Johan H. Gibcus

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.

Hi–C: A comprehensive technique to capture the conformation of genomes

We describe a method, Hi-C, to comprehensively detect chromatin interactions in the mammalian nucleus. This method is based on Chromosome Conformation Capture, in which chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, enabling selective purification of chimeric DNA ligation junctions followed by deep sequencing. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. This advance gives Hi-C the power to both explore the biophysical properties of chromatin as well as the implications of chromatin structure for the biological functions of the nucleus. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease.

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.

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.

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 Burkitts 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.

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 ∼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 Ser194 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 Ser194 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.

Cortactin expression predicts poor survival in laryngeal carcinoma

Amplification of the 11q13 region is one of the most frequent aberrations in squamous cell carcinomas of the head and neck region (HNSCC). Amplification of 11q13 has been shown to correlate with the presence of lymph node metastases and decreased survival. The 11q13.3 amplicon carries numerous genes including cyclin D1 and cortactin. Recently, we reported that FADD becomes overexpressed upon amplification and that FADD protein expression predicts for lymph node positivity and disease-specific mortality. However, the gene within the 11q13.3 amplicon responsible for this correlation is yet to be identified. In this paper, we compared, using immunohistochemical analysis for cyclin D1, FADD and cortactin in a series of 106 laryngeal carcinomas which gene correlates best with lymph node metastases and increased disease-specific mortality. Univariate Cox regression analysis revealed that high expression of cyclin D1 (P=0.016), FADD (P=0.003) and cortactin (P=0.0006) predict for increased risk to disease-specific mortality. Multivariate Cox analysis revealed that only high cortactin expression correlates with disease-specific mortality independent of cyclin D1 and/or FADD. Of genes located in the 11q13 amplicon, cortactin expression is the best predictor for shorter disease-specific survival in late stage laryngeal carcinomas.

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.

A pathway for mitotic chromosome formation

Tracking mitotic chromosome formation How cells pack DNA into fully compact, rod-shaped chromosomes during mitosis has fascinated cell biologists for more than a century. Gibcus et al. delineated the conformational transition trajectory from interphase chromatin to mitotic chromosomes minute by minute during the cell cycle. The mitotic chromosome is organized in a spiral staircase architecture in which chromatin loops emanate radially from a centrally located helical scaffold. The molecular machines condensin I and II play distinct roles in these processes: Condensin II is essential for helical winding, whereas condensin I modulates the organization within each helical turn. Science, this issue p. eaao6135 Mitotic chromosome folding involves formation of increasingly compacted helically arranged nested loop arrays. INTRODUCTION During mitosis, cells compact their chromosomes into dense rod-shaped structures to ensure their reliable transmission to daughter cells. Our work explores how cells achieve this compaction. We integrate genetic, genomic, and computational approaches to characterize the key steps in mitotic chromosome formation from the G2 nucleus to metaphase, and we identify roles of specific molecular machines, condensin I and II, in these major conformational transitions. RATIONALE We used chicken DT-40 cells expressing an analog-sensitive CDK1 to produce cell cultures that synchronously enter mitosis. We collected cells at key time points during mitotic entry; analyzed chromosome organization by microscopy, chromosome conformation capture, and polymer simulations; and delineated a pathway of mitotic chromosome formation. We used engineered cell lines to study the function of condensin complexes, which are critical for mitotic chromosome formation. We fused condensin I and II subunits to plant auxin-inducible degron domains, thus enabling their rapid depletion in late G2 just before mitotic entry. These cell lines allowed us to determine the roles of condensin I and II in specific steps of the mitotic chromosome morphogenesis pathway. RESULTS Our analysis of G2 chromosomes reveals hallmarks of interphase chromosomes, including topologically associating domains and compartments. Upon entry into prophase, this organization is lost within minutes, and by late prophase, chromosomes are folded as arrays of consecutive loops condensed around a central axis. These loops project with random but mutually correlated angles from the axis. During prometaphase, the loop array undergoes two major reorganizations. First, it acquires a helical arrangement of loops. Polymer simulations of Hi-C data show that the centrally located axis acquires a helical twist so that consecutive loops emanate as the steps of a spiral staircase. Second, the chromatin loops become nested with ~400-kb outer loops split up by ~80-kb inner loops. As prometaphase proceeds, chromosomes shorten through progressive helical winding, with the numbers of loops per turn increasing. As a result, the size of a helical turn grows from ~3 Mb (~40 loops) to ~12 Mb (~150 loops). Depletion of condensin I or II before mitotic entry revealed their differing roles in mitotic chromosome formation. Either condensin can mediate loop array formation. However, condensin II is required for the helical twisting of the scaffold from which loops emanate, whereas condensin I modulates the size and arrangement of nested inner loops. CONCLUSION We describe a pathway of mitotic chromosome folding that unifies many previous observations. In prophase, condensins mediate the loss of interphase organization and the formation of arrays of consecutive loops. In prometaphase, chromosomes adopt a spiral staircase–like structure with a helically arranged axial scaffold of condensin II at the bases of chromatin loops. The condensin II loops are further compacted by condensin I into clusters of smaller nested loops that are additionally collapsed by chromatin-to-chromatin attractions. The combination of nested loops distributed around a helically twisted axis plus dense chromatin packing achieves the 10,000-fold compaction of chromatin into linearly organized chromosomes that is required for accurate chromosome segregation when cells divide. A pathway for mitotic chromosome formation. In prophase, condensins mediate the loss of interphase chromosome conformation, and loop arrays are formed. In prometaphase, the combined action of condensin I (blue spheres in the bottom diagram) and II (red spheres) results in helically arranged nested loop arrays. 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.

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

Hodgkins 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