Developing a peptide to disrupt cohesin head domain interactions.

Cohesin mediates the 3-D structure of chromatin and is involved in maintaining genome stability and function. The cohesin core comprises Smc1 and Smc3, elongated-shaped proteins that dimerize through globular domains at their edges, called head and hinge. ATP binding to the Smc heads induces their dimerization and the formation of two active sites, while ATP hydrolysis results in head disengagement. This ATPase cycle is essential for driving cohesin activity. We report on the development of the first cohesin-inhibiting peptide (CIP). The CIP binds Smc3 in vitro and inhibits the ATPase activity of the holocomplex. Treating yeast cells with the CIP prevents cohesin's tethering activity and, interestingly, leads to the accumulation of cohesin on chromatin. CIP3 also affects cohesin activity in human cells. Altogether, we demonstrate the power of peptides to inhibit cohesin in cells and discuss the potential application of CIPs as a therapeutic approach.

UMSBP2 is chromatin remodeler that functions in regulation of gene expression and suppression of antigenic variation in trypanosomes.

Universal Minicircle Sequence binding proteins (UMSBPs) are CCHC-type zinc-finger proteins that bind the single-stranded G-rich UMS sequence, conserved at the replication origins of minicircles in the kinetoplast DNA, the mitochondrial genome of kinetoplastids. Trypanosoma brucei UMSBP2 has been recently shown to colocalize with telomeres and to play an essential role in chromosome end protection. Here we report that TbUMSBP2 decondenses in vitro DNA molecules, which were condensed by core histones H2B, H4 or linker histone H1. DNA decondensation is mediated via protein-protein interactions between TbUMSBP2 and these histones, independently of its previously described DNA binding activity. Silencing of the TbUMSBP2 gene resulted in a significant decrease in the disassembly of nucleosomes in T. brucei chromatin, a phenotype that could be reverted, by supplementing the knockdown cells with TbUMSBP2. Transcriptome analysis revealed that silencing of TbUMSBP2 affects the expression of multiple genes in T. brucei, with a most significant effect on the upregulation of the subtelomeric variant surface glycoproteins (VSG) genes, which mediate the antigenic variation in African trypanosomes. These observations suggest that UMSBP2 is a chromatin remodeling protein that functions in the regulation of gene expression and plays a role in the control of antigenic variation in T. brucei.

It's all in the numbers: Cohesin stoichiometry.

Cohesin, a structural maintenance of chromosome (SMC) complex, organizes chromatin into three-dimensional structures by threading chromatin into loops and stabilizing long-range chromatin interactions. Four subunits in a 1:1:1:1 ratio compose the cohesin core, which is regulated by auxiliary factors that interact with or modify the core subunits. An ongoing debate about cohesin's mechanism of action regards its stoichiometry. Namely, is cohesin activity mediated by a single complex or cooperation between several complexes that organize into dimers or oligomers? Several investigations that used various experimental approaches have tried to resolve this dispute. Some have convincingly demonstrated that the cohesin monomer is the active unit. However, others have revealed the formation of cohesin dimers and higher-order clusters on and off chromosomes. Elucidating the biological function of cohesin clusters and determining what regulates their formation are just two of the many new questions raised by these findings. We briefly review the history of the argument about cohesin stoichiometry and the central evidence for cohesin activity as a monomer vs. an oligomer. Finally, we discuss the possible biological significance of cohesin oligomerization and present open questions that remain to be answered.

Fold-change of chromatin condensation in yeast is a conserved property.

During mitosis, chromatin is condensed and organized into mitotic chromosomes. Condensation is critical for genome stability and dynamics, yet the degree of condensation is significantly different between multicellular and single-cell eukaryotes. What is less clear is whether there is a minimum degree of chromosome condensation in unicellular eukaryotes. Here, we exploited two-photon microscopy to analyze chromatin condensation in live and fixed cells, enabling studies of some organisms that are not readily amenable to genetic modification. This includes the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, and Candida albicans, as well as a protist Trypanosoma brucei. We found that mitotic chromosomes in this range of species are condensed about 1.5-fold relative to interphase chromatin. In addition, we used two-photon microscopy to reveal that chromatin reorganization in interphase human hepatoma cells infected by the hepatitis C virus is decondensed compared to uninfected cells, which correlates with the previously reported viral-induced changes in chromatin dynamics. This work demonstrates the power of two-photon microscopy to analyze chromatin in a broad range of cell types and conditions, including non-model single-cell eukaryotes. We suggest that similar condensation levels are an evolutionarily conserved property in unicellular eukaryotes and important for proper chromosome segregation. Furthermore, this provides new insights into the process of chromatin condensation during mitosis in unicellular organisms as well as the response of human cells to viral infection.

Disrupting the MAD2L2-Rev1 Complex Enhances Cell Death upon DNA Damage.

DNA-damaging chemotherapy agents such as cisplatin have been the first line of treatment for cancer for decades. While chemotherapy can be very effective, its long-term success is often reduced by intrinsic and acquired drug resistance, accompanied by chemotherapy-resistant secondary malignancies. Although the mechanisms causing drug resistance are quite distinct, they are directly connected to mutagenic translesion synthesis (TLS). The TLS pathway promotes DNA damage tolerance by supporting both replication opposite to a lesion and inaccurate single-strand gap filling. Interestingly, inhibiting TLS reduces both cisplatin resistance and secondary tumor formation. Therefore, TLS targeting is a promising strategy for improving chemotherapy. MAD2L2 (i.e., Rev7) is a central protein in TLS. It is an essential component of the TLS polymerase zeta (ζ), and it forms a regulatory complex with Rev1 polymerase. Here we present the discovery of two small molecules, c#2 and c#3, that directly bind both in vitro and in vivo to MAD2L2 and influence its activity. Both molecules sensitize lung cancer cell lines to cisplatin, disrupt the formation of the MAD2L2-Rev1 complex and increase DNA damage, hence underlining their potential as lead compounds for developing novel TLS inhibitors for improving chemotherapy treatments.

Hit the brakes - a new perspective on the loop extrusion mechanism of cohesin and other SMC complexes.

The three-dimensional structure of chromatin is determined by the action of protein complexes of the structural maintenance of chromosome (SMC) family. Eukaryotic cells contain three SMC complexes, cohesin, condensin, and a complex of Smc5 and Smc6. Initially, cohesin was linked to sister chromatid cohesion, the process that ensures the fidelity of chromosome segregation in mitosis. In recent years, a second function in the organization of interphase chromatin into topologically associated domains has been determined, and loop extrusion has emerged as the leading mechanism of this process. Interestingly, fundamental mechanistic differences exist between mitotic tethering and loop extrusion. As distinct molecular switches that aim to suppress loop extrusion in different biological contexts have been identified, we hypothesize here that loop extrusion is the default biochemical activity of cohesin and that its suppression shifts cohesin into a tethering mode. With this model, we aim to provide an explanation for how loop extrusion and tethering can coexist in a single cohesin complex and also apply it to the other eukaryotic SMC complexes, describing both similarities and differences between them. Finally, we present model-derived molecular predictions that can be tested experimentally, thus offering a new perspective on the mechanisms by which SMC complexes shape the higher-order structure of chromatin.

Chromosome loading of cohesin depends on conserved residues in Scc3.

Cohesin is essential for sister chromatid cohesion, which ensures equal segregation of the chromatids to daughter cells. However, the molecular mechanism by which cohesin mediates this function is elusive. Scc3, one of the four core subunits of cohesin, is vital to cohesin activity. However, the mechanism by which Scc3 contributes to the activity and identity of its functional domains is not fully understood. Here, we describe an in-frame five-amino acid insertion mutation after glutamic acid 704 (scc3-E704ins) in yeast Scc3, located in the middle of the second armadillo repeat. Mutated cohesin-scc3-E704ins complexes are unable to establish cohesion. Detailed molecular and genetic analyses revealed that the mutated cohesin has reduced affinity to the Scc2 loader. This inhibits its enrichment at centromeres and chromosomal arms. Mutant complexes show a slow diffusion rate in live cells suggesting that they induce a major conformational change in the complex. The analysis of systematic mutations in the insertion region of Scc3 revealed two conserved aspartic acid residues that are essential for the activity. The study offers a better understanding of the contribution of Scc3 to cohesin activity and the mechanism by which cohesin tethers the sister chromatids during the cell cycle.

Analyzing chromosome condensation in yeast by second-harmonic generation microscopy.

Condensation is a fundamental property of mitotic chromosomes in eukaryotic cells. However, analyzing chromosome condensation in yeast is a challenging task while existing methods have notable weaknesses. Second-harmonic generation (SHG) microscopy is a label-free, advanced imaging technique for measuring the surface curve of isotropic molecules such as chromatin in live cells. We applied this method to detect changes in chromatin organization throughout the cell cycle in live yeast cells. We showed that SHG microscopy can be used to identify changes in chromatin organization throughout the cell cycle and in response to inactivation of the SMC complexes, cohesin and condensin. Implementation of this method will improve our ability to analyze chromatin structure in protozoa and will enhance our understanding of chromatin organization in eukaryotic cells.

Monomeric cohesin state revealed by live-cell single-molecule spectroscopy.

The cohesin complex plays an important role in the maintenance of genome stability. Cohesin is composed of four core subunits and a set of regulatory subunits that interact with the core subunits. Less is known about cohesin dynamics in live cells and on the contribution of individual subunits to the overall complex. Understanding the tethering mechanism of cohesin is still a challenge, especially because the proposed mechanisms are still not conclusive. Models proposed to describe tethering depend on either the monomeric cohesin ring or a cohesin dimer. Here, we investigate the role of cohesin dynamics and stoichiometry in live yeast cells at single-molecule resolution. We explore the effect of regulatory subunit deletion on cohesin mobility and found that depletion of different regulatory subunits has opposing effects. Finally, we show that cohesin exists mostly as a canonical monomer throughout the cell cycle, and its monomeric form is independent of its regulatory factors. Our results demonstrate that single-molecule tools have the potential to provide new insights into the cohesin mechanism of action in live cells.

Alternative Functional rad21 Paralogs in Fusarium oxysporum.

Cohesin, the sister chromatid cohesion complex, is an essential complex that ensures faithful sister chromatid segregation in eukaryotes. It also participates in DNA repair, transcription and maintenance of chromosome structure. Mitotic cohesin is composed of Smc1, Smc3, Scc3, and Rad21/Mcd1. The meiotic cohesin complex contains Rec8, a Rad21 paralog and not Rad21 itself. Very little is known about sister chromatid cohesion in fungal plant pathogens. Fusarium oxysporum is an important fungal plant pathogen without known sexual life cycle. Here, we describe that F. oxysporum encodes for three Rad21 paralogs; Rad21, Rec8, and the first alternative Rad21 paralog in the phylum of ascomycete. This last paralog is found only in several fungal plant pathogens from the Fusarium family and thus termed rad21nc (non-conserved). Conserved rad21 (rad21c), rad21nc, and rec8 genes are expressed in F. oxysporum although the expression of rad21c is much higher than the other paralogs. F. oxysporum strains deleted for the rad21nc or rec8 genes were analyzed for their role in fungal life cycle. Δrad21nc and Δrec8 single mutants were proficient in sporulation, conidia germination, hyphal growth and pathogenicity under optimal growth conditions. Interestingly, Δrad21nc and Δrec8 single mutants germinate less effectively than wild type (WT) strains under DNA replication and mitosis stresses. We provide here the first genetic analysis of alternative rad21nc and rec8 paralogs in filamentous fungi. Our results suggest that rad21nc and rec8 may have a unique role in cell cycle related functions of F. oxysporum.

The chromatin remodeler Chd1 regulates cohesin in budding yeast and humans.

Chd1 is a chromatin remodeler that is involved in nucleosome positioning and transcription. Deletion of CHD1 is a frequent event in prostate cancer. The Structural Maintenance of Chromosome (SMC) complex cohesin mediates long-range chromatin interactions and is involved in maintaining genome stability. We provide new evidence that Chd1 is a regulator of cohesin. In the yeast S. cerevisiae, Chd1 is not essential for viability. We show that deletion of the gene leads to a defect in sister chromatid cohesion and in chromosome morphology. Chl1 is a non-essential DNA helicase that has been shown to regulate cohesin loading. Surprisingly, co-deletion of CHD1 and CHL1 results in an additive cohesion defect but partial suppression of the chromosome structure phenotype. We found that the cohesin regulator Pds5 is overexpressed when Chd1 and Chl1 are deleted. However, Pds5 expression is reduced to wild type levels when both genes are deleted. Finally, we show a correlation in the expression of CHD1 and cohesin genes in prostate cancer patients. Furthermore, we show that overexpression of cohesin subunits is correlated with the aggressiveness of the tumor. The biological roles of the interplay between Chd1, Chl1 and SMCs are discussed.

Identifying Functional Domains in Subunits of Structural Maintenance of Chromosomes (SMC) Complexes by Transposon Mutagenesis Screen in Yeast.

Structural maintenance of chromosomes (SMC) complexes mediate higher order chromosome structures. Eukaryotic cells contain three distinct SMC complexes called cohesin, condensin, and SMC5/6, which share the same basic architecture. The core of SMC complexes contains a heterodimer of SMC proteins, a kleisin subunit, and a set of regulatory proteins that contain HEAT and Armadillo (ARM) repeat protein-protein interaction motifs. A major challenge in studying SMC proteins and their auxiliary factors is identifying their functional domains. Bioinformatics is not an efficient way to achieve this goal because of the absence of defined sequence and structural motifs. Functional domains can be identified experimentally by performing a genetic screen and isolating functional mutants. While there are several strategies to conduct a screen, the quaternary structure of SMCs makes them excellent candidates to transposon-based random insertion mutagenesis, followed by selection of dominant negative mutants. In this chapter we list the advantages of this approach in the context of SMC complexes. We provide a detailed protocol for performing the screen in S. cerevisiae and use data from our recently reported screen on the ARM repeat protein, Scc4, to demonstrate the key steps in the protocol.

Dysregulation of the cohesin subunit RAD21 by Hepatitis C virus mediates host-virus interactions.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

A new twist in the coil: functions of the coiled-coil domain of structural maintenance of chromosome (SMC) proteins.

The higher-order organization of chromosomes ensures their stability and functionality. However, the molecular mechanism by which higher order structure is established is poorly understood. Dissecting the activity of the relevant proteins provides information essential for achieving a comprehensive understanding of chromosome structure. Proteins of the structural maintenance of chromosome (SMC) family of ATPases are the core of evolutionary conserved complexes. SMC complexes are involved in regulating genome dynamics and in maintaining genome stability. The structure of all SMC proteins resembles an elongated rod that contains a central coiled-coil domain, a common protein structural motif in which two α-helices twist together. In recent years, the imperative role of the coiled-coil domain to SMC protein activity and regulation has become evident. Here, we discuss recent advances in the function of the SMC coiled coils. We describe the structure of the coiled-coil domain of SMC proteins, modifications and interactions that are mediated by it. Furthermore, we assess the role of the coiled-coil domain in conformational switches of SMC proteins, and in determining the architecture of the SMC dimer. Finally, we review the interplay between mutations in the coiled-coil domain and human disorders. We suggest that distinctive properties of coiled coils of different SMC proteins contribute to their distinct functions. The discussion clarifies the mechanisms underlying the activity of SMC proteins, and advocates future studies to elucidate the function of the SMC coiled coil domain.

The emerging roles for the chromatin structure regulators CTCF and cohesin in neurodevelopment and behavior.

Recent genetic and technological advances have determined a role for chromatin structure in neurodevelopment. In particular, compounding evidence has established roles for CTCF and cohesin, two elements that are central in the establishment of chromatin structure, in proper neurodevelopment and in regulation of behavior. Genetic aberrations in CTCF, and in subunits of the cohesin complex, have been associated with neurodevelopmental disorders in human genetic studies, and subsequent animal studies have established definitive, although sometime opposing roles, for these factors in neurodevelopment and behavior. Considering the centrality of these factors in cellular processes in general, the mechanisms through which dysregulation of CTCF and cohesin leads specifically to neurological phenotypes is intriguing, although poorly understood. The connection between CTCF, cohesin, chromatin structure, and behavior is likely to be one of the next frontiers in our understanding of the development of behavior in general, and neurodevelopmental disorders in particular.

Identification of a region in the coiled-coil domain of Smc3 that is essential for cohesin activity.

The cohesin complex plays an important role in sister chromatin cohesion. Cohesin's core is composed of two structural maintenance of chromosome (SMC) proteins, called Smc1 and Smc3. SMC proteins are built from a globular hinge domain, a rod-shaped domain composed of long anti-parallel coiled-coil (CC), and a second globular adenosine triphosphatase domain called the head. The functions of both head and hinge domains have been studied extensively, yet the function of the CC region remains elusive. We identified a mutation in the CC of smc3 (L217P) that disrupts the function of the protein. Cells carrying the smc3-L217P allele have a strong cohesion defect and complexes containing smc3-L217P are not loaded onto the chromosomes. However, the mutation does not affect inter-protein interactions in either the core complex or with the Scc2 loader. We show by molecular dynamics and biochemistry that wild-type Smc3 can adopt distinct conformations, and that adenosine triphosphate (ATP) induces the conformational change. The L217P mutation restricts the ability of the mutated protein to switch between the conformations. We suggest that the function of the CC is to transfer ATP binding/hydrolysis signals between the head and the hinge domains. The results provide a new insight into the mechanism of cohesin activity.

Identification of Functional Domains in the Cohesin Loader Subunit Scc4 by a Random Insertion/Dominant Negative Screen.

Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader-a heterodimer composed of Scc4 and Scc2 However, very little is known about the loader's mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2 Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism.

[TEACHING BIO-MEDICAL INFORMATICS TO MEDICAL STUDENTS IN THE FACULTY OF MEDICINE IN THE GALILEE - GOALS, LESSONS AND A FUTURE PERSPECTIVE].

Bioinformatics is a scientific discipline that deals with the processing of biological data by computers. In recent years, bioinformatic tools were applied to the analysis of medical databases in order to develop new pathways for diagnosis and to improve medical treatment. The best example is personalized medicine, which depends on bioinformatic analysis. Despite early assessments, bioinformatics didn't change the clinical landscape dramatically, and personalized medicine is still not a main approach in healthcare. One of the holdbacks is the knowledge gap among clinicians. Therefore, massive integration of bioinformatics into the clinic will most likely be the challenge of the new generations of clinicians to come. As part of the innovative curriculum of the newly established Faculty of Medicine in the Galilee, Bar-Ilan University, it took up the challenge of teaching bioinformatics, and by doing so, joined some of the leading medical schools worldwide. In this review we will provide a few examples for the use of bioinformatics in the clinic. Furthermore, we will describe the content of the bioinformatics course in the Faculty of Medicine in the Galilee and discuss some of the lessons learned and future perspectives.

A conserved domain in the scc3 subunit of cohesin mediates the interaction with both mcd1 and the cohesin loader complex.

The Structural Maintenance of Chromosome (SMC) complex, termed cohesin, is essential for sister chromatid cohesion. Cohesin is also important for chromosome condensation, DNA repair, and gene expression. Cohesin is comprised of Scc3, Mcd1, Smc1, and Smc3. Scc3 also binds Pds5 and Wpl1, cohesin-associated proteins that regulate cohesin function, and to the Scc2/4 cohesin loader. We mutagenized SCC3 to elucidate its role in cohesin function. A 5 amino acid insertion after Scc3 residue I358, or a missense mutation of residue D373 in the adjacent stromalin conservative domain (SCD) induce inviability and defects in both cohesion and cohesin binding to chromosomes. The I358 and D373 mutants abrogate Scc3 binding to Mcd1. These results define an Scc3 region extending from I358 through the SCD required for binding Mcd1, cohesin localization to chromosomes and cohesion. Scc3 binding to the cohesin loader, Pds5 and Wpl1 are unaffected in I358 mutant and the loader still binds the cohesin core trimer (Mcd1, Smc1 and Smc3). Thus, Scc3 plays a critical role in cohesin binding to chromosomes and cohesion at a step distinct from loader binding to the cohesin trimer. We show that residues Y371 and K372 within the SCD are critical for viability and chromosome condensation but dispensable for cohesion. However, scc3 Y371A and scc3 K372A bind normally to Mcd1. These alleles also provide evidence that Scc3 has distinct mechanisms of cohesin loading to different loci. The cohesion-competence, condensation-incompetence of Y371 and K372 mutants suggests that cohesin has at least one activity required specifically for condensation.

Structural maintenance of chromosome complexes and bone development: the beginning of a wonderful relationship?

Bone development depends on environmental, nutritional and hormonal factors. Yet, an ordered and timed activation of genes and their associated molecular pathways are central for the growth and development of healthy bones. The correct expression of genes depends on both cis- and trans-regulatory elements. Of these, the elusive role of chromatin ultrastructure is just beginning to become appreciated. Changes in the higher-order structure of chromatin are affecting the expression of genes in response to intrinsic and environmental signals. Cohesin and condensin are members of the structural maintenance of chromosome (SMC) family of protein complexes, which mediate higher-order chromatin structure by tethering distinct regions of chromatin either inter- or intra-molecularly. In recent years, SMCs had been identified for their function in the regulation of gene expression and developmental processes, whereas malfunction of cohesin or condensin has an impact on human health. However, little is known about the specific roles of SMC complexes in bone development and their possible effect on bone health. Here, we review studies that suggest an intimate link between SMCs and bone development, as well as a plausible effect, direct or indirect, on the bone health. We describe genetic syndromes associated with SMCs with distinctive bone phenotypes and identify links between SMCs and bone-related molecular pathways. Future studies of the relationship between SMCs and bone development will reveal new understandings of both the cellular and molecular roles of SMC complexes and provide new insights into the growth and developmental processes in the bone.