Publications by authors named "Alexander Kukalev"

13 Publications

  • Page 1 of 1

Comparison of the Hi-C, GAM and SPRITE methods using polymer models of chromatin.

Nat Methods 2021 May 7;18(5):482-490. Epub 2021 May 7.

Dipartimento di Fisica, Università di Napoli Federico II and INFN Napoli, Complesso Universitario di Monte Sant'Angelo, Naples, Italy.

Hi-C, split-pool recognition of interactions by tag extension (SPRITE) and genome architecture mapping (GAM) are powerful technologies utilized to probe chromatin interactions genome wide, but how faithfully they capture three-dimensional (3D) contacts and how they perform relative to each other is unclear, as no benchmark exists. Here, we compare these methods in silico in a simplified, yet controlled, framework against known 3D structures of polymer models of murine and human loci, which can recapitulate Hi-C, GAM and SPRITE experiments and multiplexed fluorescence in situ hybridization (FISH) single-molecule conformations. We find that in silico Hi-C, GAM and SPRITE bulk data are faithful to the reference 3D structures whereas single-cell data reflect strong variability among single molecules. The minimal number of cells required in replicate experiments to return statistically similar contacts is different across the technologies, being lowest in SPRITE and highest in GAM under the same conditions. Noise-to-signal levels follow an inverse power law with detection efficiency and grow with genomic distance differently among the three methods, being lowest in GAM for genomic separations >1 Mb.
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http://dx.doi.org/10.1038/s41592-021-01135-1DOI Listing
May 2021

GAMIBHEAR: whole-genome haplotype reconstruction from Genome Architecture Mapping data.

Bioinformatics 2021 Apr 8. Epub 2021 Apr 8.

Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Hannoversche Str. 28, 10115 Berlin, Germany.

Motivation: Genome Architecture Mapping (GAM) was recently introduced as a digestion- and ligation-free method to detect chromatin conformation. Orthogonal to existing approaches based on chromatin conformation capture (3C), GAM's ability to capture both inter- and intra-chromosomal contacts from low amounts of input data makes it particularly well suited for allele-specific analyses in a clinical setting. Allele-specific analyses are powerful tools to investigate the effects of genetic variants on many cellular phenotypes including chromatin conformation, but require the haplotypes of the individuals under study to be known a-priori. So far however, no algorithm exists for haplotype reconstruction and phasing of genetic variants from GAM data, hindering the allele-specific analysis of chromatin contact points in non-model organisms or individuals with unknown haplotypes.

Results: We present GAMIBHEAR, a tool for accurate haplotype reconstruction from GAM data. GAMIBHEAR aggregates allelic co-observation frequencies from GAM data and employs a GAM-specific probabilistic model of haplotype capture to optimise phasing accuracy. Using a hybrid mouse embryonic stem cell line with known haplotype structure as a benchmark dataset, we assess correctness and completeness of the reconstructed haplotypes, and demonstrate the power of GAMIBHEAR to infer accurate genome-wide haplotypes from GAM data.

Availability: GAMIBHEAR is available as an R package under the open source GPL-2 license at https://bitbucket.org/schwarzlab/gamibhear.

Maintainer: julia.markowski@mdc-berlin.de.

Supplementary Information: Supplementary information is available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab238DOI Listing
April 2021

RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation.

Mol Syst Biol 2017 10 16;13(10):946. Epub 2017 Oct 16.

Epigenetic Regulation and Chromatin Architecture, Max Delbrück Center for Molecular Medicine, Berlin, Germany

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658700PMC
http://dx.doi.org/10.15252/msb.20177754DOI Listing
October 2017

Deficiency of Cks1 Leads to Learning and Long-Term Memory Defects and p27 Dependent Formation of Neuronal Cofilin Aggregates.

Cereb Cortex 2017 01;27(1):11-23

Eukaryotic Chromatin Dynamics Group, MRC Clinical Sciences Centre, Imperial College Hammersmith Campus, London W12 0NN, UK.

In mitotic cells, the cyclin-dependent kinase (CDK) subunit protein CKS1 regulates S phase entry by mediating degradation of the CDK inhibitor p27. Although mature neurons lack mitotic CDKs, we found that CKS1 was actively expressed in post-mitotic neurons of the adult hippocampus. Interestingly, Cks1 knockout (Cks1-/-) mice exhibited poor long-term memory, and diminished maintenance of long-term potentiation in the hippocampal circuits. Furthermore, there was neuronal accumulation of cofilin-actin rods or cofilin aggregates, which are associated with defective dendritic spine maturation and synaptic loss. We further demonstrated that it was the increased p27 level that activated cofilin by suppressing the RhoA kinase-mediated inhibitory phosphorylation of cofilin, resulting in the formation of cofilin aggregates in the Cks1-/- neuronal cells. Consistent with reports that the peptidyl-prolyl-isomerase PIN1 competes with CKS1 for p27 binding, we found that inhibition of PIN1 diminished the formation of cofilin aggregates through decreasing p27 levels, thereby activating RhoA and increasing cofilin phosphorylation. Our results revealed that CKS1 is involved in normal glutamatergic synapse development and dendritic spine maturation in adult hippocampus through modulating p27 stability.
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http://dx.doi.org/10.1093/cercor/bhw354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939225PMC
January 2017

Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells.

Elife 2015 Dec 19;4. Epub 2015 Dec 19.

Epigenetic Regulation and Chromatin Architecture Group, Berlin Institute for Medical Systems Biology, Max-Delbrück Centre for Molecular Medicine, Berlin, Germany.

Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.
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http://dx.doi.org/10.7554/eLife.11215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758952PMC
December 2015

The CDK subunit CKS2 counteracts CKS1 to control cyclin A/CDK2 activity in maintaining replicative fidelity and neurodevelopment.

Dev Cell 2012 Aug;23(2):356-70

Eukaryotic Chromatin Dynamics Group, MRC Clinical Sciences Centre, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCF(SKP2) to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2(-/-) knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2(-/-) cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In vivo, Cks2(-/-) cortical progenitor cells are limited in their capacity to differentiate into mature neurons, a phenotype akin to animals lacking p27. We propose that the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.
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http://dx.doi.org/10.1016/j.devcel.2012.06.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898080PMC
August 2012

Cks1 activates transcription by binding to the ubiquitylated proteasome.

Mol Cell Biol 2010 Aug 1;30(15):3894-901. Epub 2010 Jun 1.

Eukaryotic Chromatin Dynamics Group, MRC Clinical Sciences Centre, Imperial College Hammersmith Campus, London, United Kingdom.

Cyclin-dependent kinase-associated protein 1 (Cks1) is involved in the control of the transcription of a subset of genes in addition to its role in controlling the cell cycle in the budding yeast Saccharomyces cerevisiae. By directly ligating Cks1 onto a GAL1 promoter-driven reporter, we demonstrated that Cks1 acts as a transcription activator. Using this method, we dissected the downstream events from Cks1 recruitment at the promoter. We showed that subsequent to promoter binding, Cdc28 binding is required to modulate the level of gene expression. The ubiquitin-binding domain of Cks1 is essential for implementing downstream transcription events, which appears to recruit the proteasome via ubiquitylated proteasome subunits. We propose that the selective ability of Cks1 to bind ubiquitin allows this small molecule the flexibility to bind large protein complexes with specificity and that this may represent a novel mechanism of regulating transcriptional activation.
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http://dx.doi.org/10.1128/MCB.00655-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2916402PMC
August 2010

Nuclear myosin 1 is in complex with mature rRNA transcripts and associates with the nuclear pore basket.

FASEB J 2010 Jan 3;24(1):146-57. Epub 2009 Sep 3.

Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden.

In rRNA biogenesis, nuclear myosin 1 (NM1) and actin synergize to activate rRNA gene transcription. Evidence that actin is in preribosomal subunits and NM1 may control rRNA biogenesis post-transcriptionally prompted us to investigate whether NM1 associates with and accompanies rRNA to nuclear pores (NPC). Ultracentrifugation on HeLa nucleolar extracts showed RNA-dependent NM1 coelution with preribosomal subunits. In RNA immunoprecipitations (RIPs), NM1 coprecipitated with pre-rRNAs and 18S, 5.8S, and 28S rRNAs, but failed to precipitate 5S rRNA and 7SL RNA. In isolated nuclei and living HeLa cells, NM1 or actin inhibition and selective alterations in actin polymerization impaired 36S pre-rRNA processing. Immunoelectron microscopy (IEM) on sections of manually isolated Xenopus oocyte nuclei showed NM1 localization at the NPC basket. Field emission scanning IEM on isolated nuclear envelopes and intranuclear content confirmed basket localization and showed that NM1 decorates actin-rich pore-linked filaments. Finally, RIP and successive RIPs (reRIPs) on cross-linked HeLa cells demonstrated that NM1, CRM1, and Nup153 precipitate same 18S and 28S rRNAs but not 5S rRNA. We conclude that NM1 facilitates maturation and accompanies export-competent preribosomal subunits to the NPC, thus modulating export.
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http://dx.doi.org/10.1096/fj.09-135863DOI Listing
January 2010

A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-mesenchymal transition.

Nat Cell Biol 2009 Aug 13;11(8):943-50. Epub 2009 Jul 13.

Ludwig Institute for Cancer Research, Stockholm Branch, 17177 Stockholm, Sweden.

Epithelial-mesenchymal transition (EMT) is essential for organogenesis and is triggered during carcinoma progression to an invasive state. Transforming growth factor-beta (TGF-beta) cooperates with signalling pathways, such as Ras and Wnt, to induce EMT, but the molecular mechanisms are not clear. Here, we report that SMAD3 and SMAD4 interact and form a complex with SNAIL1, a transcriptional repressor and promoter of EMT. The SNAIL1-SMAD3/4 complex was targeted to the gene promoters of CAR, a tight-junction protein, and E-cadherin during TGF-beta-driven EMT in breast epithelial cells. SNAIL1 and SMAD3/4 acted as co-repressors of CAR, occludin, claudin-3 and E-cadherin promoters in transfected cells. Conversely, co-silencing of SNAIL1 and SMAD4 by siRNA inhibited repression of CAR and occludin during EMT. Moreover, loss of CAR and E-cadherin correlated with nuclear co-expression of SNAIL1 and SMAD3/4 in a mouse model of breast carcinoma and at the invasive fronts of human breast cancer. We propose that activation of a SNAIL1-SMAD3/4 transcriptional complex represents a mechanism of gene repression during EMT.
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http://dx.doi.org/10.1038/ncb1905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769970PMC
August 2009

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation.

Cell Cycle 2008 Oct 18;7(20):3180-6. Epub 2008 Oct 18.

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase delta-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase delta and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.
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http://dx.doi.org/10.4161/cc.7.20.6813DOI Listing
October 2008

The histone acetyltransferase PCAF associates with actin and hnRNP U for RNA polymerase II transcription.

Mol Cell Biol 2008 Oct 18;28(20):6342-57. Epub 2008 Aug 18.

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.

Actin is a key regulator of RNA polymerase (pol) II transcription. In complex with specific hnRNPs, it has been proposed that actin functions to recruit pol II coactivators during the elongation of nascent transcripts. Here, we show by affinity chromatography, protein-protein interaction assays, and biochemical fractionation of nuclear extracts that the histone acetyltransferase (HAT) PCAF associates with actin and hnRNP U. PCAF and the nuclear actin-associated HAT activity detected in the DNase I-bound protein fraction could be released by disruption of the actin-hnRNP U complex. In addition, actin, hnRNP U, and PCAF were found to be associated with the Ser2/5- and Ser2-phosphorylated pol II carboxy-terminal domain construct. Chromatin and RNA immunoprecipitation assays demonstrated that actin, hnRNP U, and PCAF are present at the promoters and coding regions of constitutively expressed pol II genes and that they are associated with ribonucleoprotein complexes. Finally, disruption of the actin-hnRNP U interaction repressed bromouridine triphosphate incorporation in living cells, suggesting that actin and hnRNP U cooperate with PCAF in the regulation of pol II transcription elongation.
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http://dx.doi.org/10.1128/MCB.00766-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2577438PMC
October 2008

Two splicing isoforms of the Y-box protein ctYB-1 appear on the same mRNA molecule.

FEBS J 2007 Jan;274(1):202-11

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.

Y-box proteins constitute an evolutionarily conserved family of DNA- and RNA-binding proteins involved in the regulation of transcription and translation. In the dipteran Chironomus tentans, a homologue to the vertebrate Y-box protein YB-1 was recently characterized and designated ctYB-1. It is transferred from nucleus to cytoplasm bound to mRNA and is likely to affect translation. It appears in two size variants, p40 and p50. We further analysed the two size variants and their interaction with mRNA. Southern blot analysis, in situ hybridization and RT-PCR analysis suggested that there is just one YB-1 gene, and that the two size variants represent splicing isoforms. In a C. tentans epithelial cell line, only p40 is present, whereas both variants appear together in eight tissues from fourth-instar larvae, although in somewhat different proportions. Furthermore, the appearance of the two isoforms was studied in relation to a specific 35-40 kb mRNA transcript in the salivary glands, the Balbiani ring mRNA. Because of their exceptional size, Balbiani ring messenger ribonucleoprotein particles in nucleoplasm and Balbiani ring polysomes in cytoplasm could be identified and selectively studied. We were able to establish that both isoforms are associated with both nuclear and cytoplasmic Balbiani ring mRNA. In addition, a p50-specific antibody coimmunoprecipitated p40 from Balbiani ring polysomes, suggesting that the two splicing isoforms are located along the same Balbiani ring mRNA molecule. The functional significance of the two isoforms is being discussed.
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http://dx.doi.org/10.1111/j.1742-4658.2006.05576.xDOI Listing
January 2007

Actin and hnRNP U cooperate for productive transcription by RNA polymerase II.

Nat Struct Mol Biol 2005 Mar 13;12(3):238-44. Epub 2005 Feb 13.

Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, S-171 77, Stockholm, Sweden.

To determine the role of actin-ribonucleoprotein complexes in transcription, we set out to identify novel actin-binding proteins associated with RNA polymerase II (Pol II). Using affinity chromatography on fractionated HeLa cells, we found that hnRNP U binds actin through a short amino acid sequence in its C-terminal domain. Post-transcriptional gene silencing of hnRNP U and nuclear microinjections of a short peptide encompassing the hnRNP U actin-binding sequence inhibited BrUTP incorporation in vivo. In living cells, we found that both actin and hnRNP U are associated with the phosphorylated C-terminal domain of Pol II, and antibodies to actin and hnRNP U blocked Pol II-mediated transcription. Taken together, our results indicate that a general actin-based mechanism is implicated in the transcription of most Pol II genes. Actin in complex with hnRNP U may carry out its regulatory role during the initial phases of transcription activation.
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http://dx.doi.org/10.1038/nsmb904DOI Listing
March 2005