Renate Renkawitz-Pohl

CTCF is conserved from Drosophila to humans and confers enhancer blocking of the Fab-8 insulator

Eukaryotic transcriptional regulation often involves regulatory elements separated from the cognate genes by long distances, whereas appropriately positioned insulator or enhancer‐blocking elements shield promoters from illegitimate enhancer action. Four proteins have been identified in Drosophila mediating enhancer blocking—Su(Hw), Zw5, BEAF32 and GAGA factor. In vertebrates, the single protein CTCF, with 11 highly conserved zinc fingers, confers enhancer blocking in all known chromatin insulators. Here, we characterize an orthologous CTCF factor in Drosophila with a similar domain structure, binding site specificity and transcriptional repression activity as in vertebrates. In addition, we demonstrate that one of the insulators (Fab‐8) in the Drosophila Abdominal‐B locus mediates enhancer blocking by dCTCF. Therefore, the enhancer‐blocking protein CTCF and, most probably, the mechanism of enhancer blocking mediated by this remarkably versatile factor are conserved from Drosophila to humans.

Chromatin dynamics during spermiogenesis

The function of sperm is to safely transport the haploid paternal genome to the egg containing the maternal genome. The subsequent fertilization leads to transmission of a new unique diploid genome to the next generation. Before the sperm can set out on its adventurous journey, remarkable arrangements need to be made during the post-meiotic stages of spermatogenesis. Haploid spermatids undergo extensive morphological changes, including a striking reorganization and compaction of their chromatin. Thereby, the nucleosomal, histone-based structure is nearly completely substituted by a protamine-based structure. This replacement is likely facilitated by incorporation of histone variants, post-translational histone modifications, chromatin-remodeling complexes, as well as transient DNA strand breaks. The consequences of mutations have revealed that a protamine-based chromatin is essential for fertility in mice but not in Drosophila. Nevertheless, loss of protamines in Drosophila increases the sensitivity to X-rays and thus supports the hypothesis that protamines are necessary to protect the paternal genome. Pharmaceutical approaches have provided the first mechanistic insights and have shown that hyperacetylation of histones just before their displacement is vital for progress in chromatin reorganization but is clearly not the sole inducer. In this review, we highlight the current knowledge on post-meiotic chromatin reorganization and reveal for the first time intriguing parallels in this process in Drosophila and mammals. We conclude with a model that illustrates the possible mechanisms that lead from a histone-based chromatin to a mainly protamine-based structure during spermatid differentiation. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila

In higher organisms, the chromatin of sperm is organised in a highly condensed protamine-based structure. In pre-meiotic stages and shortly after meiosis, histones carry multiple modifications. Here, we focus on post-meiotic stages and show that also after meiosis, histone H3 shows a high overall methylation of K9 and K27 and we hypothesise that these modifications ensure maintenance of transcriptional silencing in the haploid genome. Furthermore, we show that histones are lost during the early canoe stage and that just before this stage, hyper-acetylation of histone H4 and mono-ubiquitylation of histone H2A occurs. We believe that these histone modifications within the histone-based chromatin architecture may lead to better access of enzymes and chromatin remodellers. This notion is supported by the presence of the architectural protein CTCF, numerous DNA breaks, SUMO, UbcD6 and high content of ubiquitin, as well as testes-specific nuclear proteasomes at this time. Moreover, we report the first transition protein-like chromosomal protein, Tpl94D, to be found in Drosophila. We propose that Tpl94D – an HMG box protein – and the numerous DNA breaks facilitate chromatin unwinding as a prelude to protamine and Mst77F deposition. Finally, we show that histone modifications and removal are independent of protamine synthesis.

The Drosophila insulator proteins CTCF and CP190 link enhancer blocking to body patterning

Insulator sequences guide the function of distantly located enhancer elements to the appropriate target genes by blocking inappropriate interactions. In Drosophila, five different insulator binding proteins have been identified, Zw5, BEAF‐32, GAGA factor, Su(Hw) and dCTCF. Only dCTCF has a known conserved counterpart in vertebrates. Here we find that the structurally related factors dCTCF and Su(Hw) have distinct binding targets. In contrast, the Su(Hw) interacting factor CP190 largely overlapped with dCTCF binding sites and interacts with dCTCF. Binding of dCTCF to targets requires CP190 in many cases, whereas others are independent of CP190. Analysis of the bithorax complex revealed that six of the borders between the parasegment specific regulatory domains are bound by dCTCF and by CP190 in vivo. dCTCF null mutations affect expression of Abdominal‐B, cause pharate lethality and a homeotic phenotype. A short pulse of dCTCF expression during larval development rescues the dCTCF loss of function phenotype. Overall, we demonstrate the importance of dCTCF in fly development and in the regulation of abdominal segmentation.

Replacement by Drosophila melanogaster Protamines and Mst77F of Histones during Chromatin Condensation in Late Spermatids and Role of Sesame in the Removal of These Proteins from the Male Pronucleus

ABSTRACT Chromatin condensation is a typical feature of sperm cells. During mammalian spermiogenesis, histones are first replaced by transition proteins and then by protamines, while little is known for Drosophila melanogaster. Here we characterize three genes in the fly genome, Mst35Ba, Mst35Bb, and Mst77F. The results indicate that Mst35Ba and Mst35Bb encode dProtA and dProtB, respectively. These are considerably larger than mammalian protamines, but, as in mammals, both protamines contain typical cysteine/arginine clusters. Mst77F encodes a linker histone-like protein showing significant similarity to mammalian HILS1 protein. ProtamineA-enhanced green fluorescent protein (eGFP), ProtamineB-eGFP, and Mst77F-eGFP carrying Drosophila lines show that these proteins become the important chromosomal protein components of elongating spermatids, and His2AvDGFP vanishes. Mst77F mutants [ms(3)nc3] are characterized by small round nuclei and are sterile as males. These data suggest the major features of chromatin condensation in Drosophila spermatogenesis correspond to those in mammals. During early fertilization steps, the paternal pronucleus still contains protamines and Mst77F but regains a nucleosomal conformation before zygote formation. In eggs laid by sesame-deficient females, the paternal pronucleus remains in a protamine-based chromatin status but Mst77F-eGFP is removed, suggesting that the sesame gene product is essential for removal of protamines while Mst77F removal is independent of Sesame.

The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif

We identified and characterized the don juan gene (dj) of Drosophila melanogaster. The don juan gene codes for a sperm specific protein component with an unusual repetitive six amino acid motif (DPCKKK) in the carboxy-terminal part of the protein. The expression of Don Juan is limited to male germ cells where transcription of the dj gene is initiated during meiotic prophase. But Western blot experiments indicate that DJ protein occurs just postmeiotically. Examination of transgenic flies bearing a dj-promoter-lacZ reporter construct revealed lacZ mRNA distribution resembling the expression pattern of the endogenous dj mRNA in the adult testes, whereas beta-galactosidase expression is exclusively present in postmeiotic germ cells. Thus, these observations strongly suggest that dj transcripts are under translational repression until in spermiogenesis. To study the function and subcellular distribution of DJ in spermiogenesis we expressed a chimaeric dj-GFP fusion gene in the male germline exhibiting strong GFP fluorescence in the liver testes, where only elongated spermatids are decorated. With regard to the characteristic expression pattern of DJ protein and its conspicuous repeat units possible functional roles are discussed.

A 14 bp promoter element directs the testis specificity of the Drosophila beta 2 tubulin gene.

To analyze the regulation of gene expression during male germ cell development, we investigated the testis‐specific expression of the Drosophila beta 2 tubulin gene. Germ line transformation experiments with the upstream region of the D.melanogaster beta 2 tubulin gene fused to the Escherichia coli lacZ gene resulted in the correct tissue specific expression of the reporter gene. Furthermore, we showed that the upstream sequences of the beta 2 tubulin gene of the distantly related species D.hydei can drive the expression of the lacZ gene testis specifically in D.melanogaster flies. A detailed deletion analysis showed that 53 bp of upstream and 23 bp (D.melanogaster) or 29 bp (D.hydei) of leader sequences are sufficient to confer tissue specificity. The short promoter regions contain a 14 bp motif at identical positions in both species, which acts as a position‐dependent promoter element. In vitro mutagenesis and subsequent germline transformation experiments revealed that this sequence is the only element necessary for the testis‐specific transcription of the beta 2 tubulin gene in Drosophila.

Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS)

During myogenesis in Drosophila embryos, a prominent adhesive structure is formed between precursor cells and fusion‐competent myoblasts (fcms). Here, we show that Duf/Kirre and its interaction partners Rols7 (found in founder myoblasts and growing myotubes) and Sns (found in fcms) are organized in a ring‐structure at the contact points of fcms with precursor cells, while cytoskeletal components like F‐actin and Titin are centered in this ring in both cell types. The cytoplasmic protein Blow colocalizes with the actin plugs in fcms after cell adhesion. Furthermore, the requirement of additional as yet unidentified components was demonstrated by using mammalian C2C12 myoblasts. In this study, we propose that the fusion‐restricted myogenic‐adhesive structure (FuRMAS) is pivotal in linking cell adhesion as well as local F‐actin assembly and dynamics to downstream events that ultimately lead to plasma membrane fusion. Moreover, we suggest that the FuRMAS may restrict the area of membrane breakdown. Developmental Dynamics 236:404–415, 2007.

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila.

Drosophila myoblast fusion proceeds in two steps. The first one gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. We have identified Kette as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterised by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, we show that kette interacts genetically with blown fuse (blow) which is known to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. We propose a model in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion.

WASP and SCAR have distinct roles in activating the Arp2/3 complex during myoblast fusion

Myoblast fusion takes place in two steps in mammals and in Drosophila. First, founder cells (FCs) and fusion-competent myoblasts (FCMs) fuse to form a trinucleated precursor, which then recruits further FCMs. This process depends on the formation of the fusion-restricted myogenic-adhesive structure (FuRMAS), which contains filamentous actin (F-actin) plugs at the sites of cell contact. Fusion relies on the HEM2 (NAP1) homolog Kette, as well as Blow and WASP, a member of the Wiskott-Aldrich-syndrome protein family. Here, we show the identification and characterization of schwächling – a new Arp3-null allele. Ultrastructural analyses demonstrate that Arp3schwächling mutants can form a fusion pore, but fail to integrate the fusing FCM. Double-mutant experiments revealed that fusion is blocked completely in Arp3 and wasp double mutants, suggesting the involvement of a further F-actin regulator. Indeed, double-mutant analyses with scar/WAVE and with the WASP-interacting partner vrp1 (sltr, wip)/WIP show that the F-actin regulator scar also controls F-actin formation during myoblast fusion. Furthermore, the synergistic phenotype observed in Arp3 wasp and in scar vrp1 double mutants suggests that WASP and SCAR have distinct roles in controlling F-actin formation. From these findings we derived a new model for actin regulation during myoblast fusion.