Targeting determinants of dosage compensation in Drosophila

The dosage compensation complex (DCC) in Drosophila melanogaster is responsible for up-regulating transcription from the single male X chromosome to equal the transcription from the two X chromosomes in females. Visualization of the DCC, a large ribonucleoprotein complex, on male larval polytene chromosomes reveals that the complex binds selectively to many interbands on the X chromosome. The targeting of the DCC is thought to be in part determined by DNA sequences that are enriched on the X. So far, lack of knowledge about DCC binding sites has prevented the identification of sequence determinants. Only three binding sites have been identified to date, but analysis of their DNA sequence did not allow the prediction of further binding sites. We have used chromatin immunoprecipitation to identify a number of new DCC binding fragments and characterized them in vivo by visualizing DCC binding to autosomal insertions of these fragments, and we have demonstrated that they possess a wide range of potential to recruit the DCC. By varying the in vivo concentration of the DCC, we provide evidence that this range of recruitment potential is due to differences in affinity of the complex to these sites. We were also able to establish that DCC binding to ectopic high-affinity sites can allow nearby low-affinity sites to recruit the complex. Using the sequences of the newly identified and previously characterized binding fragments, we have uncovered a number of short sequence motifs, which in combination may contribute to DCC recruitment. Our findings suggest that the DCC is recruited to the X via a number of binding sites of decreasing affinities, and that the presence of high-and moderate-affinity sites on the X may ensure that lower-affinity sites are occupied in a context-dependent manner. Our bioinformatics analysis suggests that DCC binding sites may be composed of variable combinations of degenerate motifs

Retraction notice on the article by A.S. Zotov et al. ‘Short-Term Results of Two Strategies in Thoracoscopic Ablation for Lone Atrial Fibrillation’ doi: 10.17816/clinpract110719

Editorial board of the journal informs authors about the retraction of the article Short-Term Results of Two Strategies in Thoracoscopic Ablation for Lone Atrial Fibrillation published in Journal of Clinical Practice 13(3) 2022 by A.S. Zotov, O.Yu. Pidanov, I.S. Osmanov, A.V. Troitsky, A.A. Silaev, E.R. Sakharov, V.N. Sukhotin, O.O. Shelest, R.I. Khabazov, D.A. Timashkov. The reason for the retraction is the publication ethics violation in terms of authorship criteria. Not all authors whose names appear on the article made substantial contributions to the study drafted/revised the manuscript and approved the version to be published. Retraction made on January 09, 2023 with approve from the Editor-in-Chief

Positive Selection in Gene Regulatory Factors Suggests Adaptive Pleiotropic Changes During Human Evolution

Gene regulatory factors (GRFs), such as transcription factors, co-factors and histone-modifying enzymes, play many important roles in modifying gene expression in biological processes. They have also been proposed to underlie speciation and adaptation. To investigate potential contributions of GRFs to primate evolution, we analyzed GRF genes in 27 publicly available primate genomes. Genes coding for zinc finger (ZNF) proteins, especially ZNFs with a Krüppel-associated box (KRAB) domain were the most abundant TFs in all genomes. Gene numbers per TF family differed between all species. To detect signs of positive selection in GRF genes we investigated more than 3,000 human GRFs with their more than 70,000 orthologs in 26 non-human primates. We implemented two independent tests for positive selection, the branch-site-model of the PAML suite and aBSREL of the HyPhy suite, focusing on the human and great ape branch. Our workflow included rigorous procedures to reduce the number of false positives: excluding distantly similar orthologs, manual corrections of alignments, and considering only genes and sites detected by both tests for positive selection. Furthermore, we verified the candidate sites for selection by investigating their variation within human and non-human great ape population data. In order to approximately assign a date to positively selected sites in the human lineage, we analyzed archaic human genomes. Our work revealed with high confidence five GRFs that have been positively selected on the human lineage and one GRF that has been positively selected on the great ape lineage. These GRFs are scattered on different chromosomes and have been previously linked to diverse functions. For some of them a role in speciation and/or adaptation can be proposed based on the expression pattern or association with human diseases, but it seems that they all contributed independently to human evolution. Four of the positively selected GRFs are KRAB-ZNF proteins, that induce changes in target genes co-expression and/or through arms race with transposable elements. Since each positively selected GRF contains several sites with evidence for positive selection, we suggest that these GRFs participated pleiotropically to phenotypic adaptations in humans

Recruitment of the DCC to DBF Insertions in Wild-Type Males

<p>Polytene chromosomes with FISH signals in green and anti-MSL1 signals in red showing examples of DBF insertions recruiting the DCC in wild-type males. The cytological position of DBF inserts was estimated from FISH experiments (upper image in each panel). Recruitment of the DCC was demonstrated with immunoflourescence using anti-MSL1 antibodies (lower image in each panel). Examples from all DBF inserts that showed recruitment in wild-type males are shown. A summary of the complete analysis can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020005#pgen-0020005-t002" target="_blank">Table 2</a>.</p

DNA Sequence Logos of Representative Motifs Putatively Involved in DCC Binding

<p>Logos (generated using the WebLogo software) represent degenerate motifs based on related pairs of elements from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020005#pgen-0020005-st001" target="_blank">Table S1</a>. The logos were generated by aligning all the words of related elements from specific pairs: (A) pairs 5, 6, 7, 8, 9, and 10; (B) pairs 11, 12, 13, and 14; (C) pairs 17, 18, 19, and 20; (D) 21, 22, and 23; (E) pairs 15 and 16; and (F) pairs 1, 2, and 3. Pairs 4 and 24 were not included. Motifs Ia–Ie all contain GAGA-related sequences. Motifs IIa and IIb as well as IIIa–IIIc are also related to each other.</p

High-Affinity DCC Binding Fragments Recruit Partial and Inactive Complexes in <i>msl-3¹</i> and <i>mof¹</i> Mutants

<p>Examples of DCC recruitment to DBF inserts in females expressing MSL2 and carrying mutations in <i>msl-3¹</i> (<i>w</i>/<i>w; msl-3¹,</i> [w+ Hsp83 MSL2]/+) or <i>mof¹</i> (<i>mof¹</i>/<i>mof¹;</i> +; [w+ Hsp83 MSL2]/+). Anti-MSL1 staining is shown in red and DNA staining in blue. Arrows indicate the position of the inserts and X indicates the X chromosome. Inserts DBF12-B-99EF (A), DBF12-B-85A (B), DBF9-B-96C (C), DBF9B-98F (D), DBF6-100A (E), and DBF5-95C (G) recruit partial complexes in the <i>msl-3¹</i> mutant. Inserts DBF6-63C (F), DBF5-91F (H), and DBF7-88E (I) recruit inactive complexes in the <i>mof¹</i> mutant background.</p

Increasing the Levels of the DCC Increases Recruitment to Low-Affinity Binding Fragments

<p>Polytene chromosomes showing FISH signals (top panels, green) and anti-MSL1 staining (red) in the genetic backgrounds indicated. Arrows indicate the position of the inserts and arrowheads indicate autosomal sites of DCC binding in the MSL1 and MSL2 over-expression background. Recruitment of the DCC to the DBF11-85D insert is enhanced when the levels of DCC are increased by over-expression of MSL1 and MSL2 compared to wild-type males. The DBF3-96B insert does not recruit the DCC in wild-type males, but recruitment can be seen when MSL1 and MSL2 are over-expressed.</p

Identification of New DCC Binding Fragments Using Chromatin IP

<div><p>(A) Subsection of genomic P1 phage clone filter, showing 120 clones out of a total of 9,216, hybridized with MSL1 ChIP probe (upper panel), then stripped and re-hybridized to the mock IP control (lower panel). Two examples of clones showing MSL1 enrichment chosen for further analysis are indicated by red circles. Note that clones are always spotted in duplicate on the membrane.</p><p>(B) Southern blots of digested P1 clone DNA hybridized to MSL1 ChIP (left panel) or mock ChIP (right panel). Red boxes highlight the bands enriched in the MSL1 ChIP chosen for cloning, known as DBF1–DBF14. Size in kilobases is indicated to the left of the figure. Lane order and restriction digests are as follows: Lane 1, DBF1 (BglII); 2, DBF3 (XhoI); 3, DBF5 (XhoI); 4, DBF6 (ApaLI); 5, DBF7 (BamHI); 6, DBF9 (XhoI); 7, DBF10 (EcoRI); 8, DBF11 (ApaLI); 9, DBF12 (EcoRI); 10, DBF13 (EcoRI); 11, DBF14 (EcoRI); and 12, autosomal DNA control P1 clone (EcoRI). Note that clones DBF2, DBF4, and DBF8 (false positives) have been omitted from this figure. P1 phage clone identifiers are listed in the Materials and Methods section.</p></div

The Endogenous Loci of the DCC Binding Fragments Overlap with MSL1 In Vivo

<p>Immuno-FISH experiments using the identified DBFs as probes (FISH signals in green) and staining with anti-MSL1 antibodies to visualize DCC binding (in red). Each panel consists of images of the FISH signal (top), the anti-MSL1 signal (middle), and the merge (bottom). The FISH signals from DBF9, DBF6, DBF5, and DBF12 overlap with the anti-MSL1 signals in the <i>msl-3¹</i> mutant background (<i>w</i>/<i>w; msl-3¹,</i> [w+ Hsp83 MSL2]). DBF11 overlaps partially with MSL1 staining in this background. DBF1 and DBF7 overlap with weak anti-MSL signals in the <i>mof¹</i> mutant background (<i>mof¹</i>/<i>mof¹;</i> +; [w+ Hsp83 MSL2]/+). FISH signals from the endogenous loci of DBF3, DBF10, and DBF13 do not overlap with MSL1 staining in either the <i>msl-3¹</i>or <i>mof¹</i> background. However, DBF10 does overlap with MSL1 staining in females carrying two copies of the NOPU insert (2 × NOPU, Figure1), whereas DBF3 and DBF13 overlap with MSL staining in wild-type males (WT).</p