of germ cell development, they are likely to provide valuable insights into the biology of the germ line in general.

The first microRNAs were discovered some 20 years ago, but only a small fraction of the microRNA-encoding genes have been described in detail yet. Here we report the molecular analysis of a computationally predicted Drosophila melanogaster microRNA gene, mir-282. We show that the mir-282 gene is the source of a 4.9-kb-long primary transcript with a 5′ cap and a 3′-poly(A) sequence and a mature microRNA of ∼25 bp. Our data strongly suggest the existence of an independent mir-282 gene conserved in holometabolic insects. We give evidence that the mir-282 locus encodes a functional transcript that influences viability, longevity, and egg production in Drosophila. We identify the nervous system-specific adenylate cyclase (rutabaga) as a target of miR-282 and assume that one of the main functions of mir-282 is the regulation of adenylate cyclase activity in the nervous system during metamorphosis.

Morphogenesis, the establishment of the animal body, requires the coordinated rearrangement of cells and tissues regulated by a very strictly-determined genetic program. Dorsal closure of the epithelium in the Drosophila melanogaster embryo is one of the best models for such a complex morphogenetic event. To explore the genetic regulation of dorsal closure, we carried out a large-scale RNA interference-based screen in combination with in vivo time-lapse microscopy and identified several genes essential for the closure or affecting its dynamics. One of the novel dorsal closure genes, the small GTPase activator pebble (pbl), was selected for detailed analysis. We show that pbl regulates actin accumulation and protrusion dynamics in the leading edge of the migrating epithelial cells. In addition, pbl affects dorsal closure dynamics by regulating head involution, a morphogenetic process mechanically coupled with dorsal closure. Finally, we provide evidence that pbl is involved in closure of the adult thorax, suggesting its general requirement in epithelial closure processes.

In the genome of Drosophila melanogaster, there are 19 phosphoprotein phosphatase (PPP) catalytic subunit coding genes. Seven of the novel members of the gene family turned out to be Drosophila-specific. The expression and evolution of these genes was investigated in the present study. CG11597 is a recently evolved gene that is expressed during all stages of morphogenesis in D. melanogaster. In contrast, the transcription of PpD5, PpD6, Pp1-Y1, and Pp1-Y2 genes is restricted to the pupa and imago developmental stages and to the testis of the males, just as that of the previously characterized PpY-55A and PpN58A. With the exception of the Y-localized Pp1-Y1 and Pp1-Y2, the testis-specific phosphatase genes are expressed in X/0 males, while none of them are expressed in XX/Y females. The mRNA of PpD5, Pp1-Y1, and PpY-55A were detected in the developing cysts by in situ hybridization, in contrast with the PpD6 transcript that was found in the distal ends of elongating spermatids. The latter localization suggests post-meiotic expression. The comparison of PPP genes in five Drosophila species revealed that the sequence of the six testis-specific phosphatases changed more rapidly than that of the housekeeping phosphatases. Our results support the “faster male” hypothesis. On the other hand, the male-biased expression of the six genes remained conserved during evolution despite the fact that Pp1-Y1, Pp1-Y2, and PpD6 moved from autosomes to the Y chromosome. Interestingly, the PpD6 gene was found to be Y-linked only in Drosophila ananassae.