Rules of engagement for condensins and cohesins guide mitotic chromosome formation.

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at ~1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

The little skate genome and the evolutionary emergence of wing-like fins.

Skates are cartilaginous fish whose body plan features enlarged wing-like pectoral fins, enabling them to thrive in benthic environments1,2. However, the molecular underpinnings of this unique trait remain unclear. Here we investigate the origin of this phenotypic innovation by developing the little skate Leucoraja erinacea as a genomically enabled model. Analysis of a high-quality chromosome-scale genome sequence for the little skate shows that it preserves many ancestral jawed vertebrate features compared with other sequenced genomes, including numerous ancient microchromosomes. Combining genome comparisons with extensive regulatory datasets in developing fins-including gene expression, chromatin occupancy and three-dimensional conformation-we find skate-specific genomic rearrangements that alter the three-dimensional regulatory landscape of genes that are involved in the planar cell polarity pathway. Functional inhibition of planar cell polarity signalling resulted in a reduction in anterior fin size, confirming that this pathway is a major contributor to batoid fin morphology. We also identified a fin-specific enhancer that interacts with several hoxa genes, consistent with the redeployment of hox gene expression in anterior pectoral fins, and confirmed its potential to activate transcription in the anterior fin using zebrafish reporter assays. Our findings underscore the central role of genome reorganization and regulatory variation in the evolution of phenotypes, shedding light on the molecular origin of an enigmatic trait.

Cohesin-mediated loop anchors confine the locations of human replication origins.

DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.

Systematic evaluation of chromosome conformation capture assays.

Chromosome conformation capture (3C) assays are used to map chromatin interactions genome-wide. Chromatin interaction maps provide insights into the spatial organization of chromosomes and the mechanisms by which they fold. Hi-C and Micro-C are widely used 3C protocols that differ in key experimental parameters including cross-linking chemistry and chromatin fragmentation strategy. To understand how the choice of experimental protocol determines the ability to detect and quantify aspects of chromosome folding we have performed a systematic evaluation of 3C experimental parameters. We identified optimal protocol variants for either loop or compartment detection, optimizing fragment size and cross-linking chemistry. We used this knowledge to develop a greatly improved Hi-C protocol (Hi-C 3.0) that can detect both loops and compartments relatively effectively. In addition to providing benchmarked protocols, this work produced ultra-deep chromatin interaction maps using Micro-C, conventional Hi-C and Hi-C 3.0 for key cell lines used by the 4D Nucleome project.

Hi-C 3.0: Improved Protocol for Genome-Wide Chromosome Conformation Capture.

The intricate folding of chromatin enables living organisms to store genomic material in an extremely small volume while facilitating proper cell function. Hi-C is a chromosome conformation capture (3C)-based technology to detect pair-wise chromatin interactions genome-wide, and has become a benchmark tool to study genome organization. In Hi-C, chromatin conformation is first captured by chemical cross-linking of cells. Cells are then lysed and subjected to restriction enzyme digestion, before the ends of the resulting fragments are marked with biotin. Fragments within close 3D proximity are ligated, and the biotin label is used to selectively enrich for ligated junctions. Finally, isolated ligation products are prepared for high-throughput sequencing, which enables the mapping of pair-wise chromatin interactions genome-wide. Over the past decade, "next-generation" sequencing has become cheaper and easier to perform, enabling more interactions to be sampled to obtain higher resolution in chromatin interaction maps. Here, we provide an in-depth guide to performing an up-to-date Hi-C procedure on mammalian cell lines. These protocols include recent improvements that increase the resolution potential of the assay, namely by enhancing cross-linking and using a restriction enzyme cocktail. These improvements result in a versatile Hi-C procedure that enables the detection of genome folding features at a wide range of distances. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Fixation of nuclear conformation Basic Protocol 2: Chromosome conformation capture Basic Protocol 3: Hi-C sequencing library preparation.

A pathway for mitotic chromosome formation.

Mitotic chromosomes fold as compact arrays of chromatin loops. To identify the pathway of mitotic chromosome formation, we combined imaging and Hi-C analysis of synchronous DT40 cell cultures with polymer simulations. Here we show that in prophase, the interphase organization is rapidly lost in a condensin-dependent manner, and arrays of consecutive 60-kilobase (kb) loops are formed. During prometaphase, ~80-kb inner loops are nested within ~400-kb outer loops. The loop array acquires a helical arrangement with consecutive loops emanating from a central "spiral staircase" condensin scaffold. The size of helical turns progressively increases to ~12 megabases during prometaphase. Acute depletion of condensin I or II shows that nested loops form by differential action of the two condensins, whereas condensin II is required for helical winding.

Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization.

The molecular mechanisms underlying folding of mammalian chromosomes remain poorly understood. The transcription factor CTCF is a candidate regulator of chromosomal structure. Using the auxin-inducible degron system in mouse embryonic stem cells, we show that CTCF is absolutely and dose-dependently required for looping between CTCF target sites and insulation of topologically associating domains (TADs). Restoring CTCF reinstates proper architecture on altered chromosomes, indicating a powerful instructive function for CTCF in chromatin folding. CTCF remains essential for TAD organization in non-dividing cells. Surprisingly, active and inactive genome compartments remain properly segregated upon CTCF depletion, revealing that compartmentalization of mammalian chromosomes emerges independently of proper insulation of TADs. Furthermore, our data support that CTCF mediates transcriptional insulator function through enhancer blocking but not as a direct barrier to heterochromatin spreading. Beyond defining the functions of CTCF in chromosome folding, these results provide new fundamental insights into the rules governing mammalian genome organization.

Hi-C 2.0: An optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation.

Chromosome conformation capture-based methods such as Hi-C have become mainstream techniques for the study of the 3D organization of genomes. These methods convert chromatin interactions reflecting topological chromatin structures into digital information (counts of pair-wise interactions). Here, we describe an updated protocol for Hi-C (Hi-C 2.0) that integrates recent improvements into a single protocol for efficient and high-resolution capture of chromatin interactions. This protocol combines chromatin digestion and frequently cutting enzymes to obtain kilobase (kb) resolution. It also includes steps to reduce random ligation and the generation of uninformative molecules, such as unligated ends, to improve the amount of valid intra-chromosomal read pairs. This protocol allows for obtaining information on conformational structures such as compartment and topologically associating domains, as well as high-resolution conformational features such as DNA loops.

The hierarchy of the 3D genome.

Mammalian genomes encode genetic information in their linear sequence, but appropriate expression of their genes requires chromosomes to fold into complex three-dimensional structures. Transcriptional control involves the establishment of physical connections among genes and regulatory elements, both along and between chromosomes. Recent technological innovations in probing the folding of chromosomes are providing new insights into the spatial organization of genomes and its role in gene regulation. It is emerging that folding of large complex chromosomes involves a hierarchy of structures, from chromatin loops that connect genes and enhancers to larger chromosomal domains and nuclear compartments. The larger these structures are along this hierarchy, the more stable they are within cells, while becoming more stochastic between cells. Here, we review the experimental and theoretical data on this hierarchy of structures and propose a key role for the recently discovered topologically associating domains.

The context of gene expression regulation.

Recent advances in sequencing technologies have uncovered a world of RNAs that do not code for proteins, known as non-protein coding RNAs, that play important roles in gene regulation. Along with histone modifications and transcription factors, non-coding RNA is part of a layer of transcriptional control on top of the DNA code. This layer of components and their interactions specifically enables (or disables) the modulation of three-dimensional folding of chromatin to create a context for transcriptional regulation that underlies cell-specific transcription. In this perspective, we propose a structural and functional hierarchy, in which the DNA code, proteins and non-coding RNAs act as context creators to fold chromosomes and regulate genes.

FADD expression as a prognosticator in early-stage glottic squamous cell carcinoma of the larynx treated primarily with radiotherapy.

PURPOSE: We recently reported on the identification of the Fas-associated death domain (FADD) as a possible driver of the chromosome 11q13 amplicon and the association between increased FADD expression and disease-specific survival in advanced-stage laryngeal carcinoma. The aim of this study was to examine whether expression of FADD and its Ser194-phosphorylated isoform (pFADD) predicts local control in patients with early-stage glottic carcinoma primarily treated with radiotherapy only. METHODS AND MATERIALS: Immunohistochemical staining for FADD and pFADD was performed on pretreatment biopsy specimens of 92 patients with T1-T2 glottic squamous cell carcinoma primarily treated with radiotherapy between 1996 and 2005. Cox regression analysis was used to correlate expression levels with local control. RESULTS: High levels of pFADD were associated with significantly better local control (hazard ratio, 2.40; 95% confidence interval, 1.04-5.55; p = 0.040). FADD overexpression showed a trend toward better local control (hazard ratio, 3.656; 95% confidence interval, 0.853-15.663; p = 0.081). Multivariate Cox regression analysis showed that high pFADD expression was the best predictor of local control after radiotherapy. CONCLUSIONS: This study showed that expression of phosphorylated FADD is a new prognostic biomarker for better local control after radiotherapy in patients with early-stage glottic carcinomas.

Rapid generation of microRNA sponges for microRNA inhibition.

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

MiR-17/106b seed family regulates p21 in Hodgkin's lymphoma.

Hodgkin's lymphoma (HL) is a B cell-derived lymphoma characterized by a minority of malignant Hodgkin Reed-Sternberg (HRS) cells that have lost their normal B cell phenotype. Alterations in the cell cycle and apoptosis pathways might contribute to their resistance to apoptosis and sustained cell cycle progression. A key player in both cell cycle arrest and apoptosis is CDKN1A, encoding p21$^{{\rm{waf/cip1}}}$ (p21). P21 is regulated by p53 and can function as a cell cycle inhibitor when in the nucleus or as an apoptosis inhibitor when localized in the cytoplasm. We observed expression of p53, p21 and p-p21 in a variable number of HRS cells in 24 of 40 cases. Expression of miR-17 and miR-106a was detected in HRS cells of 10 HL cases. MiR-17/106b seed family members, CDKN1A RNA and p21 protein levels were variable in HL cell lines. We showed effective targeting of the CDKN1A 3' UTR by miR-17/106b in HL cell lines in a luciferase reporter assay and up-regulation of p21 protein levels upon anti-miR-17 treatment of KM-H2 cells. Functional studies indicated a p21-mediated G(1) arrest after miR-17/106b down-regulation in KM-H2, whereas no G(1) arrest was observed for U-HO1 and L428. This difference could not be explained by differences in the 3' UTR, the cellular location of p21 or expression variation during cell cycle progression. A strong correlation was observed for the miR-17/106b:CDKN1A ratio and the responsiveness to miR-17 inhibition, ie a low ratio in KM-H2 and an extremely high ratio in the two unresponsive HL cell lines. In conclusion, we show that miR-17/106b regulates p21 protein levels in HL and that the effect of miR-17/106b-mediated inhibition depends on the miRNA : target gene ratio. Thus, in HL high miR-17/106b expression contributes to a dysfunctional p53 pathway and thereby also to the malignant phenotype.

Cytoplasmic p21 expression levels determine cisplatin resistance in human testicular cancer.

Platinum-based chemotherapies such as cisplatin are used as first-line treatment for many cancers. Although there is often a high initial responsiveness, the majority of patients eventually relapse with platinum-resistant disease. For example, a subset of testicular cancer patients still die even though testicular cancer is considered a paradigm of cisplatin-sensitive solid tumors, but the mechanisms of chemoresistance remain elusive. Here, we have shown that one key determinant of cisplatin-resistance in testicular embryonal carcinoma (EC) is high cytoplasmic expression of the cyclin-dependent kinase (CDK) inhibitor p21. The EC component of the majority of refractory testicular cancer patients exhibited high cytoplasmic p21 expression, which protected EC cell lines against cisplatin-induced apoptosis via CDK2 inhibition. Localization of p21 in the cytoplasm was critical for cisplatin resistance, since relocalization of p21 to the nucleus by Akt inhibition sensitized EC cell lines to cisplatin. We also demonstrated in EC cell lines and human tumor tissue that high cytoplasmic p21 expression and cisplatin resistance of EC were inversely associated with the expression of Oct4 and miR-106b seed family members. Thus, targeting cytoplasmic p21, including by modulation of the Oct4/miR-106b/p21 pathway, may offer new strategies for the treatment of chemoresistant testicular and other types of cancer.

miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes.

MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitt's lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.

Hodgkin lymphoma cell lines are characterized by a specific miRNA expression profile.

Hodgkin lymphoma (HL) is derived from preapoptotic germinal center B cells, although a general loss of B cell phenotype is noted. Using quantitative reverse transcription-polymerase chain reaction and miRNA microarray, we determined the microRNA (miRNA) profile of HL and compared this with the profile of a panel of B-cell non-Hodgkin lymphomas. The two methods showed a strong correlation for the detection of miRNA expression levels. The HL-specific miRNA included miR-17-92 cluster members, miR-16, miR-21, miR-24, and miR-155. Using a large panel of cell lines, we found differential expression between HL and other B-cell lymphoma-derived cell lines for 27 miRNA. A significant down-regulation in HL compared to non-Hodgkin lymphoma was observed only for miR-150. Next, we performed target gene validation of predicted target genes for miR-155, which is highly expressed in HL and is differentially expressed between HL and Burkitt lymphoma. Using luciferase reporter assays, we validated 11 predicted miR-155 target genes in three different HL cell lines. We demonstrated that AGTR1, FGF7, ZNF537, ZIC3, and IKBKE are true miR-155 target genes in HL.

Radiotherapy in laryngeal carcinoma: can a panel of 13 markers predict response?

OBJECTIVES/HYPOTHESIS: To find biomarkers associated with response to radiotherapy in laryngeal cancer that can be used together with clinical parameters to improve outcome prediction. METHODS: In this study, 26 patients irradiated for laryngeal carcinomas with a local recurrence within two years (cases) and 33 patients without recurrence (controls) were included. All pretreatment biopsies were arrayed onto a tissue array. Immunohistochemistry was performed for 13 biomarkers that were selected from the literature as potential predictors for radioresponse in head and neck (HN) cancer: Bcl-2, Bcl-xL, p16, p21, p27, p53, cyclin D1, HIF-1alpha, CA9, COX-2, EGFR, ki-67, and pRB. RESULTS: Univariate logistic regression models showed borderline statistically significant increased relative risks, with positivity for CA9, COX-2, and p53. Goeman's global testing revealed an overall association between outcome and the 13 markers together with clinical variables. The most important markers were CA9 and COX-2. CONCLUSIONS: In laryngeal carcinoma, hypoxia and COX-2 overexpression provide a stronger contribution to an increased risk of local recurrence after radiotherapy compared with the well-known candidate markers p53, Bcl-2, and cyclin D1. However, no robust expression profile for the prediction of radioresistance was found.

Amplicon mapping and expression profiling identify the Fas-associated death domain gene as a new driver in the 11q13.3 amplicon in laryngeal/pharyngeal cancer.

PURPOSE: Amplification of the 11q13 region is a frequent event in human cancer. The highest incidence (36%) is found in head and neck squamous cell carcinomas. Recently, we reported that the amplicon size in 30 laryngeal and pharyngeal carcinomas with 11q13 amplification is determined by unique genomic structures, resulting in the amplification of a set of genes rather than a single gene. EXPERIMENTAL DESIGN: To investigate which gene(s) drive the 11q13 amplicon, we determined the smallest region of overlap with amplification and the expression levels of all genes within this amplicon. RESULTS: Using array-based comparative genomic hybridization analysis, we detected a region of approximately 1.7 Mb containing 13 amplified genes in more than 25 of the 29 carcinomas. Quantitative reverse transcription-PCR revealed that overexpression of 8 potential driver genes including, cyclin D1, cortactin, and Fas-associated death domain (FADD), correlated significantly with DNA amplification. FADD protein levels correlated well with DNA amplification, implicating that FADD is also a candidate driver gene in the 11q13 amplicon. Analysis of 167 laryngeal carcinomas showed that increased expression of FADD (P = 0.007) and Ser(194) phosphorylated FADD (P = 0.011) were associated with a worse disease-specific survival. FADD was recently reported to be involved in cell cycle regulation, and cancer cells expressing high levels of the Ser(194) phosphorylated isoform of FADD proved to be more sensitive to Taxol-induced cell cycle arrest. CONCLUSION: Because of the frequent amplification of the 11q13 region and concomitant overexpression of FADD in head and neck squamous cell carcinomas, we hypothesize that FADD is a marker to select patients that might benefit from Taxol-based chemoradiotherapy.

Microarray amplification bias: loss of 30% differentially expressed genes due to long probe - poly(A)-tail distances.

BACKGROUND: Laser microdissection microscopy has become a rising tool to assess gene expression profiles of pure cell populations. Given the low yield of RNA, a second round of amplification is usually mandatory to yield sufficient amplified-RNA for microarray approaches. Since amplification induces truncation of RNA molecules, we studied the impact of a second round of amplification on identification of differentially expressed genes in relation to the probe - poly(A)-tail distances. RESULTS: Disagreement was observed between gene expression profiles acquired after a second round of amplification compared to a single round. Thirty percent of the differentially expressed genes identified after one round of amplification were not detected after two rounds. These inconsistent genes have a significant longer probe - poly(A)-tail distance. qRT-PCR on unamplified RNA confirmed differential expression of genes with a probe - poly(A)-tail distance >500 nucleotides appearing only after one round of amplification. CONCLUSION: Our data demonstrate a marked loss of 30% of truly differentially expressed genes after a second round of amplification. Therefore, we strongly recommend improvement of amplification procedures and importance of microarray probe design to allow detection of all differentially expressed genes in case of limited amounts of RNA.

High-resolution mapping identifies a commonly amplified 11q13.3 region containing multiple genes flanked by segmental duplications.

DNA amplification of the 11q13 region is observed frequently in many carcinomas. Within the amplified region several candidate oncogenes have been mapped, including cyclin D1, TAOS1 and cortactin. Yet, it is unknown which gene(s) is/are responsible for the selective pressure enabling amplicon formation. This is probably due to the use of low-resolution detection methods. Furthermore, the size and structure of the amplified 11q13 region is complex and consists of multiple amplicon cores that differ between different tumor types. We set out to test whether the borders of the 11q13 amplicon are restricted to regions that enable DNA breakage and subsequent amplification. A high-resolution array of the 11q13 region was generated to study the structure of the 11q13 amplicon and analyzed 29 laryngeal and pharyngeal carcinomas and nine cell lines with 11q13 amplification. We found that boundaries of the commonly amplified region were restricted to four segments. Three boundaries coincided with a syntenic breakpoint. Such regions have been suggested to be putatively fragile. Sequence comparisons revealed that the amplicon was flanked by two large low copy repeats known as segmental duplications. These segmental duplications might be responsible for the typical structure and size of the 11q13 amplicon. We hypothesize that the selection for genes through amplification of the 11q13.3 region is determined by the ability to form DNA breaks within specific regions and, consequently, results in large amplicons containing multiple genes.